Agonists of amylin receptor (hAMY3R) with improved chemical stability

Abstract

The present invention relates to polypeptides as amylin receptor (hAMY3R) agonists with improved chemical stability.

Description

Technical Field

[0001] The present invention relates to a novel method for treating a leukemia based on a human adrenomedullin fragment hAM. 15-52 A polypeptide having a sequence of SEQ ID NO: 1, which acts as an agonist of the amylin receptor (hAMY3R). In particular, the present invention relates to a polypeptide having improved chemical stability. Background Art

[0002] Obesity is a disease in which excess body fat accumulates to a certain level that has a negative impact on health. Obesity affects a large number of people worldwide and is rapidly increasing in some parts of the world. The World Health Organization (WHO) estimates that approximately 650 million people were obese worldwide in 2016. Obesity is defined as a body mass index (BMI) of more than 30. Obesity is considered a major risk factor for a variety of diseases, such as cardiovascular disease (e.g., hypertension, atherosclerosis, heart attack or stroke), NASH (non-alcoholic steatohepatitis), musculoskeletal diseases, certain cancers, depression and type II diabetes, and is therefore harmful to human health. Cardiovascular disease and diabetes are two major diseases associated with obesity. A lot of research has been done in the field of obesity to find new ways to treat obesity or obesity-related diseases and disorders.

[0003] Diabetes is a group of metabolic disorders characterized by high blood sugar levels. As of 2019, the International Diabetes Federation estimates that 463 million people worldwide have diabetes, with about half of all patients diagnosed with the disease. There are two types of diabetes, type I and type II. Type I diabetes is caused by an autoimmune response that leads to a loss of pancreatic beta cells, which in turn prevents the production of enough insulin. Type II diabetes, on the other hand, is a disease that starts with insulin resistance, where the cells are unable to respond to insulin properly, and can also lead to insulin deficiency as the condition progresses.

[0004] Calcitonin peptide family

[0005] The calcitonin peptide family consists of the hormone peptides calcitonin (CT), calcitonin gene-related peptide (CGRP), islet amyloid polypeptide (IAPP, amylin or hAMY 1-37 ) and adrenomedullin (hAM) and its precursors. hAMY 1-37 It is a 37-residue peptide hormone that is co-secreted with insulin from pancreatic beta cells and has the amino acid sequence of Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr.

[0006] Amylin inhibits glucagon release and inhibits gastric emptying, thus playing an important role in maintaining glucose homeostasis by lowering blood glucose concentrations. In addition, amylin has been shown to reduce food intake and play an important role in satiety, making it a potential candidate for the treatment of, for example, obesity and / or diabetes. hAM is a 52-residue peptide hormone expressed in all tissues with the amino acid sequence Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr. It is a potent vasodilator with positive effects on cardiovascular diseases such as myocardial infarction, limb ischemia and hypertension.

[0007] The biological activities of the calcitonin family of proteins are generally mediated by binding to the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CRLR), both of which belong to the family of G protein-coupled receptors (GPCRs) 2. These receptors can be co-expressed with different receptor-modifying proteins (RAMP1-3) to create functional receptors for individual peptides in the calcitonin family of proteins. Co-expression of CTR with RAMP1 forms the amylin and CGRP receptor (AMY1R), co-expression of CTR with RAMP2 forms the amylin receptor 2 (AMY2R), and co-expression of CTR with RAMP3 forms the amylin receptor (AMY3R). Co-expression of CRLR with RAMP1 forms the CGRP receptor (CGRP1R), co-expression of CRLR with RAMP2 forms the adrenomedullin receptor (AM1R), and co-expression of CRLR with RAMP3 forms the receptor for adrenomedullin and CGRP (AM2R).

[0008] Several natural peptides in the calcitonin protein family show considerable overlap in receptor pharmacology. 1-37 In contrast, adrenomedullin is approximately 100-fold less potent against AMY3R. 15-52 ) are almost equivalent to AMY3R and AM1R, and the EC 50 The EC value for AM1R is 1.3 nM. 50 The value is 1.1 nM (the EC 50 Values ​​are measured according to the Examples herein). On the other hand, hAMY1-37 EC for AMY3R 50 The value was 10 pM, while it had no activity against AM1R.

