Polypeptides and their uses

Abstract

Pramlintide analogs are provided that retain amylin agonist activity and have advantages such as an extended half-life and reduced tendency to fibrillate. The present invention provides polypeptides having specific sequences that are pramlintide analogs conjugated to a half-life extending moiety, such as an albumin binding moiety. The disclosed peptides can be formulated or chemically conjugated to, for example, proteins, polymeric drug carriers, or pre-drug delivery systems, which enhance the chemical and / or physical stability and / or circulatory exposure of the therapeutic moiety.

Description

[Technical Field]

[0001] The present invention relates to polypeptides that are pramlintide analogs and uses thereof. In particular, the present invention relates to polypeptides that are pramlintide analogs conjugated to a half-life extending moiety, such as an albumin binding moiety, and uses thereof. [Background technology]

[0002] Pramlintide is a synthetic analog of human amylin with three proline substitutions at positions 25, 28, and 29. As a result of these substitutions, pramlintide has a reduced tendency to form amyloid fibrils, thereby overcoming the physicochemical disadvantages of native human amylin (Non-Patent Document 1).

[0003] Pramlintide is clinically used in amylin replacement therapy and simulates the important glucoregulatory actions of amylin. These glucoregulatory actions complement those of insulin by regulating the rate of glucose appearance in the circulating blood and are achieved through three primary mechanisms: slowing the rate of gastric emptying, suppressing postprandial glucagon secretion, and suppressing food intake (Non-Patent Document 2). Pramlintide is used as an adjunct to insulin in diabetic patients who have failed to achieve desired glucose control despite optimal insulin therapy (Non-Patent Document 3).

[0004] Pharmacokinetic studies have shown that the terminal half-life of amylin in rats is approximately 13 minutes, and the half-life for pramlintide in humans is approximately 20-45 minutes (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kruger DF, Gloster MA.Pramlintide for the treatment of insulin-requiring diabetes mellitus:rationale and review of clinical data.Drugs.2004;64(13):1419-32) [Non-patent document 2] Roth JD et.al.GLP-1R and amylin agonism in metabolic disease: complementary mechanisms and future opportunities.Br J Pharmacol.2012;166(1):121-136 [Non-patent document 3] Pullman J,et.al.Pramlintide is used in the management of insulin-using patients with type 2 and type 1 diabetes.Vasc Health Risk Manag.2006;2(3):203-212 [Non-patent document 4] Roth JD et.al.GLP-1R and amylin agonism in metabolic disease: complementary mechanisms and future opportunities.Br J Pharmacol.2012;166(1):121-136 Summary of the Invention [Problem to be solved by the invention]

[0006] There remains a need for pramlintide analogs that retain amylin agonist activity and offer advantages such as extended half-life and reduced tendency to fibrillate. [Means for solving the problem]

[0007] The present invention relates to polypeptides that are pramlintide analogs conjugated to an albumin binding moiety (eg, a lipid).

[0008] Thus, in one embodiment, the amino acid sequence: Xaa(-4)-Xaa(-3)-Xaa(-2)-Xaa(-1)-Xaa1-Cys2-Asn3-Xaa4-Ala5-Thr6-C ys7-Ala8-Thr9-Gln10-Arg11-Leu12-Ala13-Xaa14-Xaa15-Xaa16-Xaa17-Hi s18-Ser19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28- Xaa29-Thr30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-amide [SEQ ID NO: 2] (In the formula, Xaa(-4) is Lys (albumin binding site) or absent; Xaa(-3) is Gly or absent; Xaa(-2) is Gly or absent; Xaa(-1) is Gly, (albumin binding site), Lys (albumin binding site) or absent; Xaa1 is Lys, Lys(albumin binding site), (albumin binding site) or absent; Xaa4 is Thr, Ile, or Ala; Xaa14 is Asn, His, Glu, 2,4-diaminobutanoic acid (Dab) or an alpha methyl amino acid; Xaa15 is Phe or Trp; Xaa16 is Leu or D-Leu (dL); Xaa17 is Val, Ser, Glu, Arg, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp), Dab, or an alpha-methyl amino acid (e.g., 2-amino-2-methylpropanoic acid [Aib]); Xaa20 is Ser, Ile, Pro, or an alpha-methyl amino acid (e.g., (S)-2-amino-3-hydroxy-2-methylpropanoic acid [αMeSer]); Xaa21 is Asn, Dab, His, Pro, Ser, Arg, Lys, Gly, Glu, Ala, Hyp, or an alpha-methyl amino acid (e.g., Aib); Xaa22 is Asn, His, Hyp, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa23 is Phe, Hyp, or an alpha methyl amino acid (e.g., (S)-2-amino-2-methyl-3-phenylpropanoic acid [αMePhe]); Xaa24 is Gly, Pro, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa25 is Pro, Ala, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa26 is Ile, D-Ile (dI), Arg, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa27 is Leu, dL, Hyp, or an alpha-methyl amino acid (e.g., Aib); Xaa28 is Pro, D-Pro (dP), Ser, Hyp, or an alpha-methyl amino acid (e.g., Aib); Xaa29 is Pro, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa31 is Asn, Glu, His, Arg, Pro, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa32 is Val, Hyp, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa33 is Gly, Pro, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa34 is Ser, Pro, His, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa35 is Asn, Pro, Arg, Glu, Dab, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa36 is Thr, Hyp, or an alpha-methyl amino acid (e.g., Aib); and Xaa37 is Tyr, Pro, Hyp, or an alpha-methyl amino acid (e.g., Aib) or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least one albumin binding moiety.

