Amylin receptor (hAMY3R) agonist with improved chemical stability

Abstract

The present invention relates to a polypeptide that acts as an amylin receptor (hAMY3R) agonist and has improved chemical stability.

Description

Technical Field

[0001] The present invention relates to a polypeptide based on the sequence of human adrenomedullin fragment hAM that acts as an amylin receptor (hAMY3R) agonist. In particular, the present invention relates to a polypeptide with improved chemical stability. 15-52

Background Art

[0002] Obesity is a medical condition in which excessive body fat has accumulated to the extent of having an adverse effect on health. Obesity affects a vast number of individuals worldwide and is increasing rapidly in certain regions of the world. The World Health Organization (WHO) estimated in 2016 that approximately 650 million people worldwide were obese. Obesity is defined as a body mass index (BMI) of over 30. Obesity is considered a major risk factor for developing various medical conditions such as cardiovascular diseases (e.g., hypertension, atherosclerosis, heart attack or stroke), NASH, musculoskeletal disorders, certain types of cancer, depression, and type II diabetes, and is thus harmful to human health. Cardiovascular diseases and diabetes are two major diseases associated with obesity. In the field of obesity, many studies have been conducted in the search for new treatments for obesity or obesity-related diseases and disorders.

[0003] Diabetes is a group of metabolic disorders characterized by high blood glucose levels. As of 2019, the International Diabetes Federation estimated that 463 million people worldwide were living with diabetes, of which approximately half of the individuals were diagnosed. Diabetes is divided into two types, namely type I diabetes and type II diabetes. Type I diabetes is caused by the pancreas being unable to produce sufficient insulin due to the loss of beta cells caused by an autoimmune response. On the other hand, type II diabetes begins with insulin resistance, where cells are unable to respond appropriately to insulin, and can lead to a shortage of insulin as the disease progresses.

[0004] Calcitonin peptide family

[0005] The calcitonin family of peptides consists of the hormone peptide calcitonin (CT), calcitonin gene-related peptide (CGRP), pancreatic amyloid polypeptide (IAPP, amylin or hAMY 1-37 ), and adrenomedullin (hAM) and their precursors. hAMY 1-37 is a 37-residue peptide hormone co-secreted with insulin from pancreatic β-cells and has the amino acid sequence 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. Amylin plays an important role in maintaining glucose homeostasis by suppressing glucagon release and inhibiting gastric emptying, thereby reducing blood glucose concentration. Furthermore, amylin has been shown to reduce food intake and plays an important role in satiety and is thus a promising candidate for treating, for example, obesity and / or diabetes. hAM is a 52-residue peptide hormone expressed in all tissues and has 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. hAM is a potent vasodilator and has shown a positive effect in cardiovascular diseases such as myocardial infarction, limb ischemia, and hypertension.

[0006] The biological activities of the calcitonin protein family are generally mediated through binding to both the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CRLR), which both belong to family 2 of the G protein-coupled receptors (GPCRs). These receptors can be co-expressed in combination with different receptor activity-modifying proteins (RAMP1-3) that generate functional receptors for the individual peptides in the calcitonin protein family. Co-expression of CTR and RAMP1 results in the formation of the amylin and CGRP receptor (AMY1R), co-expression of CTR and RAMP2 results in the amylin receptor 2 (AMY2R), and co-expression of CTR and RAMP3 results in the amylin receptor (AMY3R). Co-expression of CRLR and RAMP1 results in the formation of the CGRP receptor (CGRP1R), co-expression of CRLR and RAMP2 results in the formation of the adrenomedullin receptor (AM1R), and co-expression of CRLR and RAMP3 results in the formation of the adrenomedullin receptor (AM2R).

[0007] Some of the native peptides of the calcitonin protein family show a fair degree of pharmacological overlap between receptors. For example, adrenomedullin has an efficacy for AMY3R that is about 1 / 100 that of hAMY 1-37 when compared. The adrenomedullin fragment (hAM 15-52 ) is approximately equipotent for AMY3R and AM1R, with an EC 50 for AMY3R of 1.3 nM and an EC 50 value for AM1R of 1.1 nM (this EC 50 value is measured according to the examples in this specification). On the other hand, hAMY 1-37 has an EC 50 value for AMY3R of 10 pM but is inactive for AM1R.