[0009] Human Amylin (hAMY 1-37 Natural amylin (or amylin) has some disadvantages, such as high fibrogenic tendency, short in vivo half-life, and chemical instability at pH 7. Therefore, natural amylin is not ideal as an active ingredient in drugs. The successful amylin analog pramlintide overcomes some of the disadvantages of natural amylin and has been approved by the FDA for use in type I and type II diabetes. However, pramlintide is formulated at pH 4 and is fibrogenic at pH 7, so it may cause injection site pain when administered. 1-37 In contrast, human adrenomedullin fragment hAM 15-52 No fibrillation occurs at pH 7. Therefore, hAM 15-52 The backbone has previously been used to develop new amylin analogs (see WO 2022 / 063925 A1). 15-52 A few specific substitutions in hAM can completely eliminate its adrenomedullin receptor (AM1R) potency while enhancing its amylin receptor (hAMY3R) potency. 15-52 The advantage of this chemical approach is that such amylin receptor agonists are similar in pharmacodynamics to hAMY 1-37 Similar, but benefits from hAM 15-52 One such example is a polypeptide having the amino acid sequence KCNTATCTVQRLAEQIAQFTDKDKDNVAPPTNVGSNGHyp (SEQ ID NO: 3), whose hAMY3R EC 50 hAM1R EC was 14 nM 50 >5000nM. Although hAMY3R has high in vitro potency, low fibrosis tendency and high in vivo efficacy, the peptide is susceptible to chemical instability due to deamidation, dimerization and isomerization. The present invention solves these shortcomings of SEQ ID NO: 3, thereby providing a polypeptide with improved chemical stability and most suitable for clinical development. Summary of the invention

[0010] The present invention relates to the following discovery: by 32 and X 36The invention relates to the discovery that by replacing the disulfide bridge (-SS-) of SEQ ID NO: 3 with a methylene bridge (-S-CH2-S-), dimerization and the formation of high molecular weight products can be inhibited, thereby improving the chemical stability of SEQ ID NO: 3 without adversely affecting other properties such as potency. The invention also relates to the discovery that by replacing the aspartic acid (D) at position 25 in SEQ ID NO: 3 with glutamic acid (E), isomerization can be avoided, thereby improving the chemical stability without adversely affecting other properties such as potency.

[0011] Therefore, the first aspect of the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having up to 2 amino acid substitutions, provided that the substitutions are not present in X2-X4, X7, X 11 , X 32 or X 36-38 Any location in .

[0012] definition

[0013] In this article, the polypeptide is usually amidated at the C-terminus (-CONH2), similar to the natural peptides; amylin and adrenomedullin. However, the polypeptide of the present invention may also have a free carboxylic acid (-COOH) or another post-translational modification, such as a methyl ester (-COOMe). In a highly preferred embodiment of the present invention, the polypeptide is amidated at the C-terminus. The polypeptide of the present invention may have a free amine (-NH2), N-acylation (-NHCOR), N-methylation (-NHCH3 or -N(CH3)2), N-terminal deamination, or N-lipidation.

[0014] In this article, lipidation refers to the covalent attachment of a lipid to a polypeptide (such as C18DA (octadecanedioic acid), C20DA (eicosanedioic acid)), optionally via a linker / spacer consisting of one or more common covalent linking units (such as [γE], [OEG] or [AHX]), as shown below.

[0015]

[0016] Lipidation is often performed to improve the pharmacokinetic properties of a peptide, for example by improving metabolic stability, reducing enzymatic degradation, decreasing excretion and metabolism, all leading to a longer in vivo half-life (t 1 / 2 ) extension. The polypeptides of the invention can be lipidated or non-lipidated depending on the desired half-life. The polypeptides can be lipidated, for example at a lysine (K) residue or at the N-terminus, as exemplified herein. Preferably, the lipid (and linker) is selected from tetradecanoic acid (C14), hexadecanoic acid (C16), C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][AHX]- or C20DA[γE][γE][AHX]-. Most preferably, the lipidation is C20DA[γE]-. Most preferably, lipidation is performed at the N-terminus.

