EA049130B1 - GLP-1 AND GIP RECEPTOR COAGONISTS SUITABLE FOR ORAL DELIVERY - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to novel compounds that are glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic polypeptide (GIP) receptor agonists with a long-acting action profile suitable for oral administration to humans.

Including a sequence listing via reference

This application is filed with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference.

State of the art

Glucagon-like peptide-1 (GLP-1) is a hormone synthesized by enteroendocrine cells of the intestine and is one of two known endogenous physiological incretins. GLP-1 improves glycemic control by stimulating glucose-dependent insulin secretion in response to nutrient intake (glucose), suppresses glucagon secretion by pancreatic alpha cells, slows the rate of gastric emptying, and induces weight loss primarily by reducing food intake. Glucose-dependent insulinotropic polypeptide (GIP), the other known incretin, improves glycemic control by stimulating insulin secretion in response to nutrient intake (fat, glucose). In addition, GIP appears to improve plasma lipid profiles and stimulate calcium accumulation in bone. In contrast to GLP-1, the incretin effect of GIP is significantly reduced in patients with type 2 diabetes, although recent studies have shown that the efficacy of GIP in such patients can be restored after treatment with improved glucose control. However, the role of GIP in the regulation of systemic metabolism, beyond its direct effects on the endocrine pancreas, remains controversial, particularly due to the effect of GIP on promoting fat gain in animal models. Such findings support the notion that GIPR antagonism may provide improvements in body weight. Thus, the use of compounds acting on GIP receptors, and in particular those providing either agonism or antagonism, as a strategy to improve body weight remains a controversial topic of intense scientific research (Finan et al, TRENDS Mol Med, 2016, 22(5):359-376; Killion et al, Endo Rev, 2020, 41(1):1-21).

In rodent models, long-acting GIP analogues have been shown to reduce body weight and improve glycaemic control, albeit with comparatively less potency than GLP-1 analogues in reducing body weight (Mroz et al, Mol Metab, 2019, 20: 51-62). Moreover, GIP analogues induce body weight loss through additive/synergistic effects with GLP-1 analogues when the two components are administered (Finan et al, Sci Transl Med, 2013, 5 (209): 209ra151; N0rregaard et al, Diabetes Obes Metab, 2018, 20 (1): 60-68), and are therefore suitable candidates for expanding the pharmacological properties of GLP-1. GIPR agonism may be recruited as a redundant partner to provide GLP-1R agonism as a single-molecule co-agonist to broaden the spectrum of beneficial effects of GLP-1 on metabolism, as demonstrated in preclinical animal models, most notably in promoting body weight loss and glycemic control (Finan et al, Sci Transl Med, 2013, 5(209):209ra151; Coskun et al, Mol Metab, 2018, 18:3-14). Two different peptides with potent dual incretin receptor agonism have been translated into clinical studies using multiple doses. Clinical results have demonstrated improvements in glycemic control and body weight that exceed those obtained with comparable doses of standard specific GLP-1 agonists (Frias et al, Cell Metab, 2017, 26(2):343–352; Frias et al, Lancet, 2018, 392(10160):2180–2193), demonstrating the translational implications and therapeutic benefits of co-targeting GLP-1 and GIP receptors.

A clinical study was conducted using oral delivery of GLP-1 derivatives as a once-daily tablet of semaglutide and the permeation enhancer sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) (Hedrington & Davis, Exp. Opin. Pharmacother. 2019, 20 (2): 133-141) to improve the very low plasma concentrations and bioavailability characteristic of GLP-1 derivatives after oral administration.

GLP-1/GIP co-agonists and their potential medicinal uses have been described in several patent applications, such as WO 2010/011439, WO 2013/164483, WO 2014/192284, WO 2015/067715, WO 2015/022420, WO 2015/086728, WO 2015/086729, WO 2016/111971, WO 2020/023386, US 9745360, US 2014/162945 and US 2014/0357552. Patent applications disclosing oral delivery of GLP-1 derivatives are described, for example, in WO 2011/080103, WO 2012/080471, WO 2013/189988 and WO 2019/149880.

However, no co-agonist products have yet been approved for marketing.

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The essence of the invention

The present invention relates to single-molecule co-agonists comprising a peptide and a substituent that interact with human GLP-1 and GIP receptors with high potency and are suitable for daily oral administration to humans. This is achieved by combining certain sequence variants of the peptides with substituents via single-site acylation with a dibasic fatty acid.

One aspect of the present invention relates to a peptide having the amino acid sequence:

YX2EGTX ₆ TSDYSX ₁ 2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX28GGPX32X33X34X35X36X37X38X (SEQIDNO: 36) with optional C-terminal amide modification;

where X2 represents Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X24 represents E, Q or N;

X28 represents A or R;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X34 is G or absent;

X ₃₅ is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent.

In another aspect, the present invention relates to a peptide having the amino acid sequence:

YX2EGTX ₆ TSDYSX ₁₂ Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX2 ₈ GGPX32X33X34X35X36X37X38X (SEQ ID NO: 36) with an optional C-terminal amide modification, where X ₂ is Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X ₃₅ is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent;

and a substituent attached to the peptide via lysine (K) at position 16 or 33;

or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention relates to a method for producing the GLP1/GIP co-agonists described herein.

In a further aspect, the present invention relates to a pharmaceutical composition comprising the GLP-1/GIP co-agonist compounds described herein.

A further aspect of the present invention relates to the use in medicine of the GLP-1/GIP co-agonists described herein.

In one aspect, the present invention relates to the use of the GLP-1/GIP co-agonists described herein for the prevention or treatment of diabetes, obesity and/or liver disease.

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Description

Further, Greek letters can be represented by their symbol or the corresponding handwritten name, for example, a=alpha; b=beta; ε=epsilon; y=gamma; ω=omega, etc. Also, the Greek letter μ can be represented by μk, for example, μл= μл or μМ=μМ.

GLP-1/GIP receptor co-agonist.

The present invention relates to compounds that are agonists of the GLP-1 receptor and the GIP receptor, also referred to as GLP-1/GIP receptor co-agonists or simply co-agonists.

The term compound as used herein means a chemical compound, so compounds may contain different structural elements in addition to the minimum element defined for each compound or group of compounds. It follows that a compound may be a peptide or a derivative thereof, provided that the compound contains certain structural and/or functional elements.

The term compound is also intended to encompass pharmaceutically acceptable forms thereof, i.e., a compound as defined herein or a pharmaceutically acceptable salt, amide or ester thereof.

The term analogue generally means a peptide whose sequence is characterized by one or more changes in amino acids compared to a reference amino acid sequence. An analogue may also include amino acid extensions at the N-terminal and/or C-terminal positions and/or truncations at the N-terminal and/or C-terminal positions.

Generally, amino acid residues can be identified by their full name, their one-letter code, and/or their three-letter code. These three methods are completely equivalent.

Amino acids are molecules containing an amino group and a carboxylic acid group and optionally one or more additional groups, often called a side chain.

The term amino acid includes proteinogenic (or natural) amino acids (including 20 standard amino acids) as well as non-proteinogenic (or unnatural) amino acids. Proteinogenic amino acids are amino acids that are naturally found in proteins.

Standard amino acids are amino acids encoded by the genetic code. Non-proteinogenic amino acids are either not found in proteins or are not synthesized by the standard cellular machinery (e.g., they may be post-translationally modified). Non-limiting examples of non-proteinogenic amino acids include Aib (α-aminoisobutyric acid or 2-aminoisobutyric acid), norleucine, norvaline, and the D-isomers of proteinogenic amino acids.

It should be further understood that each amino acid in the peptides for which the optical isomer is not indicated is understood to be the L-isomer (unless otherwise indicated).

The GLP-1/GIP receptor co-agonists described herein comprise or consist of a peptide and a substituent as defined below. In some embodiments, the peptide is a synthetic peptide designed to optimize the activity of the GLP-1 and GIP receptors. Compounds have been identified that have suitable receptor binding activity for both the GLP-1 receptor and the GIP receptor, as demonstrated in the examples herein.

The compounds additionally exhibit an increased half-life, which is obtained due to the substituent containing a fatty acid group.

In some embodiments, the carboxy terminus of the peptide comprises a -COOH group. In some embodiments, the compounds may optionally include an amide group (C(=O) _NH2 ) at the C-terminus, which is a naturally occurring modification with the substitution of -OH to -NH2, such as observed in natural exendin-4.

Peptide.

The GLP-1/GIP receptor co-agonists described herein provide a peptide and substituent as defined below, wherein the substituent is attached to the peptide backbone via an amino acid residue.

In some embodiments, the amino acid sequence of the peptide is:

YX2EGTX6TSDYSX12X13LEX16QAAX20X21FX23X24WLLX28GX30X31X32X33X34X35X36X37X ₃₈ X39 (SEQ ID NO: 47) with an optional amide modification at the C-terminus, where _X2 is Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

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X ₂₁ represents A or E;

X ₂₃ represents I or V;

X24 represents E, Q or N;

X28 represents A or R;

X ₃₀ is G or absent;

X ₃₁ is P or absent;

X32 is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X35 is A or absent;

X36 is P or absent;

X37 is P or absent;

X38 is P or absent;

X ₃₉ is S or absent.

In some embodiments, the amino acid sequence of the peptide is:

YX2EGTX6TSDYSX12X13LEX ₁ 6QAAX20X21FX23X24WLLX28GGPX32X33X34X35X36X37X38X (SEQ ID NO: 36) with optional C-terminal amide modification;

where X2 represents Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X24 represents E, Q or N;

X28 represents A or R;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X34 is G or absent;

X35 is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent.

In one embodiment, X ₃₉ is absent.

In one embodiment, X ₃₈ and X ₃₉ are absent. In one embodiment, X ₃₇ , X ₃₈ and X ₃₉ are absent. In one embodiment, X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent. In one embodiment, X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent. In one embodiment, X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X38 and X39 are absent. In one embodiment, X33, X34, X35, X36, X37, X38 and X39 are absent. In one embodiment, X ₃₂ , X ₃₃ , X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent. In one embodiment, _X31 , _X32 , _X33 , X34, _X35 , _X36 , _X37 , _X38 and _X39 are absent. In one embodiment _{, X30} , _X31 , _X32 , _X33 , _X34 , _X35 , _X36 , _X37 , _X38 and _X39 are _absent . In further such embodiments, _X32X33X34X35 is SSGA. In further such embodiments _{, X32X33X34X35} is ESGA. In further such embodiments _{, X32X33X34X35} is SKGA. In a further embodiment thereof _{, the} peptide _is characterized _{by an} amide modification _of the C-terminus.

In one embodiment, the amino acid sequence of the peptide is: YX2EGTX ₆ TSDYSX ₁ 2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX ₂₈ GGPSSGAPPPS (SEQ ID NO:

37), where X ₂ represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X24 represents E, Q or N;

X28 represents A or R.

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In one embodiment, the amino acid sequence of the peptide is: YX2EGTX6TSDYSX12X13LEX16QAAX20X21FX23X24WLLX28GGPESGAPPPS (SEQ ID NO:

38), where X2 is Aib or A, _X6 is F or V, _X12 is I or Y, _X13 is Y, A, L or I, _X16 is K or E, _X20 is Q, R, E, H, _X21 is A or E, _X23 is I or V, X24 is E, Q or N, X28 is A or R.

In one embodiment, the amino acid sequence of the peptide is: YX2EGTX6TSDYSXi2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX28GGPSKrAPPPS (SEQ ID NO:

39), where X2 represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X28 represents A or R.

In one embodiment, X2 is Aib. In one embodiment, X2 is A. In one embodiment, _X6 is F. In one embodiment, _X6 is V. In one embodiment, _X12 is I. In one embodiment, _X12 is Y. In one embodiment, _X13 is Y. In one embodiment, _X13 is A. In one embodiment, _X13 is L. In one embodiment, _X13 is I. In one embodiment, _X16 is K. In one embodiment, X16 is E. In one embodiment, _X20 is Q. In one embodiment, X20 is R. In one embodiment, X20 is E. In one embodiment, _X20 is H. In one embodiment, _X21 is A. In one embodiment, _X21 is E. In one embodiment, _X23 is I. In one embodiment, _X23 is V. In one embodiment, X24 is E. In one embodiment, X24 is Q. In one embodiment, X ₂₄ is N. In one embodiment, X ₂₈ is A. In one embodiment, X ₂₈ is R. In one embodiment, X ₃₀ is G. In one embodiment, X ₃₁ is P.

In one embodiment, X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is selected from the group consisting of LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE, and LLEEQAAREFIE. In one embodiment, X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is LLEKQAAREFIN. In one embodiment, X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is LLEKQAAREFIE. In one embodiment, X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is LLEKQAAQEFIE. In one embodiment, X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is LLEEQAAREFIE.

In a further embodiment, the amino acid sequence of the peptide is any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 33, 34, and 35.

In one embodiment, the amino acid sequence of the peptide is any of SEQ ID NO: 10, 22, or 25.

In one embodiment, the amino acid sequence of the peptide is the sequence of SEQ ID NO.: 25.

In one embodiment, the amino acid sequence of the peptide is any of SEQ ID NOs: 18, 20, 23, 24.

In one embodiment, the amino acid sequence of the peptide is the sequence of SEQ ID NO.: 33, 34 or 35.

In such additional embodiments, the peptide is characterized by an amide modification at the C-terminus.

Derivatives.

In some embodiments, the GLP-1 and GIP receptor agonists comprise or consist of a substituent as defined below covalently linked to a peptide.

Such compounds can be called peptide derivatives because they are obtained by covalently linking a substituent to the peptide backbone.