[0008] Human amylin (hAMY 1-37Or amylin) has several drawbacks such as a high fibrillation tendency, a short in vivo half-life, and chemical instability at pH 7. Therefore, native amylin is not optimal for use as a pharmaceutical active ingredient. Some of the drawbacks of native amylin have been overcome by the successful amylin analog pramlintide, which is approved by the FDA for use in type I and II diabetes. However, pramlintide fibrillates at pH 7 and is formulated at pH 4, which can cause pain at the injection site upon administration. hAMY 1-37 In contrast, the human adrenomedullin fragment hAM 15-52 does not fibrillate at pH 7. Therefore, the hAM 15-52 scaffold has been previously utilized in the development of new amylin analogs (see Patent Document 1). Some specific substitutions in hAM 15-52 completely abolish its adrenomedullin receptor (AM1R) potency while enhancing its amylin receptor (hAMY3R) potency, thereby converting hAM 15-52 into a selective amylin receptor agonist. The advantage of this chemical strategy is that such an amylin receptor agonist is pharmacologically similar to hAMY 1-37 but has the advantage of the low fibrillation tendency inherent to hAM 15-52 . One such example is a polypeptide having the amino acid sequence KCNTATCTVQRLAEQIAQFTDKDKDNVAPPTNVGSNGHyp (SEQ ID NO: 3), where the hAMY3R EC 50 is 14 nM and the hAM1R EC 50 is >5000 nM. Despite its high in vitro potency against hAMY3R, its low fibrillation tendency, and its high in vivo efficacy, this peptide was found to be susceptible to the effects of chemical instability due to deamidation, dimerization, and isomerization. The present invention addresses these drawbacks of SEQ ID NO: 3 in order to provide a polypeptide having improved chemical stability optimal for clinical development.

Prior Art Documents

Patent Document

[0009]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention relates to the finding that by substituting asparagine (N) at positions X3, X 32 and X 36 of SEQ ID NO: 3 with leucine (L), alanine (A), and alanine (A), respectively, and thereby avoiding deamidation, the chemical stability of SEQ ID NO: 3 can be improved without adversely affecting other properties such as efficacy. The present invention further relates to the finding that by substituting the disulfide bridge (-S-S-) of SEQ ID NO: 3 with a methylene bridge (-S-CH2-S-), and thereby suppressing dimerization and the formation of high molecular weight products, the chemical stability of SEQ ID NO: 3 can be improved without adversely affecting other properties such as efficacy. The present invention further relates to the finding that by substituting aspartic acid (D) at position 25 of SEQ ID NO: 3 with glutamic acid (E), and thereby avoiding isomerization, the chemical stability can be improved without adversely affecting other properties such as efficacy.

Means for Solving the Problems

[0011] Accordingly, in a first aspect, the present invention relates to a polypeptide comprising the amino acid sequence KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative thereof having at most two amino acid substitutions, provided that the substitutions do not occur at any of positions X2 - X4, X7, X 11 , X 32 or X 36~38 of SEQ ID NO: 4.

[0012] Definition

[0013] In the context of the present invention, the polypeptide is generally amidated at the C-terminus (-CONH2), similar to amylin and adrenomedullin, which are natural peptides. However, the polypeptides of the present invention may also have any of another post-translational modification such as a free carboxylic acid (-COOH) or a methyl ester (-COOMe). In a highly preferred embodiment of the present invention, the polypeptide is amidated at the C-terminus. The polypeptides according to the present invention may have a free amine (-NH2), may be N-acylated (-NHCOR), may be N-methylated (-NHCH3 or -N(CH3)2), may be deaminated at the N-terminus, or may be N-lipidated.

[0014] In the context of the present invention, lipidation refers to the covalent attachment of a lipid to a polypeptide, such as C18DA (octadecanedioic acid), C20DA (icosanedioic acid), etc., optionally via a linker / spacer consisting of one or more commonly used covalently attached units such as [γE], [OEG] or [AHX], as shown below.

Chemical formula

[0015] Lipidation is typically carried out to improve the pharmacokinetic profile of the polypeptide, for example, by improving metabolic stability, reducing enzymatic degradation, and decreasing excretion and metabolism. Overall, the in vivo half-life (t 1 / 2) results in an extension. The polypeptide according to the present invention can be lipidated or non-lipidated according to the desired half-life. The polypeptide 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 the list consisting of myristic acid (C14), palmitic 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, the lipidation is C20DA[γE]-. Most preferably, the lipidation is carried out at the N-terminus.