[0009] In another embodiment, the amino acid sequence: Xaa(-4)-Xaa(-3)-Xaa(-2)-Xaa(-1)-Xaa1-Cys2-Asn3-Xaa4-Ala5-Thr6-C ys7-Ala8-Thr9-Gln10-Arg11-Leu12-Ala13-Xaa14-Xaa15-Xaa16-Xaa17-Hi s18-Ser19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28- Xaa29-Thr30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-amide [SEQ ID NO: 2] (In the formula, Xaa(-4) is Lys (linker-lipid) or absent; Xaa(-3) is Gly or absent; Xaa(-2) is Gly or absent; Xaa(-1) is Gly, (linker-lipid), Lys (linker-lipid) or absent; Xaa1 is Lys, Lys(linker-lipid), (linker-lipid) or absent; Xaa4 is Thr, Ile, or Ala; Xaa14 is Asn, His, Glu, 2,4-diaminobutanoic acid (Dab) or an alpha methyl amino acid; Xaa15 is Phe or Trp; Xaa16 is Leu or D-Leu (dL); Xaa17 is Val, Ser, Glu, Arg, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp), Dab, or an alpha-methyl amino acid (e.g., 2-amino-2-methylpropanoic acid [Aib]); Xaa20 is Ser, Ile, Pro, or an alpha-methyl amino acid (e.g., (S)-2-amino-3-hydroxy-2-methylpropanoic acid [αMeSer]); Xaa21 is Asn, Dab, His, Pro, Ser, Arg, Lys, Gly or Glu, Ala, Hyp or an alpha methyl amino acid (e.g., Aib); Xaa22 is Asn, His, Hyp, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa23 is Phe, Hyp, or an alpha methyl amino acid (e.g., (S)-2-amino-2-methyl-3-phenylpropanoic acid [αMePhe]); Xaa24 is Gly, Pro, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa25 is Pro, Ala, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa26 is Ile, D-Ile (dI), Arg, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa27 is Leu, dL, Hyp, or an alpha-methyl amino acid (e.g., Aib); Xaa28 is Pro, D-Pro (dP), Ser, Hyp, or an alpha-methyl amino acid (e.g., Aib); Xaa29 is Pro, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa31 is Asn, Glu, His, Arg, Pro, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa32 is Val, Hyp, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa33 is Gly, Pro, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa34 is Ser, Pro, His, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa35 is Asn, Pro, Arg, Glu, Dab, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa36 is Thr, Hyp, or an alpha methyl amino acid (e.g., Aib); Xaa37 is Tyr, Pro, Hyp, or an alpha-methyl amino acid (e.g., Aib) or a pharmaceutically acceptable salt thereof.

[0010] In yet another embodiment, a polypeptide as set forth in Table 4 is provided.

[0011] In yet another aspect, there is provided a pharmaceutical composition comprising a polypeptide, lipidated polypeptide, or pharmaceutically acceptable salt of the invention and a pharmaceutically acceptable excipient.

[0012] In another aspect, a method of treating a disease or disorder in a subject is provided, comprising administering a polypeptide, lipidated polypeptide, pharmaceutically acceptable salt, or pharmaceutical composition of the invention.

[0013] In a further aspect, there is provided a method for producing a polypeptide or lipidated polypeptide described herein.

[0014] In a further aspect, there is provided an article of manufacture comprising a polypeptide, lipidated polypeptide, pharmaceutically acceptable salt, or pharmaceutical composition of the invention.

[0015] In a further aspect, there is provided a kit comprising a polypeptide, lipidated polypeptide, pharmaceutically acceptable salt or pharmaceutical composition of the invention, optionally further comprising instructions for use.

[0016] Aspects and embodiments of the present invention are set out with particularity in the appended claims. These and other aspects and embodiments of the present invention are also described herein.

[0017] Brief Description of Sequence Listing

[0018] [Table 1]

[0019] [Table 2]

[0020] [Table 3]

[0021] [Table 4]

[0022] [Table 5]

[0023] [Table 6]

[0024] [Table 7]

[0025] Table 1: The square brackets [ ] between the two cysteine ​​residues (cys2 and cys7) indicate the presence of an intramolecular disulfide bridge. DETAILED DESCRIPTION OF THE INVENTION

[0026] The inventors have observed that pramlintide conjugated to an albumin-binding moiety, such as a lipid, has insufficient stability (e.g., increased fibrillation tendency of pramlintide) under conditions required for pharmaceutical formulation. The present invention is based, at least in part, on findings that the polypeptides (e.g., lipidated polypeptides) described herein can exhibit improved stability (e.g., reduced or no fibrillation tendency) compared to such pramlintide conjugates.

[0027] For example, the inventors have found that when pramlintide is conjugated to a lipid to increase its half-life, its tendency to form fibrils also increases. Thus, the polypeptides (e.g., lipidated polypeptides) described herein may provide the benefit of an extended half-life compared to pramlintide, without the fibril-forming tendency of alternative lipidated pramlintide analogs. The peptides disclosed herein can be formulated or chemically conjugated to, for example, proteins, polymeric drug carriers, or pre-drug delivery systems, which enhance the chemical and / or physical stability and / or circulatory exposure of the therapeutic moiety. The inventors have further found that the polypeptides (e.g., lipidated polypeptides) described herein may exhibit improved physical and / or chemical stability compared to human amylin or pramlintide. Furthermore, the polypeptides (e.g., lipidated polypeptides) described herein may have similar or improved selectivity for human amylin (hAMYR) compared to pramlintide.

[0028] Throughout this specification, amino acid positions of a polypeptide (eg, a lipidated polypeptide) are numbered according to the corresponding position in pramlintide, which has the sequence shown in SEQ ID NO:1.

[0029] Throughout this specification, amino acids are referred to by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). In the case of certain less common or unnatural amino acids (i.e., amino acids other than those encoded by the 20 standard mammalian genetic codes), unless they are referred to by their full name, frequently used three-letter or four-letter codes are used for those residues, such as αMeSer ((S)-2-amino-2-methyl-3-phenylpropanoic acid), αMePhe ((S)-2-amino-2-methyl-3-phenylpropanoic acid), Aib (2-amino-2-methylpropanoic acid), Dab (2,4-diaminobutanoic acid), and γ-Glu (γ-glutamic acid).

[0030] In embodiments of any aspect of the invention, a polypeptide of the invention (eg, a lipidated polypeptide) is an isolated polypeptide (eg, an isolated lipidated polypeptide).

[0031] Albumin binding site The polypeptides of the present invention comprise at least one albumin binding moiety. Without being bound by theory, it is believed that the albumin binding moiety protects the polypeptide from clearance and degradation, thereby extending the half-life of the polypeptide. As used herein, "albumin binding moiety" refers to a compound that binds to albumin. Exemplary albumin binding moieties suitable for use in the polypeptides of the present invention include lipids (e.g., fatty acid derivatives), albumin-binding peptides, albumin-binding proteins, or small molecule ligands that bind to albumin. Optionally, the albumin binding moiety is a lipid, such as a lipid described herein.

[0032] A polypeptide of the invention may comprise one or more albumin binding moieties (e.g., lipids), for example 1, 2 or 3 albumin binding moieties. In a preferred embodiment, a polypeptide of the invention comprises only one albumin binding moiety (e.g., lipid).