One aspect of the present invention relates to a compound comprising a peptide and a substituent,

- 5 049130 where the amino acid sequence of the peptide is:

¥X2EGTX ₆ TSD¥SXi2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX ₂₈ GX3oX3iX32X33X34X35X36X37X ₃₈ X39 (SEQ ID NO: 47) with an optional C-terminal amide modification, where X2 is Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X24 represents E, Q or N;

X28 represents A or R;

X ₃₀ is G or absent;

X ₃₁ is P or absent;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X ₃₅ is A or absent;

X36 is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent;

and wherein the substituent is attached to the peptide via lysine (K) at position 16 or 33;

or a pharmaceutically acceptable salt thereof.

One embodiment of the present invention relates to a compound comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:

YX2EGTX ₆ TSDYSX ₁₂ Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX2 ₈ GGPX32X33X34X35X36X37X38X (SEQ ID NO: 36) with an optional C-terminal amide modification, where X2 is Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X21 represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R;

X32 is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X35 is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent,

X ₃₈ is P or absent,

X ₃₉ is S or absent;

and wherein the substituent is attached to the peptide via lysine (K) at position 16 or 33;

or a pharmaceutically acceptable salt thereof.

In further embodiments, the peptide may be defined as described herein above.

Deputy.

In one embodiment, the substituents described herein are attached to the peptide described herein via a lysine (K) residue at position 16 or 33.

In one embodiment, the substituent is attached to the peptide via the epsilon amino group of lysine (K), if said lysine is included at position 16 or 33.

In one embodiment, the substituent is a chemical structure covalently attached to the peptide that is capable of forming non-covalent complexes with plasma albumin, thereby ensuring circulation of the co-agonist in the bloodstream, as well as the effect of prolonging the time of action of the co-agonist due to the fact that the complex of the co-agonist and albumin is slowly eliminated only through renal clearance.

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In one embodiment, the substituent comprises a fatty acid group. In such an embodiment, the fatty acid group comprises a carbon chain that comprises at least 8 consecutive -CH2- groups. In one embodiment, the fatty acid group comprises at least 10 consecutive -CH2- groups, such as at least 12 consecutive -CH2- groups, at least 14 consecutive -CH2- groups, at least 16 consecutive -CH ₂ - groups, or such as at least 18 consecutive -CH ₂ - groups.

In one embodiment, the fatty acid moiety comprises 8-20 consecutive CH2- groups. In one embodiment, the fatty acid moiety comprises 10-18 consecutive _-CH2- groups. In one embodiment, the fatty acid moiety comprises 12-18 consecutive _-CH2- groups. In one embodiment, the fatty acid moiety comprises 14-18 consecutive -CH2- groups.

In some embodiments, the substituent is comprised of multiple elements, such as a prolonging element and one or more linker elements. In one embodiment, the term prolonging agent is used to describe the fatty acid group that is the terminal portion of the substituent responsible for prolonging the half-life of the compound.

In one embodiment, the prolonging agent (Prot) may be defined as:

Chem. 1: HOOC-(CH ₂ ) _n -CO-*, where n is an integer in the range 8-20, which can also denote a dibasic C(n+2)-acid, or as:

O o „LA·

Chem. 1b: ’ where n is an integer in the range 8–20.

In one embodiment, the substituent further comprises one or more linker elements. In some embodiments, the linker elements are linked to each other and the prolonging agent via amide bonds and are designated as Z (see further below).

As further defined herein below, the number of linker elements may be no more than 3, designated -Z1-Z2-Z3-, where Z1 is linked to a prolonging agent (Prot) and the last Z element is linked to a peptide, in which case the substituent may be designated as Prot-Z1-Z2-Z3-. Thus, the symbol * above indicates the point of attachment to Z1, which in the case of an amide bond is a nitrogen atom. In one embodiment, where Z1 is a bond (see below), the symbol * indicates the point of attachment to the nitrogen atom of the adjacent Z element.

In one embodiment, the substituent is defined as Prot-Z1-Z2-Z3-, where Prot- is selected from Chem. 1, Chem. 1b, and where n is an integer in the range 16-20.

In a particular embodiment, in Chem. 1 or Chem. 1b, n is 14, 15, 16, 17, 18, 19, or 20.

In a particular embodiment, in Chem. 1 or Chem. 1b, n is 14, 15, 16, 17, or 18.

In a specific embodiment in Chem. 1 or Chem. 1b, n is 14, 16, or 18.

In a particular embodiment, in Chem. 1 or Chem. 1b, n is 16, 17, 18, 19, or 20.

In a specific embodiment, in Chem. 1 or Chem. 1b, n is 16, 18, or 20.

In a specific embodiment, in Chem. 1 or Chem. 1b, n is 18 or 20.

In a specific embodiment, the prolonging agent (Prot) is a C16 dibasic acid or a C18 dibasic acid.

In a specific embodiment, the prolonging agent (Prot) is a C18 dibasic acid or a C20 dibasic acid.

In a specific embodiment, the prolonging agent (Prot) is a C16, C18 dibasic acid or a C20 dibasic acid.

As used herein, the term "linkage" means a covalent bond. If a linker element Z1-Z3 is defined as a linkage, this is equivalent to the situation where said linker element is absent. The statement herein below that any of Z1-Z3 is a linkage can also be read as any of Z1-Z3 is absent, i.e., the preceding element Z is covalently linked to the following element Z, which is not a linkage (or absent).

In some embodiments, the linker elements Z1-Z3 are individually selected from chemical moieties capable of forming amide bonds, including amino acid-like moieties such as Glu, yGlu (also referred to as gamma-Glu or gGlu and defined as *-NH-CH(COOH) _-CH2 - _CH2 -CO-*), ε-Lys (also referred to as epsilon-Lys or eLys and defined as *-NH( _CH2 ) ₄ -CH( _NH2 )-CO-*), Ser, Ala, Thr, Ado, Aeep and Aeeep and additional moieties described below.

In one embodiment, Z1 is selected from yGlu, Glu, or a linkage. In one embodiment, Z1 is yGlu.

In one embodiment, Z2 and Z3 are independently selected from Glu, ε-Lys, yGlu, Gly, Ser, Ala, Thr, Ado, Aeep, Aeeep and a bond.

Glu, Gly, Ser, Ala, Thr are amino acid residues well known from the level

- 7 049130 equipment.

ε-Lys is defined by Chem. 2: *-NH-(CH ₂ ) ₄ -CH(NH ₂ )-CO-*, which can also be described by:

_NH2

Chem. 2b:

yGlu is defined as Chem. 3: *-NH-CH(COOH)-(CH ₂ ) ₂ -CO-*, which can also be described as:

Chem. 3b: ·

Ado is defined by Chem. 4: *-NH-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-CH ₂ -CO-*, which can also be designated as 8-amino-3,6-dioxaoctanoic acid and which can also be described by:

Chem. 4b: ·

Aeer is defined under Chem. 5: *NH-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ CO*, which can also be described under:

Chem. 5b:

Aeeer is defined as Chem. 6: *NH-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ CO*, which may also be described as:

Chem. 6b:

In one embodiment, Z2 and Z3 are independently selected from Glu, ε-Lys, yGlu, Gly, Ala, Ado, Aeep, Aeeep and a bond.

In one embodiment, Z2 and Z3 are independently selected from Glu, ε-Lys, yGlu, Gly, Ala, Ado, and a bond.

In one embodiment, Z2 and Z3 are independently selected from Glu, ε-Lys, yGlu, Gly, Ado, and a bond.

In one embodiment, Z2 and Z3 are independently selected from ε-Lys, yGlu, Gly, Ado, and a bond.

In one embodiment, Z2 and Z3 are independently selected from ε-Lys, yGlu, Ado, and a bond.

In one embodiment, Z2 and Z3 are ε-Lys or Ado. In one embodiment, Z2 and Z3 are Ado. In one embodiment, Z2 and Z3 are ε-Lys.

In one embodiment, the substituent is selected from substituents A, B, C, D and E, defined below.

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In one embodiment, the substituent is selected from substituents A, B, and C, defined as follows:

Deputy # Prot Z1 Z2 Z3 A Dibasic C 18-acid vGlu Ado Ado IN Dibasic C 18-acid vGlu sLys sLys WITH Dibasic C20 acid vGlu sLys sLys D Dibasic C16 acid vGlu Ado Ado E Dibasic C16 acid vGlu sLys sLys

In some embodiments, the substituent is covalently attached to the lysine residue of the co-agonist by acylation, i.e., via an amide bond formed between the carboxylic acid group of the substituent and the epsilon-amino group of the lysine residue.

In one embodiment, the substituent is covalently attached to the lysine residue at position 16 of the peptide backbone by acylation, i.e., via an amide bond formed between the carboxylic acid group of the substituent and the epsilon-amino group of the lysine residue.

In one embodiment, the substituent is covalently attached to the lysine residue at position 33 of the peptide backbone by acylation, i.e., via an amide bond formed between the carboxylic acid group of the substituent and the epsilon-amino group of the lysine residue.

Coagonists may exist in different stereoisomeric forms having the same molecular formula and sequence of linked atoms, but differing only in the three-dimensional orientation of their atoms in space. The stereoisomerism of the exemplified coagonists is indicated in the experimental section in the names as well as in the structures using standard nomenclature. Unless otherwise indicated, the present invention pertains to all stereoisomeric forms of the derivative described.

Functional activity, which is the activation of receptors.

The functional activity of the GLP-1/GIP receptor agonists described herein can be tested in vitro as described herein in Example 2.

The term half-maximal effective concentration (EC50) generally refers to the concentration that induces a response midway between the baseline and the maximal response, based on the dose-response curve. The EC ₅₀ is used as a measure of the potency of a compound and is the concentration at which 50% of its maximal effect is observed.

Thus, the potency of the compounds in vitro can be determined as described in this document and the EC50 determined. The lower the EC50 value, the higher the potency.

To characterize such compounds, it may be useful to further consider in vitro potency values relative to native hormones for each receptor.

In vitro efficacy can be determined, for example, in a medium containing membranes expressing the relevant GLP-1 receptor and/or GIP and/or in an assay using whole cells expressing the relevant GLP-1 receptor and/or GIP.

For example, the functional response of the human GLP-1 and/or GIP receptor can be measured in a reporter gene assay, such as in a stably transfected BHK cell line that expresses the human GLP-1 and/or GIP receptor and contains DNA encoding a cAMP-responsive element (CRE) linked to a promoter and a gene encoding firefly luciferase (CRE-luciferase). When cAMP is produced as a result of activation of the GLP-1 and/or GIP receptor, this in turn leads to expression of luciferase. Luciferase can be detected by adding luciferin, which is converted enzymatically to oxyluciferin and induces bioluminescence that is measured as a reporter of in vitro potency. One example of such an assay is described in Example 2 described herein. Since compounds may include a substituent designed to bind albumin, it is also important to note that receptor activity may be affected by the presence or absence of human serum albumin (HSA) in the assay medium. A decrease in compound potency in the presence of HSA, as indicated by an increase in EC50 compared to the EC50 in the absence of HSA, indicates the interaction of the compounds with HSA and determines the long-acting profile in vivo.

In one embodiment, the compounds exhibit potent in vitro effects that include activation of human GLP-1 and GIP receptors.

- 9 049130

In one embodiment, the compounds are capable of activating human GLP-1 and GIP receptors in vitro with an EC50 of less than 20 pM according to the CRE-luciferase reporter assays described in Example 2 herein, when performed without HSA.

In one embodiment, the compounds have an in vitro potency at the human GLP-1 and GIP receptors, determined using the method of Example 2, corresponding to an EC50 of 100 pM or less, more preferably less than 50 pM, or most preferably less than 20 pM.

In one embodiment, the EC ₅₀ in the human GLP-1 and GIP receptor activation assays is in both cases 1-25 pM, such as 1-20 pM, such as 1-15 pM, or such as 110 pM.

Pharmacokinetic properties.

The pharmacokinetic properties of co-agonist compounds can be further determined in vivo using pharmacokinetic (PK) studies. Animal models such as mouse, rat, monkey, dog or pig can be used to determine such characteristics.

In such studies, animals are typically administered a single dose of the drug either intravenously (i.v.), subcutaneously (s.c.), or orally (p.o.) in an appropriate formulation. Blood samples are collected at predetermined time points after dosing and the samples are analyzed for drug concentration using an appropriate quantitative assay. Based on these measurements, plasma concentration-time profiles for the test compound are plotted and so-called non-compartmental pharmacokinetic analysis of the data is performed. An important parameter is the terminal half-life, since a long half-life indicates that less frequent administration of the compound may be possible. The terminal half-life (t ¹ / ₂ ) in vivo can be measured using a suitable model, such as after i.v. administration to minipigs, as described in Example 3; or after p.o. administration to dogs, as described in Example 4.

In one embodiment, _the terminal half-life is the half-life (t ^1/2 ) in vivo in minipigs following intravenous administration, such as described in Example 3 herein.

In one embodiment, the terminal half-life in the minipigs is at least 24 hours, such as at least 30 hours, or such as at least 40 hours.

In one embodiment, _the terminal half-life is the half-life (t ^1/2 ) in vivo in dogs after p.o. administration, such as described in Example 4 herein.

In one embodiment, the terminal half-life in dogs is at least 24 hours, such as at least 40 hours, or such as at least 50 hours.

Pharmaceutically acceptable salts.

In some embodiments, the co _- agonists described herein are provided in the form of a pharmaceutically acceptable salt. Salts are, for example, formed by a chemical reaction between a base and an acid, such as: { _2NH3 + _H2SO4 }( _{NH4 )2SO4 .} The salt may be a basic salt, an acidic salt, or may be neither (i.e., a neutral salt). Basic salts form hydroxydiones and hydronium ions in salts of acids in water. Salts of the co-agonists may be formed by adding cations or anions in a reaction with anionic or cationic groups, respectively. Such groups may be in the peptide and/or in a substituent of the derivatives. Non-limiting examples of anionic groups include any free carboxylic acid groups in the substituent, if present, 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 groups at internal acidic amino acid residues such as Asp and Glu.