[0017] In this paper, EC 50 The EC value is used as a measure of the agonist potency for a given receptor. 50 Values ​​are a measure of the concentration of a compound required to achieve half of its maximal activity in a particular assay.

[0018] In this article, the polypeptide or its derivative may be in the form of a pharmaceutically acceptable salt. Therefore, pharmaceutically acceptable salts are intended to include any salt commonly used in peptide formulations. Such salts include acid addition salts and basic salts, for example, examples can be found in Remington's Pharmaceutical Sciences (17th edition). Similarly, hAM 15-52 Various solvates of the analogs or their pharmaceutically acceptable salts are also within the scope of the present invention.

[0019] In this document, unless otherwise indicated, amino acids are L-amino acids (L-stereoisomers, natural amino acids). The abbreviation Hyp refers to L-hydroxyproline. Substitutions in derivatives can be substitutions of natural amino acids as well as non-natural amino acids, including L- and D-stereoisomers. Preferably, substitutions in variants are conservative substitutions of conservative amino acids. The group of conservative amino acids can be defined as:

[0020] G, A, V, L, I, P (aliphatic or cyclic),

[0021] S, C, T, M (hydroxyl or sulfur-containing)

[0022] F, Y, W (aromatic)

[0023] H, K, R (alkaline)

[0024] D, E, N, Q (acid or amide)

[0025] In this article, it should be understood that the cysteine ​​at position X2 and X7 is covalently linked via a bridge, such as a disulfide bridge (-SS-) or a methylene bridge (-S-CH2-S-). Most preferably, the cysteine ​​at position X2 and X7 is covalently linked via a methylene bridge (-S-CH2-S-).

[0026] In this context, the term "treatment" should be understood in the broadest sense as prevention, amelioration or treatment. Thus, treatment is also intended to include preventive treatment. DETAILED DESCRIPTION

[0027] Aspect 1-Peptides

[0028] A polypeptide having the amino acid sequence KCNTATCTVQRLAEQIAQFTDKDKDNVAPPTNVGSNGHyp (SEQ ID NO: 3) has previously been shown to have high in vitro potency against hAMY3R (i.e., hAMY3R EC 50 0.014 nM), and has a higher selectivity for hAMY3R than hAMY1R (i.e., hAM1R EC 50 >5000 nM) and is non-fibrogenic. In addition, the inventors have found that SEQ ID NO:3 also has high in vivo efficacy. Despite its positive properties, the present invention arises from the discovery that SEQ ID NO:3 is susceptible to chemical instability, which poses a significant concern for the long-term stability of drug candidates. The present invention relates to methods for improving the chemical stability of SEQ ID NO:3 and structurally related polypeptides thereof.

[0029] The inventors found that the chemical instability of SEQ ID NO: 3 is caused by deamidation, dimerization and isomerization. SEQ ID NO: 3 contains seven potential deamidation sites (i.e., positions X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X3 26 , X 32 and X 36 N; position X 10 , X 15 , X 18 Q), and found three of them (i.e., positions X3, X 32 and X 36) is a hot spot for deamidation, as shown in the following Scheme 1. Therefore, the present inventors found that, as shown in Scheme 1, by replacing asparagine (N) at positions 3, 32 and 36 in SEQ ID NO: 3 with leucine (L) and alanine (A) to prevent deamidation, the chemical stability of SEQ ID NO: 3 can be significantly improved without adversely affecting the potency (see Example 1, Table 1).

[0030]

[0031] The present inventors have further discovered that another reason for the chemical instability of SEQ ID NO:3 can be attributed to isomerization. In particular, the present inventors have discovered that X in SEQ ID NO:3 25 The aspartic acid (D) at position 1 is slightly more susceptible to structural and chiral isomerization, as shown in Scheme 2 below, which also reduces the yield of the synthesis.