[0033] The albumin binding moiety (e.g., lipid) can be attached to an amino acid residue of a polypeptide. In some embodiments, the albumin binding moiety (e.g., lipid) is attached to the amino acid residue via a linker. In alternative embodiments, the albumin binding moiety (e.g., lipid) is attached directly to the amino acid residue without an intervening linker. The albumin binding moiety (e.g., lipid) can be attached to the amino acid residue via an ester, sulfonyl ester, thioester, amide, amine, or sulfonamide. Thus, it will be understood that the albumin binding moiety (e.g., lipid) or linker comprises an acyl group, sulfonyl group, N atom, O atom, or S atom that forms an ester, sulfonyl ester, thioester, amide, amine, or sulfonamide moiety. Optionally, the acyl group in the albumin binding moiety (e.g., lipid) or linker forms an amide or ester moiety with the amino acid residue. Thus, in a preferred embodiment, the albumin binding moiety (e.g., lipid) is attached to an acylation site on the amino acid residue.

[0034] The albumin binding moiety (e.g., lipid) can be attached to any residue (e.g., εN of a lysine residue) at positions Xaa-4 to Xaa37 of the polypeptide. In some embodiments, the albumin binding moiety (e.g., lipid) is attached to the side chain of an amino acid residue in the polypeptide, e.g., εN of a lysine residue. In some embodiments, the albumin binding moiety (e.g., lipid) is attached to the N-terminus of the polypeptide (e.g., lysine at the N-terminus of the polypeptide).

[0035] In some embodiments, the albumin binding moiety (e.g., lipid) is attached to the N-terminus of the polypeptide (e.g., to a lysine at the N-terminus of the polypeptide). In some embodiments, the albumin binding moiety (e.g., lipid) is attached to an amino acid residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1, or Xaa1 (e.g., to the εN of a lysine residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1, or Xaa1). In preferred embodiments, the albumin binding moiety (e.g., lipid) is attached to Xaa-4, Xaa-1, or Xaa1 (either the N-terminus or a side chain of Xaa-4, Xaa-1, or Xaa1).

[0036] lipids In a preferred embodiment, the albumin binding moiety is a lipid. Thus, the polypeptide of the present invention can contain at least one lipid (referred to herein as a "lipidated polypeptide"). Without being bound by theory, it is believed that the lipid functions as an albumin binding moiety to protect the polypeptide from clearance and degradation, thereby extending the half-life of the polypeptide. The lipid also regulates the effectiveness of the compound as an agonist of amylin (calcitonin) receptor.

[0037] In some embodiments, the polypeptide comprises at least one lipidated amino acid residue. In some embodiments, the polypeptide comprises at least two lipidated amino acid residues. In preferred embodiments, the polypeptide contains only one lipidated amino acid residue. A lipid may be attached to an amino acid residue of the polypeptide. In some embodiments, the lipid is attached to the amino acid residue via a linker (referred to herein as a "linker-lipid"). In alternative embodiments, the lipid is attached directly to the amino acid residue without an intervening linker. A lipid may be attached to the amino acid residue via an ester, sulfonyl ester, thioester, amide, amine, or sulfonamide. Thus, it will be understood that the lipid or linker comprises an acyl group, sulfonyl group, N atom, O atom, or S atom that forms an ester, sulfonyl ester, thioester, amide, amine, or sulfonamide moiety. Optionally, the acyl group in the lipid or linker forms an amide or ester moiety with the amino acid residue. Thus, in preferred embodiments, the lipid is attached to an acylation site on the amino acid residue.

[0038] The lipid can be attached to any residue in positions Xaa-4 to Xaa-37 of the polypeptide (e.g., the εN of a lysine residue). In some embodiments, the lipid is attached to the side chain of an amino acid residue in the polypeptide, e.g., the εN of a lysine residue. In some embodiments, the lipid is attached to the N-terminus of the polypeptide (e.g., a lysine at the N-terminus of the polypeptide).

[0039] In some embodiments, the lipid is attached to the N-terminus of the polypeptide (e.g., to a lysine at the N-terminus of the polypeptide). In some embodiments, the lipid is attached to an amino acid residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1, or Xaa1 (e.g., to the εN of a lysine residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1, or Xaa1). In preferred embodiments, the lipid is attached to Xaa-4, Xaa-1, or Xaa1 (either the N-terminus or a side chain of Xaa-4, Xaa-1, or Xaa1).

[0040] In an embodiment of any aspect of the present invention, the lipid may comprise a hydrocarbon chain having 10 to 26 C atoms, e.g., 14 to 24 C atoms, e.g., 16 to 22 C atoms. For example, the hydrocarbon chain may contain 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 C atoms. In a preferred embodiment, the lipid has 18 to 20 C atoms. In particular, the lipid may have 18 or 20 C atoms. The hydrocarbon chain may be linear or branched, saturated or unsaturated. Furthermore, it may comprise a functional group, e.g., a carboxylic acid group, at the end of the lipophilic chain, which may or may not be protected during synthesis.

[0041] Optionally, the lipid comprises a dicarboxylic acid. For example, the lipid may comprise a C12 diacid, a C14 diacid, a C16 diacid, a C17 diacid, a C18 diacid, a C19 diacid, or a C20 diacid. In a preferred embodiment, the lipid comprises a C18 diacid or a C20 diacid.

[0042] Linker The albumin binding moiety (e.g., a lipid) can be attached to the polypeptide via a linker. In embodiments of any aspect of the present invention, the linker can include one or more residues of any naturally occurring or non-naturally occurring amino acid. The linker can include a combination of residues as a single unit or a repeating unit. For example, the linker can include multiple combinations of residues as a single unit or a repeating unit, each of which independently includes Glu, γ-Glu, Lys, ε-Lys, Asp, β-Asp, Gaba, β-Ala(3-aminopropanoyl), 02Oc (2-(2-(2-aminoethoxy)ethoxy)acetic acid), PEG2 (3-(2-(2-aminoethoxy)ethoxy)propanoic acid), PEG4 (1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid), PEG8 (1-amino-3 The amino acid may be a residue of PEG (1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosanoic-27-oic acid), or PEG (1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid). γ-Glu and β-Asp refer to amino acids in which the alpha-amino group and side chain carboxyl group participate in peptide bond formation. ε-Lys refers to an amino acid in which the epsilon-amino group and carboxyl group of lysine participate in peptide bond formation.

[0043] In some embodiments, the linker comprises a residue of γ-Glu, e.g., γGlu, γGlu-γGlu, γGlu-(O2Oc)-(O2Oc), or γGlu-(PEG2)-(PEG2). In some embodiments, the linker consists of γGlu, γGlu-γGlu, γGlu-(O2Oc)-(O2Oc), or γGlu-(PEG2)-(PEG2).

[0044] In some embodiments of any aspect of the invention, the polypeptide comprises any one of the linker and lipid combinations shown in any one of the columns of Table 2.