[0016] In the context of the present invention, EC 50 values are used as a measure of agonist potency at a given receptor. EC 50 values are a measure of the concentration of the compound required to achieve half of the maximum activity of the compound in a particular assay.

[0017] In the context of the present invention, the polypeptide or its derivative can be in the form of a pharmaceutically acceptable salt. Thus, pharmaceutically acceptable salts are intended to include any salts commonly used in the formulation of peptides. Such salts include both acid addition salts and basic salts, examples of which can be found, for example, in Remington´s Pharmaceutical Sciences, 17th edition. Similarly, various solvates of hAM 15-52 analogues or their pharmaceutically acceptable salts are also within the scope of the present invention.

[0018] In the context of the present invention, unless otherwise specified, all amino acids are L-amino acids (L-stereoisomers, natural amino acids). The abbreviation Hyp refers to L-hydroxyproline. Substitutions in derivatives can be substitutions for natural and non-natural amino acids, including L- and D-stereoisomers. Preferably, substitutions in mutants are conservative substitutions to conservative amino acids. The group of conservative amino acids can be defined as follows: G, A, V, L, I, P (aliphatic or cyclic), S, C, T, M (containing hydroxy or sulfur) F, Y, W (aromatic) H, K, R (basic) D, E, N, Q (acidic or amide)

[0019] In the context of the present invention, it should be understood that the cysteines at positions X2 and X7 are covalently linked by a crosslink such as a disulfide bridge (-S-S-) or a methylene bridge (-S-CH2-S-). Most preferably, the cysteines at positions X2 and X7 are covalently linked by a methylene bridge (-S-CH2-S-).

[0020] In the context of this specification, the term "treatment" should be understood in the broadest sense as prevention, amelioration, or therapy. Thus, treatment is also intended to include prophylactic treatment.

Mode for Carrying Out the Invention

[0021] Detailed Description of the Invention

[0022] Aspect 1 - Polypeptide

[0023] A polypeptide having the amino acid sequence KCNTATCTVQRLAEQIAQFTDKDKDNVAPPTNVGSNGHyp (SEQ ID NO: 3) has high in vitro potency against hAMY3R (i.e., hAMY3R EC of 0.014 nM 50 ) and high selectivity for hAMY3R over hAM1R (i.e., >5000 nM hAM1R EC50 ) and has been previously shown not to fibrillate. Furthermore, the inventors have found that SEQ ID NO: 3 also has high in vivo efficacy. Despite its favorable properties, the present invention arises from the discovery that SEQ ID NO: 3 is chemically labile, which poses a significant concern regarding the long-term stability of drug candidates. The present invention relates to a method for improving the chemical stability of SEQ ID NO: 3 and structurally related polypeptides.

[0024] The inventors have found that the chemical lability of SEQ ID NO: 3 is caused by deamidation, dimerization, and isomerization. SEQ ID NO: 3 contains seven potential deamidation sites (i.e., N at positions X3, X 26 , X 32 , and X 36 and Q at positions X 10 , X 15 , X 18 ), and three of them (i.e., positions X3, X 32 and X 36 ) have been found to be hotspots for deamidation, as shown in Scheme 1 below. Therefore, the inventors have found that by substituting asparagine (N) at positions 3, 32, and 36 of SEQ ID NO: 3 with leucine (L) and alanine (A), as shown in Scheme 1, the chemical stability of SEQ ID NO: 3 can be significantly improved without adversely affecting the potency (see Example 1, Table 1).

[0025]

Chemical formula

[0026] The inventors have further found that a further cause of the chemical lability of SEQ ID NO: 3 can be attributed to isomerization. In particular, the inventors have found that aspartic acid (D) at position X 25 of SEQ ID NO: 3 is slightly prone to structural and chiral isomerization, as shown in Scheme 2 below, which also results in a decrease in the yield in synthesis.

[0027]

Chem.

[0028] Therefore, the present invention further relates to the finding that by substituting aspartic acid (D) at position X of SEQ ID NO: 3 with glutamic acid (E), isomerization can be prevented without adversely affecting the efficacy, and the overall synthetic yield increases (see Example 1, Table 1). 25 Position asparagine acid (D) is replaced by glutamic acid (E), it can prevent isomerization without adversely affecting the efficacy, and the overall synthetic yield increases (see Example 1, Table 1).

[0029]

Chem.