Non-limiting examples of cationic groups include any free amino groups in the substituent, if present, 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 at internal basic amino acid residues such as His, Arg, and Lys.

In a specific embodiment, the peptide or derivative is in the form of a pharmaceutically acceptable salt.

Methods of obtaining.

Coagonists can be prepared, for example, by classical peptide synthesis, such as solid phase peptide synthesis using t-Boc or Fmoc protecting groups, or other well-known techniques, see, for example, 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 Fmoc Solid Phase Peptide Synthesis, Edited by W.C. Chan and P.D. White, Oxford University Press, 2000.

Alternatively, the compounds can be produced by recombinant methods, for example by culturing a host cell containing a DNA sequence encoding

- 10 049130 peptide sequence, and capable of expressing the peptide in a suitable nutrient medium under conditions that allow expression of the peptide. Non-limiting examples of host cells suitable for expressing these peptides are Escherichia coli, Saccharomyces cerevisiae, and BHK or CHO mammalian cell lines.

Coagonists that include unnatural amino acids and/or covalently attached substituents can be prepared as described in the experimental section.

Specific examples of methods for obtaining a number of co-agonists are included in the experimental section.

A further aspect of the present invention relates to a method for producing the peptides described herein.

A further aspect of the present invention relates to a method for producing the GLP1/GIP co-agonists described herein.

In one embodiment, the method for preparing a compound described herein includes a solid-phase peptide synthesis step. The substituent may be incorporated sequentially during the solid-phase peptide synthesis or prepared separately and attached via a lysine residue after peptide synthesis.

In one embodiment, the compounds are prepared by a two-step process in which two peptide fragments are ligated after a substituent has been attached to one of the peptide fragments.

Pharmaceutical compositions.

In a further aspect, the present invention relates to a pharmaceutical composition comprising a GLP-1/GIP receptor co-agonist as described herein. Compositions comprising the compound or a pharmaceutically acceptable salt thereof and optionally one or more pharmaceutically acceptable excipients can be prepared according to methods known in the art.

The term excipient in a broad sense means any component other than the active therapeutic ingredient(s). An excipient may be an inert substance, an inactive substance and/or a substance that is inactive in terms of therapy. An excipient may serve various purposes, such as a carrier, a carrier medium, a filler, a binder, a lubricant, a glidant, a disintegrant, a flow control agent, a crystallization inhibitor, a solubilizer, a stabilizer, a coloring agent, a flavoring agent, a surfactant, an emulsifier or a combination thereof, and/or to improve the administration and/or to improve the absorption of the active substance. The amount of each excipient used may vary within the ranges typical in the art. Methods and excipients that can be used to formulate oral dosage forms are described in Handbook of Pharmaceutical Excipients (e.g. 8th edition, Sheskey et al., Eds., American Pharmaceuticals Association and Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2017) and any later editions) and Remington: The Science and Practice of Pharmacy (e.g. 22nd edition, Remington and Allen, Eds., Pharmaceutical Press (2013) and any later editions).

In one embodiment, the pharmaceutical composition may be a solid formulation, such as a lyophilized or spray-dried composition, which can be used as is or to which solvents and/or diluents are added by the physician or patient prior to use. In a further embodiment, the pharmaceutical composition may be a solid formulation consisting of an active ingredient, a salt of N-[8-(2-hydroxybenzoyl)amino]caprylate and one or more additional excipients known in the art, such as by using any one or more of the formulations described in WO 2012/080471, WO 2013/189988 or WO 2019/149880.

Alternatively, the pharmaceutical composition is a liquid formulation, such as an aqueous formulation. Liquid formulations suitable for injection can be prepared using techniques conventional in the pharmaceutical industry that include dissolving and mixing suitable ingredients to produce the desired final product. Thus, according to one procedure, the GLP-1/GIP co-agonist described herein is dissolved in a suitable buffer at a suitable pH. The composition can be sterilized, for example, by sterilizing filtration.

Pharmaceutical indications for use.

A further aspect of the present invention relates to the use of the GLP-1/GIP receptor co-agonist compounds described herein as a medicament.

In one embodiment, the compounds described herein are intended for use in the following therapeutic treatments:

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

- 11 049130 (ii) delaying or preventing the progression of a diabetic disease, such as progression to type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to type 2 diabetes requiring insulin administration, delaying or preventing insulin resistance and/or delaying the progression of type 2 diabetes not requiring insulin administration to type 2 diabetes requiring insulin administration;

(iii) prevention and/or treatment of eating disorders such as obesity, for example by reducing food intake, reducing body weight, suppressing appetite, inducing a feeling of satiety; treating or preventing binge eating disorder, bulimia nervosa and/or antipsychotic or steroid-induced obesity; reducing gastric motility; delaying gastric emptying; increasing physical mobility and/or preventing and/or treating obesity-related pathologies such as osteoarthritis and/or urinary incontinence;

(iv) weight maintenance after successful weight loss (induced by medication or diet and exercise), i.e. preventing weight gain after successful weight loss;

(v) prevention and/or treatment of liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.

In one embodiment, the compounds are intended for use in a method for preventing and/or treating diabetes and/or obesity.

In one embodiment, the compounds are for use in a method of treating diabetes and/or obesity.

In one embodiment, the compounds are for use in a method of treating or preventing type 2 diabetes.

In one embodiment, the compounds are for use in a method of treating type 2 diabetes.

In one embodiment, the compounds are for use in a method of treating or preventing obesity.

In one embodiment, the compounds are for use in a method of treating obesity.

In one embodiment, the compounds are for use in a method of weight management. In one embodiment, the compounds are for use in a method of appetite reduction. In one embodiment, the compounds are for use in a method of reducing food intake.

Implementation options

1. A compound comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:

¥X2EGTX ₆ TSD¥SX ₁ 2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX ₂₈ GX3oX3iX32X33X34X35X36X37X ₃₈ X39 (SEQ ID NO: 47) with an optional C-terminal amide modification, where X2 is Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R;

X ₃₀ is G or absent;

X ₃₁ is P or absent;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X34 is G or absent;

X ₃₅ is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent;

and wherein the substituent is attached to the peptide via lysine (K) at position 16 or 33;

or a pharmaceutically acceptable salt thereof.

2. A compound according to embodiment 1, comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:

- 12 049130 ¥X2EGTX ₆ TSD¥SX ₁ 2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX ₂₈ GGPX32X33X34X35X36X37X38X (SEQ ID NO: 36) with an optional amide modification of the C-terminus, where X2 is Aib or A;

X6 represents F or V;

X _{1 2} represents I or Y;

X _{1 3} represents Y, A, L, or I;

X _{1 6} represents K or E;

X _{2 0} represents Q, R, E, H;

X2 1 represents A or E;

X _{2 3} represents I or V;

X2 4 represents E, Q or N;

X28 represents A or R;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X ₃₅ is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent, X ₃₈ is P or absent;

X ₃₉ is S or absent;

and wherein the substituent is attached to the peptide via lysine (K) at position 16 or 33;

or a pharmaceutically acceptable salt thereof.

3. The connection according to embodiment 1, wherein X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

4. The connection according to embodiment 1, wherein X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

5. The connection according to embodiment 1, wherein X ₃₂ , X ₃₃ , X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

6. The connection according to embodiment 1, wherein X ₃₀ , X ₃₁ , X ₃₂ , X ₃₃ , X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

7. A compound according to any one of embodiments 1-6, wherein the peptide is characterized by an amide modification at the C-terminus.

8. A connection according to any of the previous embodiments 1, 2 or 7, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is selected from the group consisting of SSGA, ESGA and SKGA.

9. A connection according to any of the previous embodiments 1, 2 or 7, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is an SSGA.

10. A connection according to any of the previous embodiments 1, 2 or 7, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ represents ESGA.

11. A connection according to any of the previous embodiments 1, 2 or 7, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is SKGA.

12. The compound according to embodiment 1, wherein the amino acid sequence of the peptide is:

YX2EGTX ₆ TSDYSXi2Xi3LEXi ₆ QAAX2oX2iFX23X24WLLX ₂₈ GGPSSGAPPPS (SEQ Ш NO:

37), where X ₂ represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X16 represents K or E;

X20 represents Q, R, E, H;

X ₂₁ represents A or E;

X23 represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R.

13. The compound according to embodiment 1, wherein the amino acid sequence of the peptide is:

YX2EGTX ₆ TSDYSX ₁ 2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX ₂₈ GGPESGAPPPS (SEQ Ш NO:

38), where X ₂ represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

- 13 049130

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X21 represents A or E;

X23 represents I or V;

X24 represents E, Q or N;

X28 represents A or R.

14. The compound according to embodiment 1, wherein the amino acid sequence of the peptide is:

¥X2EGTX ₆ TSD¥SX ₁ 2Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX ₂₈ GGPSKrAPPPS (SEQ Ш NO:

39), where X2 represents Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X28 represents A or R.

15. A connection according to any of the previous embodiments, wherein X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is selected from the group consisting of LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and LLEEQAAREFIE.

16. The compound according to embodiment 1, wherein the amino acid sequence of the peptide is any of SEQ ID NOs: 1-27 or 33-35.

17. The compound according to embodiment 16, wherein the peptide is characterized by an amide modification at the C-terminus.

18. A compound according to any of the previous embodiments, wherein the compound activates human GLP-1 and GIP receptors in vitro with an _EC50 of less than 20 pM when measured without HSA in the CRE-luciferase reporter assays described in Example 2.

19. The compound of any of the previous embodiments, wherein the compound has a half-life in minipigs of at least 35 hours.

20. A compound according to any of the previous embodiments wherein the substituent is attached via 16Lys.

21. A compound according to any of the previous embodiments wherein the substituent is attached via 33Lys.

22. A compound according to any of the previous embodiments, wherein the substituent comprises at least one prolonging agent.

23. The compound according to embodiment 22, wherein the prolonging agent is a fatty acid group.

24. The compound of embodiment 23, wherein the prolonging agent is a diacid defined under Chem. 1: HOOC-( _CH2 ) _n -CO-, wherein n is an integer in the range of 8-20, such as n=14, 16, or 18.

25. The compound of embodiment 23, wherein the prolonging agent is a diacid defined under Chem. 1: HOOC-( _CH2 ) _n -CO-, wherein n is an integer in the range 8-20, such as n=16 or 18.

26. A compound according to any of the previous embodiments, wherein the substituent comprises at least one linker element.

27. A compound according to embodiment 26, wherein the substituent comprises no more than three linker elements.

28. The compound of embodiment 27, wherein the substituent comprises no more than three linker elements designated as -Z1-Z2-Z3-, where -/1- is linked to the prolonging agent and -Z3- is linked to the peptide.

29. A compound according to any of the previous embodiments, wherein the substituent is:

Prot-Zl-Z2-Z3-, where Prot is a C16-C20 dibasic acid;

Z1 represents yGlu or bond;

Z2 is εLys, yGlu, or Ado; and

Z3 represents εLys or Ado.

- 14 049130

30. The compound according to embodiment 29, wherein -Zl- is -yGlu-.

31. The compound according to embodiments 29 or 30, wherein -Z2-Z3- is -Ado-Ado-.

32. The compound according to embodiments 29, 30, or 31, wherein -Z2-Z3- is -sLys-sLys-.

33. A compound according to any one of embodiments 1-17, wherein the substituent is selected from the group consisting of:

A:

WITH: .

34. A compound according to any one of embodiments 1-17, wherein the substituent is selected from the group consisting of:

35. A compound according to any one of the previous embodiments 1-17, wherein the substituent is attached to the peptide via the epsilon amino group of lysine (K).

36. The compound according to embodiment 1, wherein the compound is selected from the group consisting of the following:

- 15 049130

- 16 049130

- 17 049130

- 19 049130

- 20 049130

- 21 049130

- 22 049130

- 23 049130

- 24 049130

- 25 049130

- 26 049130

- 27 049130

- 28 049130

- 29 049130

37. The compound according to embodiment 1, wherein the compound is selected from the group consisting of the following:

- 30 049130

- 31 049130

38. The compound according to embodiment 1, wherein the compound is selected from the group consisting of the following:

- 32 049130

- 33 049130

- 34 049130

39. The connection according to embodiment 1, wherein the connection is:

- 35 049130

40. The connection according to embodiment 1, wherein the connection is:

41. A compound according to any of the previous embodiments for use as a medicinal product.

42. A pharmaceutical composition comprising a compound according to any one of the previous embodiments 1-40.

43. The composition according to embodiment 42, wherein said composition is an aqueous liquid.

44. The composition according to embodiment 42, wherein said composition is a solid composition.

45. A pharmaceutical composition according to any one of embodiments 42-44 for the prevention and/or treatment of diabetes and/or obesity.

46. The pharmaceutical composition according to any one of embodiments 42-44 for the prevention and/or treatment of liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation and/or fatty liver infiltration.

47. A method for the prevention and/or treatment of diabetes and/or obesity, comprising administering to patients

- 36 049130 tu of a pharmaceutically active amount of a compound according to any one of embodiments 1-40.

48. A method for preventing and/or treating liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation and/or fatty liver disease, comprising administering to a patient a pharmaceutically active amount of a compound according to any one of embodiments 1-40.

49. A peptide, wherein the amino acid sequence of the peptide is:

YX2EGTX6TSDYSX32X13LEX36QAAX20X21FX23X24WLLX28GX30X31X32X33X34X35X36X37X ₃₈ X39 (SEQ ID NO: 47) with optional amide modification of the C-terminus;

where X2 represents Aib or A;

X6 represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R;

X ₃₀ is G or absent;

X ₃₁ is P or absent;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X ₃₅ is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent.