[0045] The linker can be attached to the amino acid residue via an ester, sulfonyl ester, thioester, amide, amine, or sulfonamide. It will be understood, therefore, that the linker optionally includes an acyl group, sulfonyl group, N atom, O atom, or S atom that forms an ester, sulfonyl ester, thioester, amide, amine, or sulfonamide moiety. Optionally, the acyl group in the linker forms an amide or ester moiety with the amino acid residue. Thus, in a preferred embodiment, the linker is attached to an acylation site on the amino acid residue.

[0046] The linker can be attached to a site at the N-terminus of the lipidated polypeptide (eg, an acylation site) or to the epsilon amino group "εN" of a residue in the lipidated polypeptide, eg, the εN of a lysine residue.

[0047] In some embodiments, the polypeptide comprises a combination of a linker, a lipid, and an acylation site shown in any one of the columns of Table 2.

[0048] [Table 8]

[0049] [Table 9]

[0050] [Table 10]

[0051] The linker can be attached to any residue in Xaa-4 to Xaa-37 of the polypeptide (e.g., εN of a lysine residue). In some embodiments, the linker is attached to the side chain of an amino acid residue in the polypeptide, e.g., εN of a lysine residue. In some embodiments, the linker is attached to the N-terminus of the polypeptide (e.g., lysine at the N-terminus of the polypeptide).

[0052] In some embodiments, the linker is attached to the N-terminus of the polypeptide (e.g., to a lysine at the N-terminus of the polypeptide). In some embodiments, the linker is attached to an amino acid residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1, or Xaa1 (e.g., to the εN of a lysine residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1, or Xaa1). In preferred embodiments, the linker is attached to Xaa-4, Xaa-1, or Xaa1 (either the N-terminus or a side chain of Xaa-4, Xaa-1, or Xaa1).

[0053] In some embodiments, the linker is attached to a site (e.g., an acylation site) selected from the N-terminus of the polypeptide, the εN of the lysine at position Xaa(1) "1K", the εN of the lysine at position Xaa(-1) "-1K", or the εN of the lysine at position Xaa(-4) "-4K".

[0054] Amino Acid Substitutions and Modifications Polypeptides of the invention (eg, lipidated polypeptides) can contain one or more amino acid modifications or substitutions compared to the pramlintide sequence [SEQ ID NO: 1].

[0055] In some embodiments, a polypeptide (e.g., a lipidated polypeptide) comprises one or more non-proteinogenic amino acids. Non-proteinogenic amino acids can include alpha-methyl amino acids, D-enantiomers of naturally occurring amino acids, 2,4-diaminobutanoic acid (Dab), and (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp). In some embodiments, a polypeptide (e.g., a lipidated polypeptide) comprises one or more non-proteinogenic amino acids at positions 14-37, optionally at one or more of 14, 17, or 20-37. In some embodiments, a polypeptide (e.g., a lipidated polypeptide) comprises one or more alpha-methyl amino acids at positions 14-37, optionally at one or more alpha-methyl amino acids at positions 14, 17, or 20-37. Polypeptides (e.g., lipidated polypeptides) comprising one or more alpha-methyl amino acids at positions 17, 21, or 23 are particularly preferred. Representative examples of alpha-methyl amino acids include 2-amino-2-methylpropanoic acid (Aib), alpha-methylglutamine (αMeGlu), alpha-methylphenylalanine (αMePhe or αMeF), alpha-methylleucine (αMeLeu), and alpha-methylserine (αMeSer). Thus, in certain embodiments, the alpha-methyl amino acid can be Aib, αMeGlu, αMePhe, αMeLeu, or αMeSer, or any combination thereof. In preferred embodiments, a polypeptide (e.g., a lipidated polypeptide) comprises at least one alpha-methyl amino acid, optionally selected from Aib, αMePhe, and αMeSer. References herein to αMePhe and αMeF refer to (S)-2-amino-2-methyl-3-phenylpropanoic acid. References herein to αMeSer refer to (S)-2-amino-3-hydroxy-2-methylpropanoic acid. In preferred embodiments, the alpha-methyl amino acid is Aib, αMePhe, or αMeSer. [ka]

[0056] In some embodiments, a polypeptide (e.g., a lipidated polypeptide) comprises one or more non-proteinogenic amino acids at positions 14-37 selected from the group consisting of 2,4-diaminobutanoic acid (Dab), (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp), D-leucine (dL), D-isoleucine (dI), and D-proline (dP). [ka]

[0057] In some embodiments, the polypeptide (eg, lipidated polypeptide) does not contain (2S)-2-aminohexanedioic acid (Aad) and / or does not contain Aad at positions 14-37.

[0058] In some embodiments, a polypeptide (e.g., a lipidated polypeptide) does not contain Aib at one or more of positions 15, 16, 17, 19, or 20. In alternative embodiments, a polypeptide (e.g., a lipidated polypeptide) contains Aib at one or more of positions 15, 16, 17, 19, or 20 and at least one different non-proteinogenic amino acid (e.g., an alpha-methyl amino acid that is not Aib) at positions 14-37.

[0059] In some embodiments, a polypeptide (eg, a lipidated polypeptide) comprises one or more natural amino acid substitutions or modifications compared to the pramlintide sequence [SEQ ID NO: 1].

[0060] In a preferred embodiment, a polypeptide (e.g., a lipidated polypeptide) includes one or more of the following naturally occurring amino acid substitutions or modifications: deletion 1K (Δ1K), Ile4, Ala4, Glu14, His14, Trp15, Arg17, Ser17, Glu17, Pro20, Ile20, His21, Ala21, Glu21, Gly21, Lys21, Pro21, Arg21, Ser21, His22, Pro24, Ala25, Arg26, Ser28, His31, Glu31, Pro31, Arg31, His34, Pro33, Pro34, Glu35, Arg35, Pro35, and Pro37.

[0061] It will be understood that a polypeptide (eg, a lipidated polypeptide) may contain a combination of non-proteinogenic amino acids and natural amino acid substitutions or modifications compared to the pramlintide sequence [SEQ ID NO: 1].

[0062] In some embodiments, a polypeptide (eg, a lipidated polypeptide) that is a pramlintide analog, or a pharmaceutically acceptable salt thereof, is provided, comprising any of the amino acid sequence alterations shown in Table 3.

[0063] [Table 11]

[0064] [Table 12]

[0065] [Table 13]

[0066] [Table 14]

[0067] In one aspect, there is provided a polypeptide (e.g., a lipidated polypeptide) or a pharmaceutically acceptable salt thereof that is a pramlintide analog, having an alpha methyl amino acid at position 23. In a preferred embodiment, the alpha methyl amino acid is αMePhe.