[0032]

[0033] Therefore, the present invention further relates to the following discovery: by 25 Substitution of aspartic acid (D) at position 1 with glutamic acid (E) can prevent isomerization and increase the overall synthetic yield, as shown in Scheme 3 below, without adversely affecting potency (see Example 1, Table 1).

[0034]

[0035] Therefore, the first aspect of the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0036] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having up to 2 amino acid substitutions, provided that the substitutions are not present in X2-X4, X7, X 11 , X 32 or X 36-38 Any location in .

[0037] Preferably, the derivative of SEQ ID NO: 4 has 1 amino acid substitution.Most preferably, no substitutions are present in SEQ ID NO: 4.

[0038] Therefore, in a preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0039] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having one amino acid substitution, provided that the substitution is not present in X2-X4, X7, X 11 , X 32 or X 36-38 Any location in .

[0040] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0041] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp].

[0042] The inventors further discovered that the chemical instability of SEQ ID NO: 3 can be attributed to dimerization to some extent, as shown in the following Scheme 3. Therefore, the present invention further relates to the following discovery: by replacing the disulfide bridge (-SS-) formed between the cysteines at positions X2 and X7 in SEQ ID NO: 3 with a methylene bridge (-S-CH2-S-), dimerization can be inhibited, thereby improving chemical stability (see Example 1, Table 1).

[0043]

[0044] Therefore, in one embodiment of the present invention, the cysteines in X2 and X7 are covalently linked via a disulfide bridge (-SS-). In the most preferred embodiment of the present invention, the cysteines in X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-) to minimize dimerization and prevent the formation of high molecular weight products.

[0045] Therefore, in a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0046] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having up to 2 amino acid substitutions, provided that the substitutions are not present in X2-X4, X7, X 11 , X 32 or X 36-38 and wherein the cysteines in X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0047] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0048] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having one amino acid substitution, provided that the substitution is not present in X2-X4, X7, X 11 , X 32 or X 36-38 and wherein the cysteines in X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0049] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0050] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4), wherein the cysteines in X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0051] The polypeptide of the present invention can be lipidated with various lipids depending on the desired polypeptide half-life. Preferably, the lipid is selected from tetradecanoic acid (C14)-, hexadecanoic acid (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][AHX]- or C20DA[γE][γE][AHX]-. Most preferably, the lipid is C20DA[γE]-. The polypeptide can be lipidated, for example, at a lysine residue (K) or at the N-terminus. Most preferably, the polypeptide is lipidated at the N-terminus. In the most preferred embodiment, the N-terminus is lipidated with C20DA[γE]-.

[0052] Therefore, in a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0053] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having up to 2 amino acid substitutions, provided that the substitutions are not present in X2-X4, X7, X 11 , X32 or X 36-38 wherein the cysteines in X2 and X7 are covalently linked via a disulfide bridge (-SS-) or a methylene bridge (-S-CH2-S-), most preferably a methylene bridge (-S-CH2-S-), and further wherein the polypeptide is lipidated with a lipid selected from the group consisting of tetradecanoic acid (C14)-, hexadecanoic acid (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE ][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][AHX]- or C20DA[γE][γE][AHX]-, most preferably C20DA[γE]-.

[0054] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0055] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a derivative thereof having one amino acid substitution, provided that the substitution is not present in X2-X4, X7, X 11 , X 32 or X 36-38wherein the cysteines in X2 and X7 are covalently linked via a disulfide bridge (-SS-) or a methylene bridge (-S-CH2-S-), most preferably a methylene bridge (-S-CH2-S-), and further wherein the polypeptide is lipidated with a lipid selected from the group consisting of tetradecanoic acid (C14)-, hexadecanoic acid (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γ E][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][AHX]- or C20DA[γE][γE][AHX]-, most preferably C20DA[γE]-.