50. A peptide, wherein the amino acid sequence of the peptide is:

YX2EGTX ₆ TSDYSX ₁₂ Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX2 ₈ GGPX32X33X34X35X36X37X38X (SEQ ID NO: 36) with optional C-terminal amide modification;

where X ₂ represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X23 represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R;

X ₃₂ is E, S, or absent;

X ₃₃ is S, K, or absent;

X ₃₄ is G or absent;

X ₃₅ is A or absent;

X ₃₆ is P or absent;

X ₃₇ is P or absent;

X ₃₈ is P or absent;

X ₃₉ is S or absent.

51. The peptide of embodiment 49, wherein X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

52. The peptide of embodiment 49, wherein X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

53. The peptide according to embodiment 49, wherein X ₃₂ , X ₃₃ , X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

54. The peptide of embodiment 49, wherein X ₃₀ , X ₃₁ , X ₃₂ , X ₃₃ , X ₃₄ , X ₃₅ , X ₃₆ , X ₃₇ , X ₃₈ and X ₃₉ are absent.

55. The peptide according to embodiments 49 or 50, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is selected from the group consisting of SSGA, ESGA and SKGA.

- 37 049130

56. The peptide according to embodiment 55, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is SSGA.

57. The peptide according to embodiment 55, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is ESGA.

58. The peptide according to embodiment 55, wherein X ₃₂ X ₃₃ X ₃₄ X ₃₅ is SKGA.

59. The peptide according to any one of embodiments 47-58, wherein the peptide is characterized by an amide modification at the C-terminus.

60. The peptide according to embodiment 49, wherein the amino acid sequence of the peptide is:

¥X2EGTX ₆ TSDYSXi2Xi3LEXi ₆ QAAX2oX2iFX23X24WLLX ₂₈ GGPSSGAPPPS (SEQ GO NO:

37), where X2 represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R.

61. The peptide according to embodiment 48, wherein the amino acid sequence of the peptide is:

¥X2EGTX ₆ TSD¥SXi2Xi3LEX ₁₆ QAAX ₂ oX2iFX23X24WLLX ₂₈ GGPESGAPPPS (SEQ GO NO:

38), where X ₂ represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X21 represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R.

62. The peptide according to embodiment 49, wherein the amino acid sequence of the peptide is:

YX2EGTX ₆ TSDYSX ₁₂ Xi3LEX ₁₆ QAAX2oX2iFX23X24WLLX2 ₈ GGPSKrAPPPS (SEQ ID NO:

39), where X2 represents Aib or A;

X ₆ represents F or V;

X ₁₂ represents I or Y;

X ₁₃ represents Y, A, L, or I;

X ₁₆ represents K or E;

X ₂₀ represents Q, R, E, H;

X ₂₁ represents A or E;

X ₂₃ represents I or V;

X ₂₄ represents E, Q or N;

X ₂₈ represents A or R.

63. The peptide according to embodiment 49, wherein the amino acid sequence of the peptide is any of SEQ ID NOs: 1-27 or 33-35.

64. The peptide according to embodiment 63, wherein the peptide is characterized by an amide modification at the C-terminus.

65. The peptide according to any one of the previous embodiments 49-64, wherein X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is selected from the group consisting of LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and LLEEQAAREFIE.

66. The peptide of any one of previous embodiments 49-64, wherein the peptide activates human GLP-1 and GIP receptors in vitro with an _EC50 of less than 20 pM when measured without HSA in the CRE-luciferase reporter assays described in Example 2.

- 38 049130

67. The peptide according to any one of the previous embodiments 49-64, wherein X ₁₆ is K.

68. The peptide according to any one of the previous embodiments 49-64, wherein X33 is K.

69. The peptide according to embodiment 49, wherein the amino acid sequence of the peptide is any of SEQ ID NO: 15, 28, 29, 31, 32 or 43.

70. The peptide according to embodiment 49, wherein the amino acid sequence of the peptide is SEQ ID NO: 31.

71. A method for producing a compound according to any of the previous embodiments 1-40.

72. A method for producing a peptide according to any of the previous embodiments 49-64.

73. A method for producing a peptide according to any of the previous embodiments 49-70.

Methods and examples

List of abbreviations.

The following abbreviations are used hereafter in alphabetical order.

Ac: acetyl.

Ado (also called OEG): 8-amino-3,6-dioxaoctanoic acid.

Aib: α-aminoisobutyric acid.

API: Active Pharmaceutical Ingredient.

AUC: area under the curve.

BG: blood glucose level.

BHK: newborn hamster kidney.

Boc: tert-butyloxycarbonyl.

BW: body weight.

CAS: Chemical Abstracts Service.

Cl-HOBt: 6-chloro-1-hydroxybenzotriazole.

DCM: dichloromethane.

DIC: diisopropylcarbodiimide.

DIPEA: N.N-diisopropylethylamine.

DMEM: Dulbecco's modified Eagle's medium.

DPBS: Dulbecco's phosphate-buffered saline.

EDTA: ethylenediaminetetraacetic acid.

ELISA: enzyme-linked immunosorbent assay.

equiv.: molar equivalent.

FBS: fetal bovine serum.

Fmoc: 9-fluorenylmethyloxycarbonyl.

GIP: glucose-dependent insulinotropic polypeptide.

GIPR: glucose-dependent insulinotropic polypeptide receptor.

GLP-1: glucagon-like peptide-1.

GLP-1R: glucagon-like peptide-1 receptor. h: hours.

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol.

HPLC: High Performance Liquid Chromatography.

HSA: human serum albumin.

I/br: intraperitoneally.

IPGTT: intraperitoneal glucose tolerance test.

IV: intravenously.

LCMS: liquid chromatography mass spectrometry.

LYD: Landrace-Yorkshire-Duroc.

MeCN: acetonitrile.

MeOH: methanol.

mM: millimolar. mmol: millimoles. min: minutes.

Mtt: 4-methyltrityl.

MW: molecular weight.

nM: nanomolar.

NMP: 1-methylpyrrolidin-2-one.

OEG: 8-amino-3,6-dioxaoctanoic acid (also called Ado).

- 39 049130

OtBu: tert-butyl ether.

Oxyma Pure®: ethyl ester of cyanohydroxyiminoacetic acid.

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

PBS: phosphate buffered saline.

PD: pharmacodynamics.

PK: pharmacokinetics.

pM: picomolar.

RP: Reversed Phase.

RP-HPLC: Reversed Phase High Performance Liquid Chromatography. rpm: revolutions per minute.

RT: room temperature.

Rt: retention time.

S/c: subcutaneous.

SD: standard deviation.

SEC-HPLC: size-exclusion high-performance liquid chromatography.

SEM: standard error of the mean.

SNAC: Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate.

SPPS: solid-phase peptide synthesis.

tBu: tert-butyl.

TFA: trifluoroacetic acid.

TIS: triisopropylsilane.

Trt: triphenylmethyl or trityl.

Trx: tranexamic acid.

General methods of obtaining.

Methods for solid-phase peptide synthesis (SPPS methods, including methods for removing the amino acid protecting group, methods for cleaving the peptide from the resin and purifying it), as well as methods for detecting and characterizing the resulting peptide (LCMS methods) are described below in this document.

The resins used to prepare the C-terminal peptide amides were HRink Amide-ChemMatrix resin (loading, e.g., 0.5 mmol/g). The recommended standard, unless otherwise specifically stated, are the Fmoc-protected amino acid derivatives used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, FmocGln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, FmocLys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, FmocTrp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Aib-OH, etc., available from, for example, AAPPTEC, Anaspec, Bachem, ChemImpex, Iris Biotech, Midwest Biotech, Gyros Protein Technologies or Novabiochem. Unless otherwise stated, natural L-forms of amino acids were used. The N-terminal amino acid was protected by a Boc group at the alpha-amino group, either by using a reagent with a pre-incorporated Boc group (e.g., Boc-Tyr(tBu)-OH for peptides with Tyr at the N-terminus) or by replacing the N-terminal Fmoc protecting group with a Boc protecting group after incorporation of the amino acid at the N-terminus of the peptide.

In the case of coupling of the modular albumin binding moiety using SPPS, the following appropriately protected building blocks were used, such as, but not limited to, Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH), Boc-Lys(Fmoc)-OH, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester, nonadecanedioic acid mono-tert-butyl ester, eicosanedioic acid mono-tert-butyl ester, tetradecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester. All of the following operations were performed on a synthesis scale of 0.1-0.2 mmol.

1. Synthesis of resin-linked protected peptide backbone.

Method: SPPS_A.

SPPS was performed using Fmoc-based chemistry on a Protein Technologies SymphonyX solid phase peptide synthesis system using the manufacturer's provided protocols with some modifications. Stirring was achieved by periodic nitrogen sparging. Stepwise assembly was accomplished using the following steps: 1) allowing the resin to pre-swell in DMF; 2) removing the Fmoc protecting group using 20% (v/v) piperidine in DMF for two 10 min treatments; 3) washing with DMF to remove piperidine; 4) performing the coupling reaction with the Fmoc-amino acid by adding the Fmoc-amino acid (12 equiv.) and Oxyma Pure® (12 equiv.), each reagent as a 0.6 M solution in DMF, followed by adding DIC (12 equiv.) as a 1.2 M solution in DMF, followed by adding DMF to reduce the final concentration of each component to 0.3 M, then mixing

- 40 049130 coupling for 0.5-4 h; 4) washing with DMF to remove excess reagents; 5) final washes with DCM upon completion of assembly. Some amino acids, such as but not limited to amino acids following the sterically hindered amino acid (e.g., Aib), were coupled with extended reaction times (e.g., 4 h) to ensure completion of the reaction.

Method: SPPS_B.

The protected peptidyl resin was synthesized according to the Fmoc strategy on an Applied Biosystems 431A solid phase peptide synthesis system using standard Fmoc protocols provided by the manufacturer. Mixing was accomplished by vortexing and periodic nitrogen sparging. Stepwise assembly was accomplished using the following steps: 1) activation of the Fmoc-amino acid by dissolving the Fmoc-amino acid as a solid (10 equiv.) in Cl-HOBt (10 equiv.) as a 1 M solution in NMP, then adding DIC (10 equiv.) as a 1 M solution in NMP, then mixing while proceeding to steps 2-3; 2) removal of the Fmoc protecting group using 20% (v/v) piperidine in NMP for one treatment for 3 min, then a second treatment for 15 min; 3) washes with NMP to remove the piperidine; 4) addition of the activated Fmoc amino acid solution to the resin, then mixing for 45-90 min; 4) washes with NMP to remove excess reagents; 5) final washes with DCM upon completion of assembly. The standard protected amino acid derivatives listed above were supplied in pre-weighed cartridges (e.g., from Midwest Biotech) and the non-standard derivatives were weighed manually. Some amino acids, such as but not limited to amino acids following a sterically hindered amino acid (e.g., Aib), were subjected to a double coupling reaction to ensure completion of the reaction, i.e., after the first coupling reaction (e.g., 45 min), the resin was dried, additional reagents (Fmoc-amino acid, DIC, Cl-HOBt) were added, and the mixture was allowed to react again (e.g., 45 min).

Method: SPPS_C.

SPPS was performed using Fmoc chemistry on a PreludeX solid phase peptide synthesis system using the manufacturer's provided protocols with some modifications. Mixing was accomplished by shaking at 350 rpm and periodic nitrogen sparging. Stepwise assembly was performed using the following steps: 1) allowing the resin to pre-swell in DMF; 2) removing the Fmoc protecting group using 20% (v/v) piperidine in DMF for a single run at 70°C for 3 min; 3) washing with DMF to remove the piperidine; 4) performing the Fmoc-amino acid coupling reaction by adding the Fmoc-amino acid (12 equiv.) and Oxyma Pure® (12 equiv.), each as a 0.4 M solution in DMF, followed by the addition of DIC (12 equiv.) as a 1.2 M solution in DMF, then mixing for 5 min at 70°C; 4) washes with DMF to remove excess reagents; 5) final washes with DCM upon completion of assembly. Some amino acids, such as, but not limited to, amino acids following the sterically hindered amino acid (e.g., Aib), were coupled with extended reaction times (e.g., 15 min) to ensure completion of the reaction.

2. Attachment of a substituent to the resin-bound protected peptide backbone.

Method: SC_A.

The Mtt protecting group on N-epsilon-lysine was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each, followed by washes with DCM and DMF. Acylation was performed on a Protein Technologies SymphonyX solid phase peptide synthesis system using the protocols described in method SPPS_A using stepwise addition of building blocks such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedicarboxylic acid mono-tert-butyl ester, and eicosanedicarboxylic acid mono-tert-butyl ester.

Method: SC_B.

The Mtt protecting group on N-epsilon-lysine was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each, followed by washes with DCM and DMF. Acylation was performed on an Applied Biosystems 431A Solid Phase Peptide Synthesis System using the protocols described in Method SPPS_B using stepwise addition of building blocks such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedicarboxylic acid mono-tert-butyl ester, and eicosanedicarboxylic acid mono-tert-butyl ester.

Method: SC_C.

The Mtt protecting group on N-epsilon-lysine was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each, followed by washing with DCM and DMF. Acylation was performed on a Protein Tech solid-phase peptide synthesis system.

- 41 049130 nologies PreludeX using the protocols described in the SPPS_C method using stepwise addition of building blocks such as, but not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester, octadecanedicarboxylic acid mono-tert-butyl ester, and eicosanedicarboxylic acid mono-tert-butyl ester.

3. Cleavage of resin-bound peptide and purification.

Method: CP_A.

After completion of the side chain synthesis, the peptidyl resin was washed with DCM and dried, then treated with TFA/water/TIS (95:2.5:2.5 v/v/v) for about 2 h, followed by precipitation with diethyl ether. The pellet was washed with diethyl ether, dissolved in a suitable solvent (e.g., water/MeCN 2:1), and allowed to settle until all unstable adducts were destroyed. Purification was accomplished by preparative reversed-phase HPLC (Waters 2545 binary gradient module, Waters 2489 UV/Vis detector, Waters III fraction selector) on a Phenomenex Luna C8(2) column (10 μm particle size, 100 Å pore size, 250 x 21.2 mm linear dimensions). Separation of impurities and elution of the product were accomplished using an increasing gradient of MeCN in water containing 0.1% TFA. The appropriate fractions were checked for identity and purity by analytical LCMS. Fractions containing pure desired product were pooled and lyophilized to yield the TFA salt of the peptide as a white solid.