[0030] Therefore, in a first aspect, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative having at most two amino acid substitutions, provided that the substitutions are not present at any of positions X2 - X4, X7, X 11 、X 32 or X 36~38 Position.

[0031] Preferably, the derivative of SEQ ID NO: 4 has one amino acid substitution. Most preferably, there is no substitution in SEQ ID NO: 4.

[0032] Therefore, in a preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative having one amino acid substitution, provided that the substitution is not present at any of positions X2 - X4, X7, X 11 、X 32 or X 36~38 Position.

[0033] In a more preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] or a pharmaceutically acceptable salt thereof.

[0034] The inventors further found that, as shown in Scheme 3 below, the chemical instability of SEQ ID NO: 3 can be attributed to dimerization to some extent. Therefore, the present invention further relates to the finding that dimerization can be suppressed and chemical stability can be improved by replacing the disulfide bridge (-S-S-) formed between the cysteines at positions X2 and X7 of SEQ ID NO: 3 with a methylene bridge (-S-CH2-S-) (see Example 1, Table 1).

[0035]

Chemical formula

[0036] Therefore, in one embodiment of the present invention, the cysteines at X2 and X7 are covalently bonded via a disulfide bridge (-S-S-). In the most preferred embodiment of the present invention, the cysteines at X2 and X7 are covalently bonded via a methylene bridge (-S-CH2-S-) to minimize dimerization and prevent the formation of high molecular weight products.

[0037] Therefore, in a more preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative thereof having at most two amino acid substitutions, provided that the substitutions are not present at any of positions X2-X4, X7, X 11 , X 32 or X 36~38 and the cysteines at X2 and X7 are covalently bonded via a methylene bridge (-S-CH2-S-).

[0038] In an even more preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative having one amino acid substitution, provided that there is no substitution at any of the positions of X2 to X4, X7, X 11 , X 32 or X 36~38 , and the cysteines at positions X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0039] In an even more preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, wherein the cysteines at positions X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0040] The polypeptide according to the present invention can be lipidated with various lipids according to the desired half-life of the polypeptide. Preferably, the lipid is selected from the list consisting of myristic acid (C14)-, palmitic 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, 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]-.

[0041] Accordingly, in an even more preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative having at most two amino acid substitutions, provided that the substitutions are not at any of positions X2 to X4, X7, X 11 , X 32 or X 36~38 . The cysteines at positions X2 and X7 are covalently linked via a disulfide bridge (-S-S-) or a methylene bridge (-S-CH2-S-), most preferably a methylene bridge (-S-CH2-S-), and further the polypeptide is lipidated with a lipid selected from the list 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]-.

[0042] In an even more preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] (SEQ ID NO: 4) or a pharmaceutically acceptable salt thereof, or a derivative having one amino acid substitution, provided that the substitution is not at any of positions X2 to X4, X7, X 11 , X 32 or X 36~38Regarding its derivatives that are not present in any of the positions, the cysteines of X2 and X7 are covalently linked via a disulfide bridge (-S-S-) or a methylene bridge (-S-CH2-S-), most preferably a methylene bridge (-S-CH2-S-), and furthermore the polypeptide is lipidated with a lipid selected from the list 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]-.

[0043] In a highly preferred embodiment, the present invention relates to a polypeptide comprising the amino acid sequence: KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] or a pharmaceutically acceptable salt thereof, wherein the cysteines at X2 and X7 are covalently linked via a disulfide bridge (-S-S-) or a methylene bridge (-S-CH2-S-), most preferably a methylene bridge (-S-CH2-S-), and further the polypeptide is lipidated with a lipid selected from the list 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]-.

[0044] In any of the above embodiments where the polypeptide is lipidated, most preferably, the lipidation is N-terminal lipidation, and most preferably the cysteines at X2 and X7 are covalently linked via a methylene bridge (-S-CH2-S-).

[0045] In an even more highly preferred embodiment, the present invention has the following structure:

Chemical formula

[0046] In an even more highly preferred embodiment, the invention relates to the following structure:

Chemical formula

[0047] In any of the above embodiments, preferably, the C-terminus is amidated (-CONH2).

[0048] In a most preferred embodiment, the present invention relates to a polypeptide having the following structure, or a pharmaceutically acceptable salt thereof.