[0056] In a highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0057] KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp], wherein the cysteines in X2 and X7 are covalently linked via a disulfide bridge (-SS-) or a methylene bridge (-S-CH2-S-), most preferably a methylene bridge (-S-CH2-S-), and further wherein the polypeptide is lipidated with a lipid selected from the group consisting of tetradecanoic acid (C14)-, hexadecanoic acid (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][O EG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γ E][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][AHX]- or C20DA[γE][γE][AHX]-, most preferably C20DA[γE]-.

[0058] In any of the above embodiments, wherein the polypeptide is lipidated, most preferably the lipidation is N-terminal lipidation, and most preferably, the cysteines in X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0059] In a more highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the following structure:

[0060]

[0061] wherein the polypeptide is lipidated with a lipid selected from the group consisting of tetradecanoic acid (C14)-, hexadecanoic acid (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[ γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG]- , C20DA[γE][γE][OEG][OEG]-, C20DA[γE][AHX]- or C20DA[γE][γE][AHX]-, most preferably C20DA[γE]-.

[0062] In an even more highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the structure:

[0063]

[0064] wherein the polypeptide is lipidated at the N-terminus with a lipid selected from the group consisting of tetradecanoic acid (C14)-, hexadecanoic acid (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA [γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG] -, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][AHX]- or C20DA[γE][γE][AHX]-, most preferably C20DA[γE]-.

[0065] In any of the above embodiments, the C-terminus is preferably amidated (-CONH2).

[0066] In a most preferred embodiment, the present invention relates to a polypeptide having the structure,

[0067] or a pharmaceutically acceptable salt thereof.

[0068] Aspect II - Medical Uses

[0069] In a second aspect, the present invention relates to the use of the polypeptide of the first aspect as a medicament. More specifically, the second aspect of the present invention relates to the use of the polypeptide of the first aspect for treating, preventing or ameliorating a disease, condition or disorder selected from the following: excessive food intake, overweight, obesity, bulimia, Prader-Willi syndrome, dyslipidemia, metabolic diseases / conditions, type I or type II diabetes, impaired glucose tolerance, insulin resistance syndrome and / or NASH, preferably obesity, diabetes, NASH or a combination thereof, most preferably obesity and / or diabetes.

[0070] Aspect III - Pharmaceutical Compositions

[0071] In a third aspect, the present invention relates to a pharmaceutical composition comprising one or more polypeptides of the first aspect and / or its medical use in treating, preventing or improving the disease, illness or disorder of the second aspect. According to conventional techniques in the art, the pharmaceutical composition may include a pharmaceutically acceptable carrier (carrier) and / or one or more excipients, such as those disclosed in 'Remington: Essentials of Pharmaceutics' (Linda A.Felton, Pharmaceutics press 2012). Suitable formulations include, but are not limited to, tablets, pills, capsules, emulsions, suspensions, sustained release agents, solutions or lyophilized powders for dissolving before administration. It should be understood that different routes of administration may be selected according to the dosage form. Such routes of administration may include, but are not limited to, oral administration, parenteral administration (intravenous (IV), subcutaneous (SC), intradermal (ID) and intramuscular (IM)) or inhalation. Preferably, the route of administration is parenteral administration. Most preferably, the route of administration is subcutaneous administration.

[0072] Aspect IV - Treatment Methods

[0073] In a fourth aspect, the invention relates to a method for treating a human or animal subject using one or more polypeptides of the first aspect or the pharmaceutical composition of the third aspect, wherein the human or animal subject is diagnosed with, suffers from, or is at risk of developing one or more diseases, conditions or disorders of the second aspect. The method comprises administering one or more compounds of the first aspect or the pharmaceutical composition of the third aspect in an effective therapeutic amount to treat the disease, condition or disorder mentioned in the third aspect.