[0068] In a preferred embodiment of any aspect in which a polypeptide (e.g., a lipidated polypeptide) comprises an alpha methyl amino acid (e.g., αMePhe) at position 23, the polypeptide (e.g., a lipidated polypeptide) comprises the following combination of modifications: 14E, 17R, 23αMePhe; Δ1K, 14E, 17R, 23αMePhe; 14Dab, 23αMePhe, 31E; 17Aib, 23αMePhe; 17R, 23αMePhe, 31E; 23αMePhe, 31E; 23αMePhe, 31R; or 23αMePhe, 35R Contains one of the following:

[0069] In one aspect, a polypeptide (e.g., a lipidated polypeptide) or a pharmaceutically acceptable salt thereof that is a pramlintide analog is provided, having at least two Aib residues. In a preferred embodiment, the polypeptide (e.g., a lipidated polypeptide) contains Aib at at least two of positions 17 and 20 to 37. In a particularly preferred embodiment, the polypeptide (e.g., a lipidated polypeptide) contains Aib at positions 21, 26, 27, 28, 29, 31, 32, 33, 34, and 35.

[0070] In a preferred embodiment of any aspect in which the polypeptide (e.g., lipidated polypeptide) comprises at least two Aib residues, the polypeptide (e.g., lipidated polypeptide) comprises the following combination of modifications: 14H, 17Aib, 21Aib, 31E; 17Aib, 21Aib; 17Aib, 21Aib, 37P; 17Aib, 26Aib; 17Aib, 27Aib; 17Aib, 28Aib; 17Aib, 29Aib; 17Aib, 31Aib; 17Aib, 32Aib; 17Aib, 33Aib; 17Aib, 34Aib; 17Aib, 35Aib; 17R, 21Aib, 31Aib; 17R, 21Aib, 35Aib; 21Aib, 26Aib; 21Aib, 27Aib; 21Aib, 28Aib; 21Aib, 31Aib; 21Aib, 33Aib; 21Aib, 34Aib; 21Aib, 35Aib; 21Aib, 36Aib; or 21Aib, 37Aib Contains one of the following:

[0071] In one aspect, there is provided a polypeptide (e.g., a lipidated polypeptide) or a pharmaceutically acceptable salt thereof that is a pramlintide analog, having an alpha methyl amino acid at position 21. In a preferred embodiment, the alpha methyl amino acid is Aib.

[0072] In a preferred embodiment of any aspect in which a polypeptide (e.g., a lipidated polypeptide) comprises an alpha methyl amino acid (e.g., Aib) at position 21, the polypeptide (e.g., a lipidated polypeptide) comprises the following combination of modifications: 14E, 21Aib; 14H, 17Aib, 21Aib, 31E; 14H, 21Aib, 35E; 14H, 21Aib; 16dL, 21Aib; 17Aib, 21Aib, 37P; 17Aib, 21Aib; 17E, 21Aib; 17R, 21Aib, 31Aib; 17R, 21Aib, 31E; 17R, 21Aib, 31R; 17R, 21Aib, 35Aib; 17R, 21Aib; 17S, 21Aib, 31H; 17S, 21Aib, 31P; 17S, 21Aib, 31R; 17S, 21Aib, 33P; 17S, 21Aib, 35P; 17S, 21Aib; 21Aib, 24Hyp, 25A, 28S, 21Aib, 24P, 25A, 28S, 31Dab; 21Aib, 24P, 25A, 28S, 35Dab, 21Aib, 24P, 25A, 28S; 21Aib, 26Aib; 21Aib, 26dI; 21Aib, 27Aib; 21Aib, 27dL; 21Aib, 28Aib; 21Aib, 28dP; 21Aib, 31Aib; 21Aib, 31E; 21Aib, 31H; 21Aib, 31R;Δ1K, 21Aib, 33Aib; 21Aib, 34Aib; 21Aib, 35Aib; 21Aib, 35E; 21Aib, 35R; 21Aib, 36Aib; 21Aib, 37Aib; 21Aib, 37P; or 21Aib Contains one of the following:

[0073] In one aspect, there is provided a polypeptide (e.g., a lipidated polypeptide) or a pharmaceutically acceptable salt thereof that is a pramlintide analog, having an alpha methyl amino acid at position 17. In a preferred embodiment, the alpha methyl amino acid is Aib.

[0074] In a preferred embodiment of any aspect in which a polypeptide (e.g., a lipidated polypeptide) comprises an alpha methyl amino acid (e.g., Aib) at position 17, the polypeptide (e.g., a lipidated polypeptide) comprises the following combination of modifications: 14H, 17Aib; -1G, -2G, 17Aib; 17Aib, 23αMePhe; 17Aib, 21Dab, 31E; 17Aib, 21Dab; 17Aib, 21Aib; 14H, 17Aib, 21Aib, 31E; 17Aib, 21Aib, 37P; 17Aib, 26Aib; 17Aib, 27Aib; 17Aib, 28Aib; 17Aib, 29Aib; 17Aib, 31Aib; 17Aib, 32Aib; 17Aib, 33Aib; 17Aib, 34Aib; or 17Aib, 35Aib Contains one of the following:

[0075] Pharmacokinetics The polypeptides of the present invention (e.g., lipidated polypeptides) may exhibit advantageous pharmacokinetic properties compared to pramlintide. For example, the polypeptides of the present invention (e.g., lipidated polypeptides) may have an extended half-life compared to pramlintide.

[0076] As used herein, the term "half-life" refers to the time required for the concentration of an isolated polypeptide in plasma to decrease to 50% of its initial level. Methods for determining the half-life of a protein are known in the art and are described in Example 4.

[0077] It will be appreciated that an increased half-life is advantageous because it allows a therapeutic protein to be administered according to a safe and convenient dosing schedule, e.g., a lower dose that can be administered less frequently. Furthermore, achieving a lower dose may provide additional benefits, such as providing an improved safety profile. In contrast, pramlintide requires frequent and inconvenient administration.

[0078] The present inventors have shown that polypeptides (e.g., lipidated polypeptides) of the present invention can have a half-life of at least 4 hours in a rat model (see Example 4). In embodiments, the polypeptides (e.g., lipidated polypeptides) have a half-life of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, or at least 14 hours in a rat model. In a preferred embodiment, the polypeptides (e.g., lipidated polypeptides) have a half-life of at least 14 hours.

[0079] Reduced fibrillation Polypeptides (e.g., lipidated polypeptides) of the invention may exhibit a reduced tendency to undergo fibrillation in pharmaceutically relevant aqueous media, particularly at pH values ​​ranging from 4 to 7, compared to lipidated pramlintide. In some embodiments, the polypeptides (e.g., lipidated polypeptides) exhibit a reduced tendency to undergo fibrillation in pharmaceutically relevant aqueous media, particularly at pH values ​​ranging from 4 to 7, compared to pramlintide that is lipidated in a similar manner, e.g., with the same lipid attached, the lipid attached via the same linker, and / or the lipid attached at the same position. Exemplary lipidated pramlintide molecules are provided in Table 1, e.g., SEQ ID NOs: 3, 4, 5, 6, 7, 112, and 113.