Chemical formula

[0049] Aspect II - Medical Use

[0050] In a second aspect, the present invention relates to a polypeptide according to the first aspect for use as a medicament. More specifically, the second aspect of the present invention is a disease, disorder or condition selected from the list consisting of overeating, excessive weight, obesity, binge eating disorder, Prader-Willi syndrome, dyslipidemia, metabolic disease / disorder, 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, and relates to a polypeptide according to the first aspect for use in treating, preventing or ameliorating a disease, disorder or condition.

[0051] Aspect III - Pharmaceutical Composition

[0052] In a third aspect, the present invention relates to a pharmaceutical composition comprising one or more polypeptides according to the first aspect and / or its medical use in the treatment, prevention, or amelioration of a disease, disorder, or condition according to the second aspect. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier (vehicle) and / or one or more excipients according to the prior art in the art, for example, as disclosed in "Remington: Essentials of Pharmaceutics", edited by Linda A. Felton, Pharmaceutical press (2012). Suitable formulations include, but are not limited to, tablets, pills, capsules, emulsions, suspensions, sustained-release formulations, solutions, or lyophilized powders intended to be dissolved before administration. It should be understood that different routes of administration can be used depending on the choice of formulation. Such routes of administration 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.

[0053] Aspect IV - Method of treatment

[0054] In a fourth aspect, the present invention relates to a method of treating a subject who is human or an animal with one or more polypeptides according to the first aspect or a pharmaceutical composition according to the third aspect, wherein the subject who is human or an animal is diagnosed with, suffering from, or at risk of developing one or more of the diseases, disorders, or conditions according to the second aspect. This method comprises administering one or more compounds according to the first aspect or a pharmaceutical composition according to the third aspect in an effective therapeutic amount to treat the disease, disorder, or condition described in the third aspect.

Examples

[0055] General protocol for the synthesis of hybrid polypeptides

[0056] Peptides were synthesized on Tentagel S RAM (Rapp polymer GmbH, Germany) with a loading of 0.23 - 0.25 mmol / g using a Syro II fully automated parallel peptide synthesizer (MultiSynTech GmbH, Germany) equipped with a heating block. The resin was treated with 40% piperidine / DMF (0.2M HOBt (1-hydroxybenzotriazole)) at 45 °C for 3 minutes, followed by treatment with 20% piperidine / DMF (0.1M HOBt) at 75 °C for 7 - 12 minutes to perform N α -Fmoc deprotection in two steps. At room temperature, N α -Fmoc-deprotected Asp, Cys, and His residues were excluded (i.e., 3 minutes with 40% piperidine / DMF (0.2M HOBt), followed by 15 minutes with 20% piperidine / DMF (0.1M HOBt)). The coupling chemical was DIC (N,N'-diisopropyl-carbodiimide) / oxyma (ethyl cyano(hydroxyimino)acetate) in DMF using a 0.5M and 6-fold molar excess of amino acid solution in DMF. Standard Fmoc-protected amino acids were used. The coupling conditions were single coupling or double coupling at 75 °C for 15 minutes, except for His and Cys residues that were double-coupled at 50 °C for 15 minutes. Fmoc-amino acids were dissolved at 0.5M in DMF containing 0.5M oxyma, except for His dissolved in NMP. The resin was washed 4 times with NMP after N α -Fmoc deprotection and 3 times after coupling.

[0057] Trityl (Trt) was used as the protecting group for cysteine, and disulfide bridges were formed on the resin by treating 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.

[0058] After synthesis, the resin was washed with DCM, dried, and treated with TFA (trifluoroacetic acid) / TES (triethylsilane) / water (95 / 2.5 / 2.5) at 42 °C for 35 minutes to cleave the polypeptide from the resin, followed by precipitation with 4 volumes of cold diethyl ether, further washing with diethyl ether, and drying.

[0059] After cleaving the peptide from the resin using standard procedures, a methylene bridge between two cysteines was formed in solution. The peptide (300 mg) was dissolved in 20 mM phosphate buffer (10 mL, pH 7.8), dithiothreitol (2 equivalents) was added, and the mixture was stirred for 10 minutes. Subsequently, sodium iodide (2 equivalents) was added, followed by a solution of diiodomethane (40 equivalents) and ethanolamine (20 equivalents) in acetonitrile (15 mL). The reaction was monitored by LCMS until completion, diluted with 100 ml of water, and the lipophilic substances were removed by extraction with diethyl ether (2 × 80 mL).