[0074] Example

[0075] General protocol for hybrid peptide synthesis

[0076] The peptides were synthesized on a TentagelS RAM (Rapp polymer GmbH, Germany) with a loading of 0.23 mmol / g to 0.25 mmol / g using a Syro II fully automated parallel peptide synthesizer (MultiSynTech GmbH, Germany) equipped with a heating block. α -Fmoc deprotection was performed in two stages, i.e., treatment of the resin with 40% piperidine / DMF (0.2 M HOBt (1-hydroxybenzotriazole)) at 45°C for 3 min, followed by treatment with 20% piperidine / DMF (0.1 M HOBt) at 75°C for 7 to 12 min. The exceptions were Asp, Cys, and His residues, which were N- α -Fmoc deprotected; i.e. treated with 40% piperidine / DMF (0.2M HOBt) for 3 minutes, followed by 20% piperidine / DMF (0.1M HOBt) for 15 minutes. The coupling chemistry was DIC (N,N'-diisopropylcarbodiimide) / Oxyma (ethyl cyano(hydroxyimino)acetate) in DMF using a 0.5M solution of the amino acid in DMF and a 6-fold molar excess. Standard Fmoc protected amino acids were used. Coupling conditions were 75°C for 15 minutes for single or double coupling. The exceptions were His and Cys residues which were double coupled at 50°C for 15 minutes. The Fmoc-amino acids were dissolved at 0.5M in DMF containing 0.5M Oxyma, except for His which was dissolved in NMP. N α The resin was washed with NMP 4 times after -Fmoc deprotection and 3 times after coupling.

[0077] Disulfide bridges were formed on the resin using trityl (Trt) as a protecting group for cysteine ​​by treatment with 1% iodine in 75% HFIP (hexafluoroisopropanol) in DCM for 1 minute. The resin was washed 3 times with 75% HFIP in DCM and then 4 times with DCM.

[0078] After synthesis, the resin was washed with DCM and dried, and the peptide was cleaved from the resin by treatment with TFA (trifluoroacetic acid) / TES (triethylsilane) / water (95 / 2.5 / 2.5) at 42°C for 35 minutes, then precipitated with 4 volumes of cold ether, further washed with ether and left to dry.

[0079] After peptide is cleaved from resin using standard procedure, the methylene bridge between two cysteines is prepared in solution. Peptide (300mg) is dissolved in 20mM phosphate buffer (10mL, pH 7.8), dithiothreitol (2 equivalents) is added, and the mixture is stirred 10 minutes. Sodium iodide (2 equivalents) is then added, followed by the acetonitrile solution (15mL) of diiodomethane (40 equivalents) and ethanolamine (20 equivalents). The reaction is monitored by LCMS until completion, diluted with 100ml water, and removed lipophilic substances by extraction with ether (2x80mL).

[0080] The crude peptide was purified by reverse phase HPLC using a Waters preparative HPLC equipped with a C8 column (ReprosilGold 200A, 5 μm, 40 mm x 250 mm), a preparative pump (waters 2545), a UV / VIS detector (Waters 2489) and a Waters fraction collector III. The mobile phase was run at room temperature with a flow rate of 50 ml / min using a buffer A (0.1% TFA in water) and a buffer B (0.1% TFA in ACN, gradient: 35% to 45% B in 20 minutes). The relevant fractions were analyzed, pooled and lyophilized. The final product was characterized by analytical UPLC-MS.

[0081] The purity and quality of the peptides were determined by analytical HPLC-MS on a Kinetex C8 column (Phenomenex, 100A, 2.6 μm, 4.6 mm x 150 mm) using a Waters Acquity HPLC system equipped with a 3100 mass spectrometer detector. Gradient elution analysis was performed using buffer A (0.3% TFA in water) and buffer B (0.3% TFA in ACN) at a temperature of 40°C. The details of the gradient are summarized below.

[0082] Waters HPLC Method (21 minute run) Acetonitrile Gradient:

[0083] 10% to 40% (0 to 2 minutes), 40% to 60% (2 minutes to 16 minutes), 60% to 90% (16.5 minutes to 18.5 minutes), 90% to 10% (18.5 to 21 minutes).