[0080] Thus, polypeptides (e.g., lipidated polypeptides) of the present invention may be suitable for formulation in acidic media (e.g., pH 4) and in neutral or near-neutral media (e.g., pH 7 or 7.4). Such polypeptides (e.g., lipidated polypeptides) may be suitable for co-formulation with, for example, insulin, various insulin analogs, and / or other therapeutic agents (e.g., antidiabetic or antiobesity agents) that require a neutral or near-neutral formulation pH.

[0081] In some embodiments, a polypeptide (e.g., a lipidated polypeptide) exhibits no detectable fibrillation at pH 4 and 37°C after about 5 hours, about 7 hours, about 9 hours, about 11 hours, about 13 hours, about 15 hours, about 17 hours, or about 20 hours under conditions such as those described in Example 3.

[0082] In preferred embodiments, the polypeptide (e.g., lipidated polypeptide) does not exhibit detectable fibrillation after about 48 hours, about 72 hours, about 96 hours, about 108 hours, about 120 hours, about 132 hours, or about 144 hours at pH 4 and 37°C, e.g., under the conditions described in Example 3. In particularly preferred embodiments, the polypeptide (e.g., lipidated polypeptide) does not exhibit detectable fibrillation after 144 hours at pH 4 and 37°C, e.g., under the conditions described in Example 3.

[0083] In some embodiments, fibril formation is detected by an increase in fluorescence intensity in a Thioflavin T fibrillation assay, for example as described in Example 3.

[0084] In preferred embodiments, the polypeptides of the invention (e.g., lipidated polypeptides) are soluble at concentrations required for therapeutic efficacy. In some embodiments, the lipidated polypeptides of the invention are soluble at a concentration of at least 1 mg / mL under the conditions described in Example 3.

[0085] Effectiveness The polypeptides (e.g., lipidated polypeptides) of the present invention are amylin receptor agonists, i.e., they can bind to one or more receptors or receptor complexes considered to be physiological receptors for human amylin and induce signal transduction by one or more receptors or receptor complexes. These include the human calcitonin receptor hCTR and complexes comprising the human calcitonin receptor hCTR and at least one of the human receptor activity-modulating proteins designated hRAMP1, hRAMP2, and hRAMP3. The complexes between hCTR and hRAMP1, hRAMP2, and hRAMP3 are designated hAMYR1, hAMYR2, and hAMYR3 (i.e., human amylin receptors 1, 2, and 3), respectively. In some embodiments, a compound is considered an amylin receptor agonist if it has agonist activity at one or more of hAMYR1, hAMYR2, and hAMYR3. For example, a compound is considered an amylin receptor agonist if it has agonist activity at hAMYR3.

[0086] The ability to induce cAMP formation as a result of binding to the relevant receptor or receptor complex is typically considered to be indicative of agonist activity. Other intracellular signaling pathways or events can also be used as readouts for amylin receptor agonist activity. These can include calcium release, arrestin recruitment, receptor internalization, kinase activation or inactivation, lipase activation, inositol phosphate release, diacylglycerol release, or nuclear transcription factor translocation.

[0087] The EC50 value can be used as a measure of agonist efficacy at a given receptor. The EC50 value is a measure of the concentration of a compound required to achieve half of the compound's maximal activity in a particular assay, such as the cAMP assay described in Example 2. In Example 2, the inventors show that certain polypeptides (e.g., lipidated polypeptides) disclosed herein exhibit greater or similar selectivity for hAMYR than hCTR, such as pramlintide, as measured using cAMP release from binding to hAMYR and hCTR. Pramlintide exhibits at least 10-fold selectivity for hAMYR compared to hCTR.

[0088] Polypeptides of the present invention (eg, lipidated polypeptides) may exhibit improved efficacy, eg, as amylin receptor agonists, compared to lipidated pramlintide.

[0089] In some embodiments, a polypeptide (e.g., a lipidated polypeptide) has at least about 1-fold selectivity for hAMYR over hCTR, optionally at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, or at least about 100-fold selectivity for hAMYR over hCTR. In preferred embodiments, a polypeptide (e.g., a lipidated polypeptide) has at least about 10-fold selectivity for hAMYR over hCTR.

[0090] In some embodiments, the polypeptide (eg, a lipidated polypeptide) has about 12-20 fold, about 14-18 fold, optionally about 16 fold selectivity for hAMYR over hCTR.

[0091] In some embodiments, the isolated polypeptide has an EC50 measured under the conditions described in Example 2 (i.e., containing 0.1% bovine serum albumin (BSA)) of less than about 1.4 nM, less than about 1.2 nM, less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.3 nM, or less than about 0.2 nM.

[0092] chemical stability Polypeptides of the invention (e.g., lipidated polypeptides) can be chemically stable, e.g., they can form an acceptable percentage of degradation products in formulations that are generated over a defined period of time by chemical pathways such as deamidation, aggregation, or oxidation.

[0093] The polypeptides of the present invention (e.g., lipidated polypeptides) can be chemically conjugated to protein or polymer drug carriers or formulated in pre-drug delivery systems, which enhance the chemical and / or physical stability and / or circulatory exposure of the polypeptides.

[0094] In some embodiments, a polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the polypeptide comprises any one of a lipid linker as shown in Table 2 and any one of a sequence alteration as shown in Table 3.

[0095] In some embodiments, a polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the polypeptide comprises a lipid linker and an amino acid sequence alteration shown in Table 4.

[0096] [Table 15]

[0097] [Table 16]

[0098] [Table 17]

[0099] [Table 18]

[0100] [Table 19]

[0101] process Polypeptides of the present invention (e.g., lipidated polypeptides) can be produced by any method known in the art. The production of polypeptides such as amylin or analogs thereof is well known in the art. Thus, polypeptides of the present invention (e.g., lipidated polypeptides) can be produced by chemical synthesis, for example, solid-phase polypeptide synthesis using t-Boc or Fmoc chemistry or other established techniques. Alternatively, they can be produced by recombinant expression of nucleic acid molecules encoding the fusion polypeptide in host cells. After synthesis, polypeptides of the present invention (e.g., lipidated polypeptides) can optionally be isolated or purified.

[0102] Treatment method In a further embodiment, the polypeptides (eg, lipidated polypeptides) of the present invention are provided in pharmaceutical compositions.