[0060] The crude peptide was purified by reverse-phase HPLC using Waters preparative HPLC with a C8 column (Reprosil Gold 200 Å, 5 μm, 40 mm × 250 mm), a preparative pump (waters2545), a UV / VIS detector (Waters2489), and a Waters fraction collector III. The mobile phase was run at a flow rate of 50 ml / min at room temperature with a gradient of buffer A (0.1% TFA in H2O) and buffer B (0.1% TFA in ACN, gradient: 35 - 45% B over 20 minutes). The relevant fractions were analyzed, pooled, and lyophilized. The properties of the final product were elucidated by analytical UPLC-MS.

[0061] The purity and mass of the peptide were determined by analytical HPLC-MS on a Kinetex C8 column (Phenomenex, 100 Å, 2.6 μm, 4.6 mm × 150 mm) using a Waters Acquity HPLC system equipped with a 3100 mass detector. Analysis was performed by gradient elution with buffer A (0.3% TFA in H2O) and buffer B (0.3% TFA in ACN) at a temperature of 40 °C. The details of the gradient are summarized below.

[0062] Waters HPLC method (run for 21 minutes) Acetonitrile gradient: 10% - 40% (0 - 2 minutes), 40% - 60% (2 minutes - 16 minutes), 60% - 90% (16.5 minutes - 18.5 minutes), 90% - 10% (18.5 - 21 minutes).

[0063] Example 1 SEQ ID NO: 4: [Chemical formula] Retention time 10.9 minutes Measured values m / z = 2193.2, m / z = 1462.6, m / z = 1096.9. Calculated value m = 4384

[0064] General protocol for cAMP assay to measure in vitro receptor activation

[0065] hAMY3-R:

[0066] Cells that stably overexpress the hAMY3 receptor were obtained from Ogeda Corporation (now Astellas Pharma Inc.), subcloned, and monoclonal cell lines with appropriate assay regions were grown, aliquoted, and frozen. An aliquot was thawed and seeded at 2000 cells / well in a 384-well format in DPBS containing 0.05% casein and 0.5 mM IBMX. Then, using human amylin (Bachem, catalog number H-7905) as a positive control, the cells were immediately stimulated with serial dilutions of the test compound for 30 minutes at room temperature. The Cisbio assay (catalog number 62AM4PEC) for Gs-coupled receptors was used to measure cAMP accumulation. For the measurement, assay reagents were added according to the manufacturer's instructions, and time-resolved fluorescence energy transfer was recorded after 1 hour.

[0067] hAM1-R:

[0068] Cells that stably overexpress the hAM1 receptor were obtained from Ogeda Corporation (now Astellas Pharma Inc.), subcloned, and monoclonal cell lines with appropriate assay regions were grown, aliquoted, and frozen. An aliquot was thawed and seeded at 8000 cells / well in a 384-well format in DPBS containing 0.05% casein and 0.5 mM IBMX. Then, using human adrenomedullin 1-52 (Bachem, catalog number H-2932) as a positive control, the cells were immediately stimulated with serial dilutions of the test compound for 30 minutes at room temperature. The Cisbio assay (catalog number 62AM4PEC) for Gs-coupled receptors was used to measure cAMP accumulation. For the measurement, assay reagents were added according to the manufacturer's instructions, and time-resolved fluorescence energy transfer was recorded after 1 hour.

[0069] General protocol for determining the physical stability of peptide analogs

[0070] The peptide was dissolved in buffer (50 mM sodium acetate at pH 4 or 50 mM sodium phosphate at pH 7) and incubated for 1 hour. The sample was then divided into 80 μl duplicates in a black 384-well plate (μ-clear, Greiner Bio-One) and mixed with thioflavin T (ThT) to a final concentration of 4 μM. The plate was sealed and placed in a plate reader (CLARIOstar, BMG) and the temperature was set to 40 °C during the experiment. Prior to each measurement, the sample was further stressed by shaking the plate at 700 rpm (linear) for 5 minutes. Fluorescence was measured every 10 minutes for 4 days by exciting ThT at 450 nm and measuring emission at 480 nm. For each peptide, the ThT signal was smoothed over time using locally weighted regression fitting (LOESS) implemented in the statistical programming environment R. For the smoothed data, the maximum ThT signal was normalized as a percentage between the maximum ThT signal for hAMY and the buffer background. Thus, high values indicate fibrillization properties similar to hAMY, and low values indicate no fibrillization.