4. Salt exchange from TFA salt to sodium salt.

Method: SX_A.

The lyophilized peptide isolated by the CP_A method was dissolved at 5-20 mg/mL in a suitable aqueous buffer (e.g., water/MeCN 4:1, 0.2 M sodium acetate) and adjusted to pH 7-8 with 1 M NaOH if necessary to achieve complete dissolution. The buffer solutions containing the peptide were salt exchanged using a Sep-Pak C18 cartridge (0.5-2 g). The cartridge was first equilibrated with 4 column volumes of isopropanol, then 4 column volumes of MeCN, then 8 column volumes of water. The peptide solution was injected into the cartridge and the eluate was reapplied to ensure complete retention of the peptide. The cartridge was washed with 4 column volumes of water, then 10 column volumes of a buffer solution (e.g., pH 7.5) containing, for example, but not limited to _{, NaHCO3} , NaOAc, or _Na2HPO4 . The column was washed with 4 column volumes of water and the peptide was eluted with 5-10 column volumes of 50-80% MeCN in water. The eluent containing the peptide was lyophilized to give the sodium salt of the peptide as a white solid, which was used as such.

General methods of detection and characterization.

LCMS Methods: Method: LCMS_A.

LCMS_A was performed on a setup consisting of an Agilent 1260 Infinity series HPLC system and an Agilent Technologies 6120 Quadrupole MS. Eluents: A: 0.05% TFA in water; B: 0.05% TFA in MeCN/water 9:1.

Analysis was performed at rt (column temperature 37°C) by injecting an appropriate volume of sample onto the column which was eluted with a gradient of A and B. Column: Phenomenex Kinetex C8, 2.6 µm, 100 A, 4.6 x 75 mm. Gradient run time: linear 10-80% B in 10 min at a flow rate of 1.0 ml/min. Detection: diode array detector set to 214 nm. MS ionization mode: APIES, positive polarity. MS mass scan range: 500-2000 amu. The most abundant isotope for each m/z value is given.

Method: LCMS_B.

LCMS_B was performed on a setup consisting of an Agilent 1260 Infinity series HPLC system and an Agilent Technologies 6120 Quadrupole MS. Eluents: A: 0.05% TFA in water; B: 0.05% TFA in MeCN/water 9:1.

Analysis was performed at rt (column temperature 37°C) by injecting an appropriate volume of sample onto the column which was eluted with a gradient of A and B. Column: Phenomenex Kinetex C8, 2.6 µm, 100 A, 4.6 x 75 mm. Gradient run time: linear 20-100% B in 10 min at a flow rate of 1.0 ml/min. Detection: diode array detector set to 214 nm. MS ionization mode: APIES, positive polarity. MS mass scan range: 500-2000 amu. The most abundant isotope for each m/z value is given.

Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will be apparent to those skilled in the art. It is therefore to be understood that the accompanying embodiments are intended to cover all such modifications and changes that fall within the true spirit of the present invention.

Example 1. Synthesis of compounds.

The compounds are further described using single-letter amino acid codes except for Aib. The substituent is included after the lysine (K) residue to which it is attached.

- 42 049130

Connection No. 1.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-aMUHO-6-[[(2S)-2-aMUHO-6-[[(4S)-4-Kap6oKCu-4-(19carboxynonadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] QAAQEFVNWLLAGGPSSGAPPPS-NH2.

SEQ ID NO: 1 with C-terminal amide modification; substituent: C; substituent position: K16.

Synthesis methods: SPPS_A; SC_B; CP_A.

Estimated molecular weight (average): 4873.5 Da.

LCMS_A: Rt=6.0 min; certain value [M+ZN] ³⁺ 1625.4, [M+4N] ⁴⁺ 1219.1.

Connection No. 2.

Y-Aib-EGTFTSDYSYYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19carboxynonadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAQEFVNWLLAGGPSSGAPPPS-NH2.

ABOUT

SEQ ID NO: 2 with C-terminal amide modification; substituent: C; substituent position: K16.

Synthesis methods: SPPS_A; SC_B; CP_A.

Estimated molecular weight (average): 4923.5 Da.

LCMS_A: Rt=6.0 min; certain value [M+ZN] ³⁺ 1641.8, [M+4N] ⁴⁺ 1237.5.

Connection No. 3.

Y-Aib-EGTFTSDYSYYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAQEFVNWLLAGGPSSGAPPPS-NH2.

SEQ ID NO: 2 with C-terminal amide modification; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_B; CP_A.

Estimated molecular weight (average): 4895.4 Da.

LCMS_A: Rt=5.7 min; certain value [M+ZN] ³⁺ 1632.7, [M+4N] ⁴⁺ 1224.6.

Connection No. 4.

Y-Aib-EGTFTSDYSYYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAQAFVNWLLAGGPSSGAPPPS-NH2.

- 43 049130

SEQ ID NO: 3 with C-terminal amide modification; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_B; CP_A.

Estimated molecular weight (average): 4837.4 Da.

LCMS_A: Rt=5.7 min; certain value [M+ZN] ³⁺ 1613.3, [M+4N]4+ 1210.1.

Connection No. 5.

Y-Aib-EGTFTSDYSYYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAQAFVNWLLAGGPSSGAPPPS-OH.

ABOUT

SEQ ID NO:3; substituent: B; substituent position: K16.

Synthesis methods: SPPS_B; SC_B; CP_A.

Estimated molecular weight (average): 4838.4 Da.

LCMS_A: Rt=5.7 min; determined value [M+3H] ³⁺ 1613.6, [M+4H]4+ 1210.4.

Connection No. 6.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFVNWLLAGGPSSGAPPPS-NH2.

SEQ ID NO: 4 with C-terminal amide modification; substituent: B; substituent position: K16. Synthesis methods: SPPS_B; SC_B; CP_A.

Estimated molecular weight (average): 4873.5 Da.

LCMS_A: Rt=5.6 min; determined value [M+3H] ³⁺ 1625.3, [M+4H]4+ 1219.1.

Connection No. 7.

Y-Aib-EGTFTSDYSIYLE-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]QAAREFVNWLLAGGPSSGAPPPS-NH2.

SEQ ID NO: 4 with C-terminal amide modification; substituent: A; substituent position: K16.

Synthesis methods: SPPS_B; SC_B; CP_A.

Estimated molecular weight (average): 4907.4 Da.

LCMS_A: Rt=6.3 min; determined value [M+3H] ³⁺ 1636.5, [M+4H]4+ 1227.9.

- 44 049130

Connection No. 8.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-aMUHO-6-[[(2S)-2-aMUHO-6-[[(4S)-4-Kap6oKCu-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl] QAAREFINWLLAGGPSSGAPPPS-NH2.

SEQ ID NO: 5 with C-terminal amide modification; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4887.5 Da.

LCMS_A: Rt=5.7 min; certain value [M+ZN] ³⁺ 1629.8, [M+4N] ⁴⁺ 1222.8.

Connection No. 9.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFIEWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 6; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_B; CP_A.

Estimated molecular weight (average): 4903.5 Da.

LCMS_A: Rt=5.8 min; certain value [M+3H] ³⁺ 1635.2, [M+4H] ⁴⁺ 1226.8.

Connection No. 10.

Y-Aib-EGTFTSDYSIYLE-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] QAAREFIEWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 6; substituent: A; substituent position: K16.

Synthesis methods: SPPS_A; SC_B; CP_A.

Estimated molecular weight (average): 4937.5 Da.

LCMS_A: Rt=6.5 min; certain value [M+3H] ³⁺ 1646.6, [M+4H] ⁴⁺ 1235.1.

Connection No. 11.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 5; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4888.5 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1630.2, [M+4H] ⁴⁺ 1222.9.

Connection No. 12.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFIEWLLRGGPSSGAPPPS-OH.

- 45 049130

Synthesis methods: SPPS_A; SC_C; CP_A.

SEQ ID NO: 7; substituent: B; substituent position: K16.

Estimated molecular weight (average): 4988.6 Da.

LCMS_A: Rt=5.7 min; certain value [M+ZN] ³⁺ 1663.6, [M+4N] ⁴⁺ 1248.0.

Connection No. 13.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl] QAAREFIEWLLRGGPESGAPPPS-OH.

SEQ ID NO: 8; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 5030.6 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1677.7, [M+4H] ⁴⁺ 1258.4.

Connection No. 14.

Y-Aib-EGTFTSDYSIALE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 9; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4796.4 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1599.6, [M+4H] ⁴⁺ 1199.8.

Connection No. 15.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 10; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A; SX_A.

Estimated molecular weight (average): 4838.5 Da.

LCMS_A: Rt=5.8 min; certain value [M+3H] ³⁺ 1613.4, [M+4H] ⁴⁺ 1210.3.

Connection No. 16.

Y-Aib-EGTFTSDYSIYLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19carboxynonadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFINWLLAGGPSSGAPPPS-OH.

- 46 049130

SEQ ID NO: 5; substituent: C; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4916.5 Da.

LCMS_A: Rt=6.0 min; certain value [M+ZN] ³⁺ 1639.6, [M+4N] ⁴⁺ 1229.9.

Connection No. 17.

YAEGTFTSDYSIYLE-K[(2S)-2-αmino-6-[[(2S)-2-αmino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 11; substituent: B; substituent position: K16.

Synthesis methods: SPPS_C; SC_A; CP_A.

Estimated molecular weight (average): 4874.5 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1625.4, [M+4H] ⁴⁺ 1219.4.

Connection No. 18.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] -QAAREFINWLLAG-OH.

SEQ ID NO: 12; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4003.6 Da.

LCMS_A: Rt=6.2 min; certain value [M+3H] ³⁺ 1335.3, [M+4H] ⁴⁺ 1001.7.

Connection No. 19.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] -QAAREFINWLLAGGP-OH.

SEQ ID NO: 13; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4157.8 Da.

LCMS_A: Rt=6.1 min; certain value [M+3H] ³⁺ 1386.6, [M+4H] ⁴⁺ 1040.3.

Connection No. 20.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl] -QAAREFINWLLAGGPSS OH.

- 47 049130

SEQ ID NO: 14; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4331.9 Da.

LCMS_A: Rt=5.9 min; certain value [M+ZN] ³⁺ 1444.7, [M+4N] ⁴⁺ 1083.7.

Connection No. 21.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl] QAAREFINWLLAGGPSSGA-OH.

SEQ ID NO: 15; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4460.0 Da.

LCMS_A: Rt=5.9 min; certain value [M+3H] ³⁺ 1487.5, [M+4H] ⁴⁺ 1116.1.

Connection No. 22.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFVNWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 16; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4824.4 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1608.9, [M+4H] ⁴⁺ 1206.9.

Connection No. 23.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFIQWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 17; substituent: B; substituent position: K16 Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4852.5 Da.

LCMS_A: Rt=5.9 min; certain value [M+3H] ³⁺ 1618.4, [M+4H] ⁴⁺ 1214.1.

Connection No. 24.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAQEFINWLLAGGPSSGAPPPS-OH.

- 48 049130

SEQ ID NO: 18; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4810.4 Da.

LCMS_A: Rt=6.0 min; certain value [M+ZN] ³⁺ 1604.0, [M+4N] ⁴⁺ 1203.4.

Connection No. 25.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] QAAEEFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 19; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4811.4 Da.

LCMS_A: Rt=6.1 min; certain value [M+3H] ³⁺ 1604.4, [M+4H] ⁴⁺ 1203.6.

Connection No. 26.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAHEFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO:20; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4819.4 Da.

LCMS_A: Rt=5.8 min; certain value [M+3H] ³⁺ 1607.1, [M+4H] ⁴⁺ 1205.5.

Connection No. 27.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFIEWLLRGGPSSGAPPPS-OH.

SEQ ID NO:21; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4938.6 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1646.9, [M+4H] ⁴⁺ 1235.4.

Connection No. 28.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFIEWLLRGGPESGAPPPS-OH.

- 49 049130

SEQ ID NO:22; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A; SX_A.

Estimated molecular weight (average): 4980.6 Da.

LCMS_A: Rt=5.8 min; certain value [M+ZN] ³⁺ 1660.7, [M+4N] ⁴⁺ 1246.0.

Connection No. 29.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] QAAQEFIEWLLAGGPSSGAPPPS-OH.

SEQ ID NO:23; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A; SX_A.

Estimated molecular weight (average): 4825.4 Da.

LCMS_A: Rt=6.1 min; certain value [M+3H] ³⁺ 1609.3, [M+4H] ⁴⁺ 1207.0.

Connection No. 30.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAHEFIEWLLAGGPSSGAPPPS-OH.

SEQ ID NO:24; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4834.4 Da.

LCMS_A: Rt=5.8 min; certain value [M+3H] ³⁺ 1612.1, [M+4H] ⁴⁺ 1209.3.

Connection No. 31.

Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFIEWLLAGGPSSGAPPPS-OH.

SEQ ID NO:25; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A; SX_A.

Estimated molecular weight (average): 4853.5 Da.

LCMS_A: Rt=5.9 min; certain value [M+3H] ³⁺ 1618.5, [M+4H] ⁴⁺ 1214.2.

Connection No. 32.

Y-Aib-EGTFTSDYSILLE-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] QAAREFIEWLLAGGPSSGAPPPS-OH.

- 50 049130

SEQ ID NO:25; substituent: A; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4887.4 Da.

LCMS_A: Rt=6.6 min; certain value [M+ZN] ³⁺ 1629.8, [M+4N] ⁴⁺ 1222.8.

Connection No. 33.