[0103] The pharmaceutical compositions of the present invention may contain one or more excipients. Pharmaceutically acceptable excipients are known in the art; see, for example, Remington's Pharmaceutical Sciences (by Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference in its entirety.

[0104] The present invention encompasses therapies comprising administering a polypeptide (e.g., a lipidated polypeptide) of the present invention to an animal, particularly a mammal, such as a human, to prevent, treat, or ameliorate symptoms associated with a disease, disorder, or infection.

[0105] Thus, the polypeptides (e.g., lipidated polypeptides) or pharmaceutical compositions of the present invention can be used, for example, in therapy to treat a disease or disorder. Also provided are methods for treating a disease or disorder, comprising administering a therapeutically effective amount of a polypeptide (e.g., lipidated polypeptide) or pharmaceutical composition of the present invention to a subject or patient in need thereof. The use or method can include administering a therapeutically effective schedule of taking the polypeptide (e.g., lipidated polypeptide) of the present invention less frequently than a therapeutically effective dosing schedule of pramlintide.

[0106] It will be appreciated that the polypeptides (e.g., lipidated polypeptides) of the present invention may be used in the treatment and / or prevention of metabolic disorders such as obesity, diabetes (e.g., type 1 or type 2 diabetes), and / or conditions associated with obesity.

[0107] Thus, the polypeptides (e.g., lipidated polypeptides) of the present invention may be used in a method for treating reproductive health complications of obesity or overweight, such as obesity, overweight, morbid obesity, pre-operative obesity, obesity-related inflammation, obesity-related gallbladder disease, sleep apnea and respiratory disorders, dyslipidemia, cartilage degeneration, osteoarthritis, or infertility in a subject, the method comprising administering a therapeutically effective amount of the polypeptide (e.g., lipidated polypeptide) to the subject.

[0108] This also provides a method for inhibiting or reducing weight gain, promoting weight loss, reducing food intake, and / or reducing excess weight, comprising administering a polypeptide of the invention (e.g., a lipidated polypeptide) to a subject.

[0109] Metabolic disorders that may be treated by the polypeptides (e.g., lipidated polypeptides) of the invention include diabetes, type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, impaired glucose tolerance (IG1), conditions associated with elevated blood glucose levels, metabolic disorders including metabolic syndrome, or hyperglycemia, e.g., abnormal postprandial hyperglycemia.

[0110] In a preferred embodiment, the polypeptides of the invention (eg, lipidated polypeptides) are used to treat type 1 or type 2 diabetes.

[0111] The polypeptides (e.g., lipidated polypeptides) or pharmaceutical compositions of the present invention can be used to treat, inhibit or reduce weight gain, promote weight loss, reduce food intake, and / or reduce excess weight.

[0112] A polypeptide (e.g., a lipidated polypeptide) or pharmaceutical composition of the present invention may be used in the treatment and / or prevention of eating disorders, Alzheimer's disease, fatty liver ("fatty liver"), renal failure, arteriosclerosis (e.g., atherosclerosis), cardiovascular disease, macrovascular disease, microvascular disease, diabetic heart (including diabetic cardiomyopathy and heart failure as diabetic complications), coronary heart disease, peripheral arterial disease or stroke, cancer, dumping syndrome, hypertension, e.g., pulmonary hypertension, or dyslipidemia, e.g., atherogenic dyslipidemia, cholecystitis, or short bowel syndrome.

[0113] The route of administration of the polypeptides (e.g., lipidated polypeptides) of the present invention or pharmaceutical compositions thereof can be, for example, oral, parenteral, by inhalation, or topical. In a preferred embodiment, the polypeptides (e.g., lipidated polypeptides) or pharmaceutical compositions thereof are administered to a subject or patient by parenteral administration. The term "parenteral," as used herein, includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. In a preferred embodiment, the polypeptides (e.g., lipidated polypeptides) or pharmaceutical compositions thereof are administered to a subject or patient by injection, such as by intravenous, subcutaneous, or intramuscular injection. In a particularly preferred embodiment, the polypeptides (e.g., lipidated polypeptides) or pharmaceutical compositions thereof are administered by subcutaneous injection. Administration by injection, such as by subcutaneous injection, offers the advantage of being more comfortable for the subject or patient and the opportunity to administer to a subject or patient outside of a hospital. In some embodiments, the polypeptides (e.g., lipidated polypeptides) or pharmaceutical compositions thereof are administered by self-administration.

[0114] In some embodiments, the subject or patient is a mammal, particularly a human.

[0115] In some embodiments, the polypeptide or pharmaceutical composition is administered to the subject in combination with insulin.

[0116] Products and Kits In another aspect, the invention provides articles of manufacture comprising a polypeptide (eg, a lipidated polypeptide) or pharmaceutical composition of the invention.

[0117] In yet another aspect, the present invention provides kits comprising a polypeptide (e.g., a lipidated polypeptide) or pharmaceutical composition of the invention. The kit may include a package containing the polypeptide (e.g., a lipidated polypeptide) or pharmaceutical composition, optionally along with instructions for use. In some embodiments, the polypeptide (e.g., a lipidated polypeptide) or pharmaceutical composition of the invention is formulated in a single-dose vial or container closure system (e.g., a pre-filled syringe). Optionally, such a container may bear a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency for manufacture, use, or sale for human administration.

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0119] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.

[0120] Unless otherwise indicated, each given nucleic acid sequence is written left to right in 5' to 3' orientation; an amino acid sequence is written left to right in amino to carboxy orientation.

[0121] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an agent" includes a plurality of such agents, a reference to "an agent" includes a reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth.

[0122] "About" can generally mean an acceptable degree of error for the quantity being measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. Optionally, the term "about" shall be understood herein as plus or minus (±) 5%, optionally ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, of the numerical value of the number with which it is used.

[0123] An embodiment described herein as "comprising" one or more features may also be considered a disclosure of a corresponding embodiment "consisting of" such features.

[0124] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0125] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format. It should also be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly not only to include the numerical values ​​expressly recited as the limits of the range, but also to include all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited.

[0126] The above-described embodiments should be understood as illustrative examples. Further embodiments are contemplated. It should be understood that any feature described with respect to any one embodiment may be used alone or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments or in any other combination of the embodiments. Furthermore, equivalents and modifications not described above may also be used without departing from the scope of the invention, as defined in the appended claims.

[0127] Other examples and variations of the polypeptides (eg, lipidated polypeptides) and methods described herein will be apparent to those of skill in the art in light of the present disclosure.

[0128] Other examples and variations are within the scope of this disclosure as set forth in the following claims.