[0071] General protocol for determining deamidation and isomerization

[0072] The peptide was dissolved in 50 mM phosphate buffer (pH 7) and incubated at 40 °C for up to 28 days. The sample was then analyzed on an Exploris 120 mass spectrometer coupled to a Vanquish Tandem UPLC (both Thermo Fisher Scientific) equipped with an Acquity BEH C18 column (1.7 um, 2.1×50 mm, 130A, Waters). The peptide was eluted using a linear gradient starting from 5% to 70% buffer B over 7.5 minutes at 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). Data were acquired in data-dependent acquisition mode with an MS1 resolution of 60,000 and an MS2 resolution of 30,000 in positive mode (top 4). Peptides were fragmented with 25% normalized collision energy. Deamidation and isomerization were quantified using the area under the curve integral.

[0073] General protocol for determining the formation of dimers and HMWP

[0074] The peptide was dissolved in 50 mM phosphate buffer (pH 7) and incubated at 40 °C for up to 28 days. The sample was then analyzed using size exclusion chromatography to quantify the amount of high molecular weight products (HMWP), a general term for covalently linked peptide dimers, trimers, and higher order multimers. Analysis was performed on a Dionex Ultimate 3000 RS UHPLC focusing chromatography system (Thermo Scientific) equipped with an Acquity Protein BEH SEC 1.7μm 4.6×300mm 125Å (Waters part number 186006506). The solvent system consisted of two mobile phases that were assumed to disrupt all non-covalent interactions between peptides, leaving only monomeric peptides and covalent HMWP to be analyzed.

[0075] Mobile phase A (all v / v%): 95% H2O, 5% MeCN (acetonitrile), 0.1% TFA (trifluoroacetic acid) Mobile phase B (all v / v%): 95% MeCN, 5% H2O, 0.1% TFA

[0076] A typical load is 1 μl of 1.0 mg / ml, and the detection signal at 215 nm should optimally be between 0.1 and 1.5 abs units. Isocratic elution was performed for 20 minutes at 60% mobile phase B and a flow rate of 0.3 ml / min. The chromatogram was integrated as HMWP (all peaks before the main peak). The amounts of the various species were reported as percentages of the area relative to the total area.

[0077] [Table 1]

[0078] Table 1 shows in vitro potency, fibrillation, deamidation (%), dimerization and HMWP formation (%), and isomerization (%). The deamidation (%) of SEQ ID NO: 3 is calculated based on the total of the polypeptides in which deamidation occurred at either position X3 or X 36 or both X3 and X 36 . From SEQ ID NO: 4 with less than 5% deamidation, positions X 15 , X 18 , and X 26 are not particularly prone to deamidation.

[0079] [Table 2]

[0080] Table 2 shows the sequences of hAM 15-52 , hAMY 1-37 , a reference peptide from the prior art (SEQ ID NO: 3) and the polypeptide according to the invention (SEQ ID NO: 4).

Claims

1. A polypeptide comprising the amino acid sequence KCLTATCTVARLAEQIAQFTDKDKENVAPPTAVGSAG[Hyp] or a pharmaceutically acceptable salt thereof, or having up to two amino acid substitutions, but ２ ~X ４ , X ７ , X １１ , X ３２ or X ３６～３８ A derivative thereof in which the substitution is not present at any of the positions.

2. The polypeptide of claim 1, wherein the derivative has one amino acid substitution.

3. The X ２ and X ７ The cysteines in ２ 2. The polypeptide of claim 1, wherein the amino acid is covalently linked via a hydroxyl group.

4. The X ２ and X ７ The cysteine ​​in ２ 2. The polypeptide of claim 1, wherein the amino acid is covalently linked via a hydroxyl group.

5. The polypeptide is 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]-, C2 2. The polypeptide of claim 1, wherein the polypeptide is lipidated with a lipid selected from the list consisting of 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]-.

6. The polypeptide of claim 5, wherein the lipid is C20DA[γE]-.

7. The polypeptide of claim 1 , wherein the N-terminus is lipidated.

8. 2. The polypeptide of claim 1, having the following structure: 【Chemical 1】

9. The C-terminus is amidated (-CONH ２ 2. The polypeptide of claim 1, wherein

10. The polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, having the following structure: 【Chemistry 2】

11. A polypeptide described in claim 10 having the following structure: 【Chemistry 3】

12. The polypeptide described in claim 10, wherein the polypeptide is a pharmaceutically acceptable salt.

13. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 12 for use as a medicament.

14. 13. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and optionally one or more excipients.

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