Y-Aib-EGTVTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO:26; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4790.4 Da.

LCMS_A: Rt=5.8 min; certain value [M+3H] ³⁺ 1597.4, [M+4H] ⁴⁺ 1198.3.

Connection No. 34.

Y-Aib-EGTFTSDYSIILE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO:27; substituent: B; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4838.5 Da.

LCMS_A: Rt=5.8 min; certain value [M+3H] ³⁺ 1613.4, [M+4H] ⁴⁺ 1210.3.

Connection No. 35.

Y-Aib-EGTFTSDYSILLE-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] QAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO: 10; substituent: A; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4872.4 Da.

LCMS_A: Rt=6.6 min; certain value [M+3H] ³⁺ 1624.9, [M+4H] ⁴⁺ 1218.9.

Connection No. 36.

Y-Aib-EGTFTSDYSIILE-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]QAAREFINWLLAGGPSSGAPPPS-OH.

- 51 049130

SEQ ID NO:27; substituent: A; substituent position: K16.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4872.4 Da.

LCMS_A: Rt=6.5 min; certain value [M+ZN] ³⁺ 1624.8, [M+4N] ⁴⁺ 1218.9.

Connection No. 37.

Y-Aib-EGTFTSDYSIYLEEQAAR-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] -FINWLLAGGP SSGAPPPSOH.

SEQ ID NO:28; substituent: B; substituent position: K21.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4888.5 Da.

LCMS_A: Rt=6.0 min; certain value [M+3H] ³⁺ 1630.3, [M+4H] ⁴⁺ 1223.0.

Connection No. 38.

Y-Aib-EGTFTSDYSIYLEE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]AAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO:29; substituent: B; substituent position: K17.

Synthesis methods: SPPS_C; SC_C; CP_A.

Estimated molecular weight (average): 4889.5 Da.

LCMS_A: Rt=6.0 min; certain value [M+3H] ³⁺ 1630.6, [M+4H] ⁴⁺ 1223.2.

Connection No. 39.

Y-Aib-EGTFTSDYSILLEEQAAR-K[(2S)-2-αmino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] -FINWLLAGGP SSGAPPPSOH.

SEQ ID NO: 30; substituent: B; substituent position: K21.

Synthesis methods: SPPS_A; SC_C; CP_A.

Estimated molecular weight (average): 4838.5 Da.

LCMS_A: Rt=6.2 min; certain value [M+3H] ³⁺ 1613.5, [M+4H] ⁴⁺ 1210.4.

Connection No. 40.

Y-Aib-EGTFTSDYS-K[(2S)-2-amino-6-[[(2S)-2-αmino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]LLEEQAAREFIEWLLAGGPSSGAPPPS-OH.

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SEQ ID NO:31; substituent: B; substituent position: K12.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4869.4 Da.

LCMS_A: Rt=5.7 min; certain value [M+ZN] ³⁺ 1623.8, [M+4N] ⁴⁺ 1218.2.

Connection No. 41.

Y-Aib-EGTFTSDYS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl] amino] hexanoyl] amino] hexanoyl] YLEEQAAREFINWLLAGGPSSGAPPPS-OH.

SEQ ID NO:32; substituent: B; substituent position: K12.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4904.4 Da.

LCMS_A: Rt=5.7 min; certain value [M+3H] ³⁺ 1635.4, [M+4H] ⁴⁺ 1226.7.

Connection No. 42.

Y-Aib-EGTFTSDYSIYLEEQAAREFINWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPSON.

SEQ ID NO: 33; substituent: B; substituent position: K33.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4930.5 Da.

LCMS_A: Rt=6.2 min; certain value [M+3H] ³⁺ 1644.2, [M+4H] ⁴⁺ 1233.4.

Connection No. 43.

Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPSON.

SEQ ID NO: 34; substituent: B; substituent position: K33.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4895.5 Da.

LCMS_A: Rt=6.3 min; certain value [M+3H] ³⁺ 1632.4, [M+4H] ⁴⁺ 1224.6.

Connection No. 44.

Y-Aib-EGTFTSDYSILLEEQAAREFINWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPSON.

- 53 049130

SEQ ID NO: 35; substituent: B; substituent position: K33.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4880.5 Da.

LCMS_A: Rt=6.2 min; certain value [M+ZN] ³⁺ 1627.5, [M+4N]4+ 1220.9.

Connection No. 45.

Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]GAPPPS-OH.

SEQ ID NO: 34; substituent: A; substituent position: K33.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4929.5 Da.

LCMS_A: Rt=6.6 min; determined value [M+3H] ³⁺ 1643.8, [M+4H]4+ 1233.1.

Connection No. 46.

Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPSOH.

SEQ ID NO: 34; substituent: E; substituent position: K33.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4867.5 Da.

LCMS_A: Rt=6.1 min; defined value [M+3H] ³⁺ 1623.1, [M+4H]4+ 1217.6.

Connection No. 47.

Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]GAPPPS-OH.

SEQ ID NO: 34; substituent: D; substituent position: K33.

Synthesis methods: SPPS_A; SC_A; CP_A.

Estimated molecular weight (average): 4901.4 Da.

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LCMS_A: Rt=6.3 min; certain value [M+3H] ³ + 1634.6, [M+4H] ⁴⁺ 1226.1.

Example 2. Functional efficiency in vitro (CRE-luciferase; whole cells).

The purpose of this example is to test the functional activity or potency of the compounds in vitro at the human GLP-1 and GIP receptors. In vitro functional potency is a measure of target receptor activation in a whole cell assay. Potency values for the derivatives of example 1 were determined as described below. Human GLP-1(7-37) and human GIP were included in the respective assays for comparison.

Principle.

In vitro functional efficacy was determined by measuring the response of the target receptor in a reporter gene assay in individual cell lines. The assay was performed using stably transfected BHK cell lines that express one of the following G protein-coupled receptors: human GLP-1 receptor or human GIP receptor; and wherein each cell line contains DNA encoding a cAMP-responsive element (CRE) linked to a promoter and the firefly luciferase gene (CRE-luciferase). Activation of the corresponding receptor results in the production of cAMP, which in turn results in the expression of luciferase protein. After completion of the reaction incubation, luciferase substrate (luciferin) is added, which results in the enzymatic conversion of luciferin to oxyluciferin and induces bioluminescence. Luminescence is measured as the readout in the assay.

Cultivation and isolation of cells.

The cell lines used in these assays were BHK cells, with the parental cell line being BHKTS13. The cell lines were derived from a clone containing the CREluciferase element and were generated by additional transfection with the corresponding human receptor to generate the corresponding cell line: BHK CRE luc2P hGLP-1R or BHK CRE luc2P hGIPR. The cells were cultured at 37°C with 5% _CO2 in cell culture medium. They were aliquoted and stored in liquid nitrogen. The cells were maintained in continuous culture and plated the day before each assay.

Materials.

The following chemicals were used in the assay: Pluronic F-68 10% (Gibco 2404), human serum albumin (HSA; Sigma A9511), 10% fetal bovine serum (FBS; Invitrogen 16140071), chicken egg white ovalbumin (Sigma A5503), DMEM without phenol red (Gibco 21063029), DMEM (Gibco 12430-054), 1 M Hepes (Gibco 15630), Glutamax 100x (Gibco 35050), G418 (Invitrogen 10131-027), hygromycin (Invitrogen 10687-010), and Steadylite plus (PerkinElmer 6016757).

Buffers.

Cell culture medium for GLP-1R consisted of DMEM with 10% FBS, 500 μg/ml G418, and 300 μg/ml hygromycin. Cell culture medium for GIPR consisted of DMEM with 10% FBS, 400 μg/ml G418, and 300 μg/ml hygromycin. Assay buffer consisted of DMEM without phenol red, 10 mM Hepes, 1x Glutamax, 1% ovalbumin, and 0.1% Pluronic F-68 supplemented with HSA at twice the final assay concentration. Assay buffer was mixed 1:1 with an equal volume of test compound in assay buffer to give the final assay concentration of HSA.

Procedure.

1) Cells were seeded at 5000 cells/well and incubated overnight in an assay plate.

2) Cells were washed once in DPBS.

3) Stock solutions of test compounds and reference compounds at concentrations in the range of 100-300 μM were diluted 1:150 in assay buffer. Compounds were then diluted 1:10 in column 1 of a 96-well deep-well dilution plate and then transferred row-wise to generate a 12-point 3.5-fold dilution curve.

4) Assay buffer (50 µl aliquot) with or without HSA was added to each well of the assay plate.

5) A 50 µl aliquot of compound or blank was transferred from the dilution plate to an assay plate containing assay buffer with or without HSA.

6) The assay plate was incubated for 3 h in an incubator with 5% _CO2 at 37°C.

7) Cells were washed once with DPBS.

8) An aliquot of 100 µl DPBS was added to each well of the assay plate.

9) A 100 µl aliquot of Steadylite plus (light sensitive) reagent was added to each well of the assay plate.

10) Each assay plate was covered with aluminum foil to protect from light and shaken at 250 rpm for 30 min at room temperature.

11) The reaction in each assay plate was read on a microtiter plate reader.

Calculations and results.

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First, regression analysis was performed on the microtiter plate reader data in Excel to calculate a logarithmic concentration scale for the x-axis based on the individual test compound stock concentrations and assay dilutions. The data were then transferred to GraphPad Prism software for plotting and statistical analysis. The software performs nonlinear regression analysis (response versus log(agonist concentration)). The EC50 values calculated by the software and expressed in pM are shown in Tables 1-3 below. At least two replicates were measured for each sample. The reported values are the average of the replicates.

Table 1

Functional efficacy against human GLP-1R and GIPR in the presence of 0% and 1% HAS

No connection hGLP-lR, CRE-Luc, 0% HSA EU ₅₀ (pM) hGLP-lR, CRE-Luc, 1% HSA EC ₅₀ (pM) hGIPR, CRE-Luc, 0% HSA EC ₅₀ (nM) hGIPR, CRE-Luc, 1% HSA EC ₅₀ (pM) hGLP-l(7-37) 8.4 6,7 H. 0. H. 0. hGIP But. H. 0. H,3 6.4 1 7,8 1659,0 2.8 172.5 2 6.0 899.7 3.8 275.4 3 8.5 626.3 6.9 229.2 4 6,6 484.3 8.5 293.8 5 9.4 624.8 14.2 362.4 6 3,4 242.7 18,5 434,0 7 4.2 513.2 28.7 686.8 8 2,3 82.1 6,7 191.1 9 3.8 278.8 10.8 508.2 10 3.9 939.9 H,3 1745,1 AND 3,3 132.3 9.9 450.2 37 2.7 94.6 H,2 297.8 12 4.0 54.4 9.0 71,5 13 7.4 204.1 13.9 257.2 14 5.4 306.2 H,6 580,1 15 5.0 129.7 7.1 137.5 38 3.1 258.9 12.4 815,1 16 3.1 321,1 13.4 803.8 17 5.2 256.6 34.9 1319,0 18 19.7 226.2 20.9 285.1 19 8.6 364.8 6.5 275.4 20 4.2 256,0 7.1 247.4 21 5.7 118.2 5.4 130.9 22 2.8 170.5 6.2 168.9 23 4.1 117.8 6,7 134.3 24 12.1 490.2 3.9 115.1 25 15,5 740.2 7.2 263,0 26 7.4 594.6 4.9 153,0 39 8.2 135.4 28.4 365.2

- 56 049130

27 4.6 127,0 6.3 131.8 28 6.2 139.1 6,6 86.5 29 16.8 378,5 3.8 101.7 30 7.3 267,5 3.2 185,0 41 1,2 31.4 4.9 79.3 42 2,3 83.5 5.9 305.7 40 6.4 333.6 9.5 500,6 31 4.2 202,0 4.7 188,5 32 5.0 981.9 7.1 678,7 43 4.0 359,5 5,6 444,5 44 4.2 240.9 5.7 365.3 33 14.8 360.7 2.9 102.8 34 6.0 186.4 8.3 212.8 35 z,o 412.6 5.2 303,0 36 4.7 413.8 5.5 356,5 45 3.9 1580,0 3.8 1220,0 46 1,5 110.8 1,8 97,0 47 2,1 154.5 3.1 155.7

n. o. = not defined.

The compounds of the present invention demonstrate effective functional activation of human GLP-1R and human GIPR receptors under given conditions.

Example 3. Pharmacokinetic study in minipigs.

The purpose of this example is to determine the in vivo half-life of the derivatives of the present invention after i.v. administration to minipigs, i.e. to prolong their residence time in the body and thus their time of action. This was performed in a pharmacokinetic (PK) study, in which the terminal half-life of the test derivative is determined. The terminal half-life is generally understood to be the period of time required for a determined plasma concentration measured after the initial distribution phase to be halved.

Study.

Female Gottingen Minipigs from Ellegaard Gottingen Minipigs (Dalmose, Denmark), approximately 7-14 months old and weighing approximately 16-35 kg, were used in the study. The minipigs were housed individually and fed strictly once daily with the SDS minipig diet (Special Diets Services, Essex, UK).

After 3 weeks of acclimatization, each animal had two indwelling central venous catheters implanted in the caudal vena cava. Animals were allowed to recover for 1 week after surgery and were then used for multiple pharmacokinetic studies with a suitable washout period between successive dosing of the derivatives.

Animals were fasted for approximately 18 h before dosing and from 0 to 4 h after dosing, but had access to water ad libitum throughout the period.

The sodium salts of the compounds of Examples 1 were dissolved to a concentration of 20-40 nmol/mL in a buffer containing 0.007% polysorbate 20, 50 mM sodium phosphate, 70 mM sodium chloride, pH 7.4. Intravenous administrations (volume typically corresponding to 1.5-2 nmol/kg, e.g., 0.1 mL/kg) of the compounds were performed through one catheter, and blood was collected at predetermined time points for up to 14 days after dosing (preferably through a different catheter). Blood samples (e.g., 0.8 mL) were collected in 8 mM EDTA buffer and then centrifuged at 4°C and 1942 g for 10 min.