[0084] Example 1

[0085] SEQ ID 4:

[0086]

[0087] Retention time 10.9 minutes

[0088] We find that m / 2=2193.2, m / 3=1462.6, m / 4=1096.9. We calculate m=4384

[0089] General Protocol for cAMP Assays to Measuring In Vitro Receptor Activation

[0090] hAMY3-R:

[0091] Cells stably overexpressing the hAMY3 receptor were obtained from Ogeda (now Astellas Pharma), subcloned, and monoclonal cell lines with an appropriate detection window were expanded, aliquoted, and frozen. An aliquot was thawed and plated in DPBS containing 0.05% casein and 0.5 mM IBMX at a density of 2000 cells / well in a 384-well plate. The cells were then immediately stimulated with graded doses of the test compound for 30 minutes at room temperature, with human amylin (Bachem, cat no. H-7905) as a positive control. cAMP accumulation was measured using the Cisbio assay for Gs-coupled receptors (cat. no. 62AM4PEC), where the detection reagent was added according to the manufacturer's instructions and time-resolved fluorescence energy transfer was recorded after one hour.

[0092] hAMl-R:

[0093] Cells stably overexpressing the hAM1 receptor were obtained from Ogeda (now Astellas Pharma), subcloned, and monoclonal cell lines with appropriate detection windows were expanded, aliquoted, and frozen. An aliquot was thawed and plated in DPBS containing 0.05% casein and 0.5 mM IBMX at a density of 8000 cells / well in 384-well plates. The cells were then immediately stimulated with graded doses of the test compound for 30 minutes at room temperature, with human adrenomedullin 1-52 (Bachem, cat no. H-2932) as a positive control. cAMP accumulation was measured using the Cisbio assay for Gs-coupled receptors (cat. no. 62AM4PEC), where the detection reagent was added according to the manufacturer's instructions, and time-resolved fluorescence energy transfer was recorded after one hour.

[0094] General protocol for determining the physical stability of peptide analogs

[0095] The peptide was dissolved in buffer (50mM sodium acetate at pH 4, or 50mM sodium phosphate at pH 7) and incubated for one hour. The sample was then divided into two replicates of 80 μl in a black 384-well plate (p-clear, Greiner Bio-One), and mixed with thioflavin T (ThT) to make a final concentration of 4 μM. The plate was sealed and placed in a plate reader (CLARIOstar, BMG), and during the experiment, the temperature was set to 40 ° C. Before each measurement, the plate was shaken at a speed of 700rpm (linear) for five minutes, and pressure was further applied to the sample. By exciting ThT at 450nm and measuring emission at 480nm, fluorescence was measured every 10 minutes for four days. For each peptide, the local polynomial regression fit (LOESS) implemented in the statistical programming environment R was used to smooth the ThT signal over time. For smooth data, the maximum ThT signal was normalized as the percentage between the maximum ThT signal of hAMY and the buffer background. Therefore, high values ​​indicate fibrotic properties similar to hAMY, whereas low values ​​indicate the absence of fibrosis.

[0096] Universal method for the determination of deamidation and isomerization

[0097] The peptides were dissolved in 50 mM phosphate buffer (pH 7) and incubated at 40 ° C for up to 28 days. The samples were then analyzed using an Explorisl20 mass spectrometer (both Thermo Fisher) coupled to a Vanquish Tandem UPLC equipped with an Acquity BEH C18 column (1.7 μm, 2.1x50 mm, 130A, Waters). The peptides were eluted using a linear gradient, starting from 5% to 70% buffer B in 7.5 minutes, with a flow rate of 0.4 mL / min (buffer B = 100% acetonitrile, containing 0.1% formic acid; buffer A = 100% water, containing 0.1% formic acid). The data were collected in a data-dependent acquisition mode in a positive mode (Top4), with an MS1 ​​resolution of 60.000 and an MS2 resolution of 30.000. The peptides were fragmented at a normalized collision energy of 25%. Deamidation and isomerization were quantified using the area under the curve.