Sampling and analysis.

Plasma was pipetted into Micronic tubes on dry ice and stored at -20°C until analysis for plasma compound concentrations using ELISA or similar antibody-based assay or LCMS. Individual plasma concentration-time profiles were analyzed using a non-compartmental model in Phoenix WinNonLin version 6.4 (Pharsight Inc., Mountain View, CA, USA) and the resulting terminal half-lives (harmonic mean) were determined.

- 57 049130

Results.

Table 2

Terminal half-life measured after intravenous administration to minipigs

The tested compounds of the present invention are characterized by very long half-life values compared to the half-life values of hGLP-1 and hGIP, which, when measured in humans, are approximately 2-4 min and 5-7 min, respectively. Based on the measured half-life values in minipigs, half-life values in humans are preliminarily determined to be sufficient for administration at least once a week when using an injection solution or for administration at least once a day when using a tablet for oral administration.

Example 5. Pharmacokinetic study in dogs.

The purpose of this example is to determine the half-life and plasma concentration in vivo of the compounds of the invention after p.o. administration to beagle dogs, i.e., the terminal half-life and concentration of the test substance that is released into the bloodstream over time. This was performed in a pharmacokinetic (PK) study that determines such parameters of the test compound. The terminal half-life is generally understood to be the period of time required for a determined plasma concentration measured after the initial distribution phase to be halved.

Obtaining compositions in tablet form.

Tablet compositions containing the test substance and SNAC (sodium N-(8-(2-hydroxybenzoyl)amino)caprylate) were prepared according to methods known to those skilled in the art by mixing the test substance with SNAC and magnesium stearate, compacted by a roller press, as described, for example, in WO 2019/149880. The amount of SNAC in the tablet composition was 100-300 mg, the amount of magnesium stearate in the tablet composition was 7.7 mg, and the target amount of each test substance in the tablet composition was 3-4 mg.

Animals, dosing and sampling.

Male Beagle dogs aged 1–7 years weighing 9–17 kg at the time of study were included in the studies. Dogs were dosed in a fasted state. Dogs were group housed in kennels (12 h light:12 h dark) and fed individually and strictly once daily with Royal Canin Medium Adult adult dog food (Royal Canin Products, subsidiary in China, or Brogaarden A/S, Denmark). Dogs were used for repeated PK studies using a suitable washout period between consecutive dosing. An appropriate acclimatization period was provided before the start of the first PK study. All animal handling, dosing, and blood sampling were performed by trained and qualified personnel. Dogs were fasted overnight before the studies and for 0–4 h post-dose. Dogs were restricted from access to water for a period of 1 h pre-dose through 4 h post-dose, but were provided with access to water ad libitum throughout the remainder of the period.

The compositions were administered by oral administration of a single dose to dogs in groups of 6-8 dogs. Tablets were administered as follows: 10 minutes prior to tablet administration, dogs were given approximately 3 nmol/kg of SEQ ID NO: 48 subcutaneously, then the tablets were placed in the back of the dog's mouth to prevent chewing. The mouth was then closed and 10 ml of drinking water was administered via syringe or tube to facilitate swallowing of the tablet.

A single blood sample was collected prior to dosing and additional samples were collected at predetermined time points post-dose, such as 5, 10, 15, 20, 30 and 45 min and 1, 1.5, 2, 4, 7, 10, 24, 48, 72, 120, 144, 168, 192, 216 h up to 240 h, to adequately cover the complete plasma concentration-time profile of test substance absorption. At each blood sampling time point, approximately 0.8 mL of whole blood was collected into 1.5 mL EDTA-coated tubes, which were gently inverted to mix the sample with EDTA. Blood samples were collected into EDTA-based buffer (8 mM) and then centrifuged at 4°C and 2000 g for 10 min. Blood plasma was pipetted into Micronic tubes on dry ice and stored at -20°C or below until analysis. Blood samples were collected routinely, i.e., via a Venflon catheter placed in the saphenous vein of the forearm on the forepaw for the first 2 h and then via a jugular vein syringe at other time points. The first few drops were allowed to drain from the Venflon to prevent heparin saline from contaminating the sample.

- 58 049130

All blood samples were collected into assay tubes containing EDTA for stabilization and stored on ice until centrifugation. Plasma was separated from whole blood by centrifugation and stored at -20°C or below until analysis.

Analysis and calculations.

Blood plasma was analyzed for the test substance using LC-MS (liquid chromatography-mass spectrometry) as known to those skilled in the art. The system consisted of either a Thermo Fisher QExactive mass spectrometer equipped with a TurboFlow 10-valve interface module system, a CTC HTS PAL autosampler, Accela 1250 pumps, and a Hot Pocket column oven; or a Thermo Fisher QExactive Plus mass spectrometer equipped with a TurboFlow valve interface module system, a TriPlus RSI autosampler, Dionex UltiMate 3000 pumps, and a Hot Pocket column oven. RP-HPLC separation was achieved using a 1:1 acetonitrile/methanol linear gradient in 1% aqueous formic acid using either a Phenomenex Onyx Monolithic C18 column (50 x 2.0 mm) at a flow rate of 0.8 mL/min at 30°C or an Agilent Poroshell 120 SB-C18 column (50 x 2.1 mm, 2.7 μm) at a flow rate of 0.4 mL/min at 60°C. Mass spectrometric analysis was performed in either positive ionization SIM or positive ionization PRM mode.

For each individual animal, the plasma concentration-time profile was analyzed using a non-compartmental model in Pharsight Phoenix WinNonLin version 6.4 or other appropriate PK analysis software and the resulting terminal half-life (t _1/2 ), maximum plasma concentration per dose (C _max /D), time to maximum plasma concentration (t _max ), and area under the curve to infinity per dose (AUC/D) were determined. Statistical analyses of pharmacokinetic studies are summarized as median (for t _max ), harmonic mean (t _1/2 ), or arithmetic mean (C _max , AUC).

Results.

Table 3

Pharmacokinetic parameters after oral administration of compositions containing test ________ substance and SNAC in tablets to beagle dogs _________

Connection No. SNAC amount (mg) Target amount of gestational substance (mg) Tmax ( ^h ) tl/2 (h) Shah/DOSE (kg/l) AUC/dose (kg*h/l) 9 100 4 2.0 50 0.24 12.1 10 100 4 1,0 36 0,20 8.4 AND 100 4 1,4 34 0.19 7.1 13 100 O 1,5 60 0.29 14.8 15 100 O 1,5 59 0.24 I,9 15 300 2.0 46 0.28 I,6 16 100 3 1,3 47 0.21 8.6 20 100 2.0 56 0.21 10.8 24 300 3 1,8 74 0.29 16.4 25 300 3 1,8 43 0,30 15.2 26 300 O 1,2* 70* 0.49* 27.4* 28 300 O 1.3** 67** 0.36** 18.6** 29 300 3 1,2** 69** 0.26** 16.6** 30 300 3 0.8 81 0.24 6.5 40 300 3 2.0 61 0.48 27,0 31 300 3 1.3** 56** 0.35** 2** 32 300 3 1.8* 80* 0.45* 25.3* 43 300 3 1,5 131 0.22 21.4

* = average data from two experiments with the same design conditions;

** = average data from three experiments with the same design conditions.

The tested compounds of the present invention demonstrate oral bioavailability in this model, as plasma compound concentrations (C _max /D>0 and AUC/D>0) were determined after oral administration. In addition, the tested compounds of the present invention are further characterized by very long half-lives compared to the half-lives of hGLP-1 and hGIP, which are approximately 2-4 min and 5-7 min, respectively, when measured in humans.

Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will be apparent to those skilled in the art. It is therefore to be understood that the accompanying embodiments are intended to cover all such modifications and changes that fall within the true spirit of the present invention.

Sequence listing <110> Novo Nordisk A/S <120> GLP-1 AND GIP RECEPTOR COAGONISTS SUITABLE FOR ORAL DELIVERY <130> 200018 <160>48 <170> PatentIn version 3.5 <210>1 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>1

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys 1 5 1015

Gln Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>2 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib

- 60 049130 <400> 2

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Tyr Tyr Leu Glu Lys 15

Gln Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro 20 25 30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 3 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 3

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Tyr Tyr Leu Glu Lys

5 10 15

Gln Ala Ala Gln Ala Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30

Ser Gly Ala Pro Pro Pro Ser 35 <210> 4 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 4

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Tyr Leu Glu Lys 15

Gln Ala Ala Arg Glu Phe Val Asn Trp Leu

Leu Ala Gly Gly Pro Ser

- 61 049130

Ser Gly Ala Pro Pro Pro Ser 35 <210> 5 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 5

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu 1 5 10 15

Lys

Gln Ala Ala Arg Glu Phe

Ile Asn Trp Leu Leu Ala Gly Gly Pro

30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 6 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 6

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu

5 10 15

Lys

Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro 20 25 30

Ser

Ser Gly Ala Pro Pro Pro Ser 35

- 62 049130 <210> 7 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 7

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu

5 10 15

Lys

Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Arg Gly Gly Pro 20 25 30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 8 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 8

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu 1 5 10 15

Lys

Gln Ala Ala Arg Glu Phe

Ile Glu Trp Leu Leu Arg Gly Gly Pro

30

Glu

Ser Gly Ala Pro Pro Pro Ser 35 <210> 9 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic

- 63 049130 <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>9

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Leu Glu Lys

5 1015

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>10 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>10

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 1015

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>11 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400> 11

Tyr Ala Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Tyr Leu Glu Lys 15

- 64 049130

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro 20 25 30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 12 <211> 29 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 12

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu

5 10 15

Lys

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly

25 <210> 13 <211> 31 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 13

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu

5 10 15

Lys

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro 20 25 30 <210> 14 <211> 33 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic

- 65 049130 <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 14

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 10 15

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro 20 25 30

Ser

Ser <210> 15 <211> 35 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 15

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Leu Leu Glu Lys 15

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu

Leu Ala Gly Gly Pro Ser 30

Ser Gly Ala <210> 16 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib

- 66 049130 <400> 16

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

Gln Ala Ala Arg Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 17 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 17

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 10 15

Gln Ala Ala Arg Glu Phe Ile Gln Trp Leu Leu Ala Gly Gly Pro Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 18 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 18

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 10 15

Gln Ala Ala Gln Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser

- 67 049130

Ser Gly Ala Pro Pro Pro Ser 35 <210> 19 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>19

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 1015

Gln Ala Ala Glu Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>20 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>20

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 510

Ser Ile Leu Leu Glu Lys 15

Gln Ala Ala H is Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser

2530

Ser Gly Ala Pro Pro Pro Ser 35

- 68 049130 <210> 21 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>21

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 1015

Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Arg Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>22 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>22

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 1015

Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Arg Gly Gly Pro Glu 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>23 <211>39 <212> PROTEIN <213> Artificial sequence <220>

- 69 049130 <223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 23

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Leu Leu Glu

Lys

Gln Ala Ala Gln Glu Phe

Ile Glu Trp Leu Leu Ala Gly Gly Pro

30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 24 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 24

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Leu Leu Glu

Lys

Gln Ala Ala His Glu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro 20 25 30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 25 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib

- 70 049130 <400> 25

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Leu Leu Glu Lys 15

Gln Ala Ala Arg Glu Phe

Ile Glu Trp Leu Leu Ala Gly Gly Pro Ser 25 30

Ser Gly Ala Pro Pro Pro Ser 35 <210> 26 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 26

Tyr Xaa Glu Gly Thr Val Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys

5 10 15

Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30

Ser Gly Ala Pro Pro Pro Ser 35 <210> 27 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 27

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Ile Leu Glu Lys 15

- 71 049130

Gln Ala Ala Arg Glu

Phe Ile Asn Trp Leu 25

Leu Ala Gly Gly Pro 30

Ser

Ser Gly Ala Pro Pro 35

Pro Ser <210> 28 <211> 39 <212> PROTEIN <213> Artificial <220>

<223> Synthetic Sequence <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 28

Tyr Xaa Glu Gly Thr

5

Phe Thr Ser Asp Tyr

Ser Ile Tyr Leu Glu 15

Glu

Gln Ala Ala Arg Lys

Phe Ile Asn Trp Leu 25

Leu Ala Gly Gly Pro 30

Ser

Ser Gly Ala Pro Pro 35

Pro Ser <210> 29 <211> 39 <212> PROTEIN <213> Artificial Sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400>29

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu

5 1015

Glu

Lys Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro 20 2530

Ser

Ser Gly Ala Pro Pro Pro Ser 35

- 72 049130 <210> 30 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 30

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Ile Leu Leu Glu

Glu

Gln Ala Ala Arg Lys Phe

Ile Asn Trp Leu Leu Ala Gly Gly Pro

30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 31 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 31

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Leu Leu Glu 1 5 10 15

Glu

Gln Ala Ala Arg Glu Phe

Ile Glu Trp Leu Leu Ala Gly Gly Pro

30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 32 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

- 73 049130 <223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 32

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr 1 5 10

Ser Lys Tyr Leu Glu

Glu

Gln Ala Ala Arg Glu Phe

Ile Asn Trp Leu Leu Ala Gly Gly Pro

30

Ser

Ser Gly Ala Pro Pro Pro Ser 35 <210> 33 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 33

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu 1 5 10 15

Glu

Gln Ala Ala Arg Glu Phe

Ile Asn Trp Leu Leu Ala Gly Gly Pro

30

Ser

Lys Gly Ala Pro Pro Pro Ser 35 <210> 34 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib

- 74 049130 <400> 34

Tyr Xaa Glu Gly Thr 1 5

Gln Ala Ala Arg Glu

Lys Gly Ala Pro Pro 35 <210> 35 <211> 39 <212> PROTEIN <213> Artificial <220>

<223> Synthetic

Phe Thr Ser Asp Tyr 10

Phe Ile Glu Trp Leu 25

Pro Ser sequence

Ser Ile Leu Leu Glu

Leu Ala Gly Gly Pro 30

Glu

Ser <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <400> 35

Tyr Xaa Glu Gly Thr

5

Gln Ala Ala Arg Glu

Lys Gly Ala Pro Pro 35

Phe Thr Ser Asp Tyr 10

Phe Ile Asn Trp Leu 25

Pro Ser

Ser Ile Leu Leu Glu

Leu Ala Gly Gly Pro 30

Glu

Ser <210> 36 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> OTHER_CHARACTERISTIC < 222> (2)..(2) < 223> Xaa is Aib or Ala <220>

< 221> OTHER_CHARACTERISTIC < 222> (6)..(6) < 223> Xaa is Phe or Val <220>

- 75 049130

<221> <222> <223> OTHER_CHARACTERISTIC (12) . . (12) Xaa is Ile or Tyr <220> <221> <222> <223> OTHER_CHARACTERISTIC (13)..(13) Xaa is Tyr, Ala , Leu or Ile <220> <221> <222> <223> OTHER_CHARACTERISTIC (16)..(16) Xaa is Lys or Glu <220> <221> <222> <223> OTHER_CHARACTERISTIC (20).. (20) Xaa is Gln, Arg , Glu or His <220> <221> <222> <223> OTHER_CHARACTERISTIC (21) . . (21) Xaa is Ala or Glu <220> <221> <222> <223> OTHER_CHARACTERISTIC (23)..(23) Xaa is Ile or Val <220> <221> <222> <223> OTHER_CHARACTERISTIC (24)..(24) Xaa is Glu, Gln or Asn <220> <221> <222> <223> OTHER_CHARACTERISTIC (28).. (28) Xaa is Ala or Arg <220> <221> <222> <223> OTHER_CHARACTERISTIC (32) .. (32) Xaa is Glu, Ser or missing <220> <221> <222> <223> OTHER_CHARACTERISTIC (33) .. (33) Xaa is Sir, Lys or missing <220> <221> <222> <223> OTHER_CHARACTERISTIC (34) . . (34) Xaa is Gly or absent <220> <221> <222> <223> OTHER_CHARACTERISTIC (35)..(35) Xaa is Ala or absent <220> <221> <222> <223> OTHER_CHARACTERISTIC (36)..(36) Xaa is Pro or absent

- 76 049130 <220>

< 221> OTHER_CHARACTERISTIC < 222> (37).. (37) < 223> Xaa is Pro or absent <220>

< 221> OTHER_CHARACTERISTIC < 222> (38).. (38) < 223> Xaa is Pro or absent <220>

< 221> OTHER_CHARACTERISTIC < 222> (39) .. (39) <223> Xaa is Ser or absent <400> 36

Tyr Xaa Glu Gly 1

Thr Xaa Thr Ser 5

Asp Tyr Ser Xaa 10

Xaa Leu Glu Xaa

Gln Ala Ala Xaa

Xaa Phe Xaa Xaa

Trp Leu Leu Xaa 25

Gly Gly Pro Xaa 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa <210> 37 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> OTHER_CHARACTERISTIC < 222> (2)..(2) < 223> Xaa is Aib or Ala <220>

< 221> OTHER_CHARACTERISTIC < 222> (6)..(6) < 223> Xaa is Phe or Val <220>

< 221> OTHER_CHARACTERISTIC < 222> (12)..(12) < 223> Xaa is Ile or Tyr <220>

< 221> OTHER_CHARACTERISTIC < 222> (13)..(13) < 223> Xaa is Tyr, Ala, Leu or Ile <220>

< 221> OTHER_CHARACTERISTIC < 222> (16)..(16) < 223> Xaa is Lys or Glu

- 77 049130 <220>

< 221> OTHER_CHARACTERISTIC < 222> (20).. (20) < 223> Xaa is Gln, Arg, Glu or His <220>

< 221> OTHER_CHARACTERISTIC < 222> (21)..(21) < 223> Xaa is Ala or Glu <220>

< 221> OTHER_CHARACTERISTIC < 222> (23)..(23) < 223> Xaa is Ile or Val <220>

< 221> OTHER_CHARACTERISTIC < 222> (24).. (24) < 223> Xaa is Glu, Gln or Asn <220>

< 221> OTHER_CHARACTERISTIC < 222> (28).. (28) < 223> Xaa is Ala or Arg < 400>37

Tyr Xaa Glu Gly Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu Xaa

5 1015

Gln Ala Ala Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>38 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> OTHER_CHARACTERISTIC < 222> (2)..(2) < 223> Xaa is Aib or Ala <220>

< 221> OTHER_CHARACTERISTIC < 222> (6)..(6) < 223> Xaa is Phe or Val <220>

< 221> OTHER_CHARACTERISTIC < 222> (12)..(12) < 223> Xaa is Ile or Tyr

- 78 049130 <220>

< 221> OTHER_CHARACTERISTIC < 222> (13)..(13) < 223> Xaa is Tyr, Ala, Leu or Ile <220>

< 221> OTHER_CHARACTERISTIC < 222> (16)..(16) < 223> Xaa is Lys or Glu <220>

< 221> OTHER_CHARACTERISTIC < 222> (20)..(20) < 223> Xaa is Gln, Arg, Glu or His < 220>

< 221> OTHER_CHARACTERISTIC < 222> (21)..(21) < 223> Xaa is Ala or Glu <220>

< 221> OTHER_CHARACTERISTIC < 222> (23)..(23) < 223> Xaa is Ile or Val <220>

< 221> OTHER_CHARACTERISTIC < 222> (24).. (24) < 223> Xaa is Glu, Gln or Asn <220>

< 221> OTHER_CHARACTERISTIC < 222> (28).. (28) < 223> Xaa is Ala or Arg < 400> 38

Tyr Xaa Glu Gly Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu

5 10 15

Xaa

Gln Ala Ala Xaa Xaa Phe Xaa Xaa

Trp Leu Leu Xaa Gly Gly Pro

30

Glu

Ser Gly Ala Pro Pro Pro Ser 35 <210> 39 <211> 39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> OTHER_CHARACTERISTIC <222> (2)..(2) <223> Xaa is Aib or Ala

- 79 049130 <220>

< 221> OTHER_CHARACTERISTIC < 222> (6)..(6) < 223> Xaa is Phe or Val <220>

< 221> OTHER_CHARACTERISTIC < 222> (12)..(12) < 223> Xaa is Ile or Tyr <220>

<221> OTHER_CHARACTERISTIC <222> (13)..(13)

<223> Xaa is Tyr, Ala, Leu or Ile <220> <221> <222> <223> <220> <221> <222> OTHER_CHARACTERISTIC (16)..(16) Xaa is OTHER_CHARACTERISTIC (20)..(20) Lys or Glu <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <400> Xaa is OTHER_CHARACTERISTIC (21) . . (21) Xaa is OTHER_CHARACTERISTIC (23)..(23) Xaa is OTHER_CHARACTERISTIC (24)..(24) Xaa is OTHER_CHARACTERISTIC (28).. (28) Xaa is 39 Gln, Ala Ile Glu, Ala Arg, Glu or or Glu or Val Gln or Asn or Arg His Tyr Xaa Glu Gly Thr Xaa Thr Ser Asp Tyr Ser 1 5 10 Xaa Xaa Leu Glu Xaa 15

Gln Ala Ala Xaa Xaa Phe Xaa Xaa

Trp Leu Leu Xaa Gly Gly Pro Ser 25 30

Lys Gly Ala Pro

Pro Pro Ser <210> 40 <211> 4 <212> PROTEIN

- 80 049130 <213> Artificial sequence <220>

<223> Synthetic <400>40

Ser Ser Gly Ala <210>41 <211>4 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400>41

Glu Ser Gly Ala <210>42 <211>4 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400>42

Ser Lys Gly Ala <210>43 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400>43

Leu Leu Glu Lys Gln Ala Ala Arg Glu Phe Ile Asn

510 <210>44 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400> 44

- 81 049130

Leu Leu Glu Lys Gln Ala Ala Arg Glu Phe Ile Glu 1 510 <210>45 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400>45

Leu Leu Glu Lys Gln Ala Ala Gln Glu Phe Ile Glu

510 <210>46 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <400>46

Leu Leu Glu Glu Gln Ala Ala Arg Glu Phe Ile Glu

510 <210>47 <211>39 <212> PROTEIN <213> Artificial sequence <220>

<223> Synthetic <220>

<221> MOD._OST.

<222> (2)..(2) <223> Aib <220>

< 221> OTHER_CHARACTERISTIC < 222> (2)..(2) < 223> Xaa is Aib or Ala <220>

< 221> OTHER_CHARACTERISTIC < 222> (6)..(6) < 223> Xaa is Phe or Val <220>

< 221> OTHER_CHARACTERISTIC < 222> (12)..(12) < 223> Xaa is Ile or Tyr <220>

< 221> OTHER_CHARACTERISTIC

- 82 049130

<222> <223> (13) .. (13) Xaa is Tyr, Ala, Leu or Ile <220> <221> <222> <223> OTHER_CHARACTERISTIC (16)..(16) Xaa is Lys or Glu <220> <221> <222> <223> OTHER_CHARACTERISTIC (20).. (20) Xaa is Gln, Arg, Glu or His <220> <221> <222> <223> OTHER_CHARACTERISTIC (21) . . (21) Xaa is Ala or Glu <220> <221> <222> <223> OTHER_CHARACTERISTIC (23)..(23) Xaa is Ile or Val <220> <221> <222> <223> OTHER_CHARACTERISTIC (24)..(24) Xaa is Glu, Gln or Asn <220> <221> <222> <223> OTHER_CHARACTERISTIC (28).. (28) Xaa is Ala or Arg <220> <221> <222> <223> OTHER_CHARACTERISTIC (30).. (30) Xaa is Gly or missing <220> <221> <222> <223> OTHER_CHARACTERISTIC (31)..(31) Xaa is Pro or missing <220> <221> <222> <223> OTHER_CHARACTERISTIC (32) .. (32) Xaa is Glu, Ser or absent <220> <221> <222> <223> OTHER_CHARACTERISTIC (33) .. (33) Xaa is Sir, Lys or absent <220> <221> <222> <223> OTHER_CHARACTERISTIC (34) . . (34) Xaa is Gly or missing <220> <221> <222> <223> OTHER_CHARACTERISTIC (35)..(35) Xaa is Ala or missing

- 83 049130 <220>

< 221> OTHER_CHARACTERISTIC < 222> (36)..(36) < 223> Xaa is Pro or absent <220>

< 221> OTHER_CHARACTERISTIC < 222> (37).. (37) < 223> Xaa is Pro or absent <220>

< 221> OTHER_CHARACTERISTIC < 222> (38).. (38) < 223> Xaa is Pro or absent <220>

< 221> OTHER_CHARACTERISTIC < 222> (39)..(39) <223> Xaa is Ser or absent <400> 47

Tyr Xaa Glu Gly Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu Xaa 1 5 10 15

Gln Ala Ala Xaa Xaa Phe Xaa Xaa

Trp Leu Leu Xaa Gly Xaa Xaa Xaa 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa <210>48 <211>29 <212> PROTEIN <213> Homo sapiens <400>48

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

5 1015

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

Claims (10)

1. A compound comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is YX2EGTX6TSDYSX12X13LEX16QAAX20X21FX23X24WLLX28GX30X31X32X33X34X35X36X37X38X39 (SEQ ID NO: 47); where X ₂ represents Aib or A; X ₆ represents F or V; X ₁₂ represents I or Y; X ₁₃ represents Y, A, L, or I; X ₁₆ represents K or E; X ₂₀ represents Q, R, E or H; X ₂₁ represents A or E; - 84 049130 X23 represents I or V; X ₂₄ represents E, Q or N; X ₂₈ represents A or R; X ₃₀ is G or absent; X ₃₁ is P or absent; X32 is E, S, or absent; X33 is S, K, or absent; X ₃₄ is G or absent; X35 is A or absent; X ₃₆ is P or absent; X37 is P or absent; X ₃₈ is P or absent; and X ₃₉ is S or absent; wherein the substituent is attached to the peptide via lysine (K) at position 16 or 33; and wherein the substituent comprises at least one HOOC-( _CH2 ) _n -CO-, wherein n is 14, 16, or 18; or a pharmaceutically acceptable salt thereof. 2. The connection according to claim 1, where X ₃₂ X ₃₃ X ₃₄ X ₃₅ is selected from the group consisting of SSGA, ESGA and SKGA. 3. The compound of claim 1, wherein the amino acid sequence of the peptide is YX ₂ EGTX ₆ TSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ WLLX ₂₈ GGPSSGAPPPS (SEQ ID NO: 37); wherein X ₂ is Aib or A; X6 represents F or V; X12 represents I or Y; X13 represents Y, A, L, or I; X16 represents K or E; X ₂₀ represents Q, R, E or H; X ₂₁ represents A or E; X ₂₃ represents I or V; X ₂₄ represents E, Q or N; X ₂₈ represents A or R. 4. A connection according to any one of the preceding paragraphs, wherein X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FX ₂₃ X ₂₄ is selected from the group consisting of LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and LLEEQAAREFIE. 5. The compound of claim 1, wherein the amino acid sequence of the peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. 6. A compound according to any one of the preceding claims, wherein the peptide comprises an amide group at the C terminus. 7. A compound according to any one of claims 1 to 6, wherein the substituent is selected from the group consisting of: - 85 049130 8. The compound of claim 1, wherein the compound is selected from the group consisting of the following: - 86 049130 - 87 049130 - 88 049130 - 89 049130 - 90 049130 - 92 049130 - 93 049130 9. The compound of claim 8, wherein the compound is - 94 049130 10. The compound of claim 8, wherein the compound is Evra Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2