[0098] General protocol for determining dimer and HMWP formation

[0099] The peptides were dissolved in 50 mM phosphate buffer (pH 7) and incubated at 40°C for up to 28 days. The samples were then analyzed using size exclusion chromatography to quantify the amount of high molecular weight products (HMWP), which is a general term for covalently linked peptide dimers, trimers, and higher multimers. The analysis was performed on an AcquityProtein BEH SEC 1.7 μm 4.6 x 300 mm HPLC was performed on a Dionex Ultimate 3000RS UHPLC Focused chromatography system (Thermo Scientific) with a 1.25 μM column (Waters part number 186006506). The solvent system consisted of two mobile phases, which were assumed to disrupt all non-covalent interactions between peptides, leaving only monomeric peptides and covalent HMWPs for analysis.

[0100] Mobile phase A (all v / v%): 95% H2O, 5% MeCN (acetonitrile), 0.1% TFA (trifluoroacetic acid)

[0101] Mobile phase B (all v / v%): 95% MeCN, 5% H2O, 0.1% TFA

[0102] The typical load is 1 μl of 1.0 mg / ml, and the detection signal at 215 nm should be optimally between 0.1 and 1.5 abs units. Isocratic elution is performed with 60% mobile phase B at a flow rate of 0.3 ml / min for 20 minutes. The chromatogram is integrated in the form of HMWP (all peaks before the main peak), and the amount of each substance is expressed as a percentage of the total area.

[0103] Table 1.

[0104]

[0105] Table 1 shows in vitro potency, fibrillation, deamidation (%), dimerization and HMWP formation (%) and isomerization (%). Deamidation (%) of SEQ ID NO: 3 is based on the 36 Position or X3 and X 36 The total number of polypeptides at positions where deamidation occurs is calculated. 15 , X 18 and X 26 The deamidation rate is less than <5%, so it is less prone to deamidation.

[0106] Table 2.

[0107]

[0108] Table 2 continued.

[0109]

[0110] Table 2 shows the hAM 15-52 、hAMY 1-37 , the sequences of a reference peptide of the prior art (SEQ ID NO: 3) and a polypeptide of the present invention (SEQ ID NO: 4).

Claims

1. A polypeptide comprising the amino acid sequence KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] or a pharmaceutically acceptable salt thereof, or a derivative thereof having up to 2 amino acid substitutions, provided that the substitutions are not present in X2-X4, X7, X 11 , X 32 or X 36-38 Any location in .

2. The polypeptide according to claim 1, wherein The derivative has 1 amino acid substitution.

3. A polypeptide as claimed in any one of the preceding claims, wherein The cysteines in X2 and X7 are covalently linked via a disulfide bridge (-SS-) or a methylene bridge (-S-CH2-S-).

4. A polypeptide as claimed in any one of the preceding claims, wherein The cysteines in X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

5. A polypeptide as claimed in any one of the preceding claims, wherein The polypeptide is lipidated with a lipid selected from the group consisting of myristoyl (C14)-, hexadecanoic (C16)-, C18DA[γE]-, C18DA[γE][γE]-, C18DA[γE][OEG]-, C18DA[γE][OEG][OEG]-, C18DA[γE][γE][OEG][OEG]-, C18DA[γE][AHX]-, C18DA[γE][γE][AHX]-, C20DA[γE]-, C20DA[γE][γE]-, C20DA[γE][OEG]-, C20DA[γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][γE][OEG][OEG]-, C20DA[γE][AHX]-, or C20DA[γE][γE][AHX]-.

6. The polypeptide according to claim 6, wherein The lipid is C20DA[γE]-.

7. A polypeptide as claimed in any one of the preceding claims, wherein The polypeptide is N-terminally lipidated.

8. A polypeptide as claimed in any one of the preceding claims, having the structure or a pharmaceutically acceptable salt thereof.

9. A polypeptide as claimed in any one of the preceding claims, wherein The C-terminus is amidated (-CONH2).

10. A polypeptide as claimed in any preceding claim for use as a medicament.

11. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and optionally one or more excipients.

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