[0129] All documents cited herein are hereby incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents. [Example]

[0130] Example 1: Production of lipidated pramlintide analog peptides Lipidated pramlintide analog peptides were synthesized as C-terminal carboxamides using Rink Amide MBHA resin (100-200 mesh). All peptides were prepared by automated synthesis using a Liberty Blue™ microwave solid-phase peptide synthesizer (CEM Corporation, NC, USA) using the Fmoc / tBu protocol. The manufacturer's protocol was applied for coupling of amino acids in DMF and deprotection of the Fmoc protecting group using piperidine (20% v / v) in DMF. Asparagine, cysteine, glutamine, and histidine were incorporated as their side-chain trityl (Trt) derivatives. Lysine was incorporated as its side-chain tert-butyloxycarbonyl (Boc) derivative. Serine, threonine, and tyrosine were incorporated as side-chain tert-butyl (tBu) ethers, and aspartate and glutamate were incorporated as their side-chain OtBu ethers. Arginine was incorporated as the side chain 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) derivative.

[0131] If subsequent chemical modification of the N-terminal lysine side chain was required, Boc-Lys(Fmoc) was incorporated. Upon completion of peptide chain elongation, coupling of albumin binding moieties, such as lipids, was performed manually using HATU as a coupling reagent in the presence of DIPEA.

[0132] The peptide was cleaved from the solid support by treatment with a mixture of TFA:TIS:EDT:thioanisole:water (90:2.5:2.5:2.5:2.5 v / v) for 4 hours with stirring at room temperature. The cleavage mixture was then filtered, concentrated in vacuo, precipitated, washed with diethyl ether, and the solid isolated by centrifugation. The crude linear peptide was dried under a stream of nitrogen, dissolved in 20% MeCN (v / v) in water with 1% TFA (v / v), and filtered. The crude linear peptide was purified using preparative RP-HPLC on a Varian SD-1 Prep Star binary pump system, monitored by UV absorbance at 210 nm, using an Xbridge C18-A stationary phase (19.0 x 250 mm, 5 micron) column eluting with a linear solvent gradient of 25 to 70% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) over 25 minutes.

[0133] The linear purified peptide was cyclized by treatment with iodine (1% w / v in methanol) for 10 minutes at room temperature, and excess iodine was reduced by treatment with ascorbic acid (1% w / v in water). The crude cyclic peptide was repurified as described above. The purified fractions were pooled, frozen, and lyophilized.

[0134] LC / MS characterization of purified peptides was performed on a Waters MassLynx 3100 platform using an XBridge C18 stationary phase (4.6 x 100 mm, 3 microns) eluting with a linear binary gradient of 10 to 90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) over 10 minutes at 1.5 mL / min at ambient temperature. Analytes were detected by both UV absorption at 210 nm and ionization using the Waters 3100 mass detector (ESI+ mode). Analytical RP-HPLC characterization was performed on an Agilent 1260 Infinity system using an Agilent Polaris C8-A stationary phase (4.6 × 100 mm, 3 microns) eluting a linear binary gradient of 10–90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) at 1.5 mL / min over 15 min at 40 °C.

[0135] Example 2: In vitro efficacy of lipidated pramlintide analog peptides in human or rat amylin or calcitonin receptor cells The functional activity of lipidated pramlintide analog peptides, such as cAMP production, was tested in 1321N1 cell lines with stable recombinant expression of human calcitonin receptor (hCTR) or human amylin receptor (calcitonin receptor co-expressed with receptor activity-modifying protein, RAMP3) (hAMYR3) or HEK cells with stable recombinant expression of rat calcitonin receptor (ratCTR) or rat amylin receptor (calcitonin receptor co-expressed with receptor activity-modifying protein, RAMP3) (ratAMYR3).

[0136] Cryopreserved cell stocks were rapidly thawed in a water bath, suspended in assay buffer (0.1% BSA (Sigma #A3059) in HBSS (Sigma #H8264) with 25 mM HEPES, pH 7.4, and 0.5 mM BMX (Sigma #17018)) and spun at 240 x g for 5 minutes. Cells were added at a batch-dependent optimized concentration (e.g., 0.125 x 10 hCTR cells). 5 cells / mL, hAMYR3 cells 0.125×10 5 cells / mL, rat CTR cells 1 × 10 5 cells / mL, rat AMYR3 2 × 10 5 The cells were resuspended in assay buffer (in cells / mL).

[0137] Test peptide stocks were prepared in DMSO, diluted in assay buffer to reach the stated concentrations, and transferred in duplicate to 384 black shallow-well microtiter assay plates (Corning #3676). Cells were added to the assay plates, incubated at room temperature for 30 minutes, and cAMP levels were measured using the cAMP Dynamic 2HTRF kit (Cisbio, catalog #62AM4PEJ) following the manufacturer's recommended two-step protocol. Plates were read on an Envision (Perkin Elmer) using an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm.

[0138] Data were converted to %Delta F as described in the manufacturer's guidelines and EC 50 The EC50 values ​​were analyzed as percent activation of the maximal amylin or calcitonin effect by a four-parameter logistic fit to determine EC50 values. The selectivity of the peptide for hAMYR versus hCTR is defined as the ratio of the EC50 values ​​at the two receptors.

[0139] All tested compounds show measurable potency at hAMYR and hCTR. Analogs that show greater than 10-fold selectivity for hAMYR versus hCTR are preferred.

[0140] [Table 20]

[0141] [Table 21]

[0142] [Table 22]

[0143] [Table 23]

[0144] [Table 24]

[0145] Example 3: Thioflavin T fibrillation assay Peptide aggregation to form fibrils is an indicator of physical instability. Fibril formation in solution poses a significant risk to the stability of injectable peptide pharmaceuticals. The thioflavin T (ThT) fibrillation assay is a useful tool for assessing the aggregation kinetics of peptides or proteins under accelerated and stressed conditions, which can be used to predict the long-term viability of compounds in solution.

[0146] ThT can selectively bind to amyloid fibrils, and the resulting complex emits a strong fluorescent signal at 482 nm when excited at 450 nm (Anal Biochem. 1989 Mar;177(2):244-9). Monitoring the change in the fluorescent signal is an established method for studying the fibril-forming potential of peptides and proteins.

[0147] ThT (purchased from Sigma Aldrich) stock solution is prepared by dissolving ThT powder in Milli-Q water and filtering to obtain a 0.25 mM solution. The concentration of the solution is 36 mM. -1 cm -1 The intensity is measured at 412 nm using an extinction coefficient of 0.05%. The test peptides were dissolved at 1 mg / mL in 25 mM sodium acetate buffer, pH 4.0.

[0148] A 100 μL aliquot of peptide solution and a 5 μL aliquot of ThT solution were placed in a clean, black-bottom fluorescent 96-well plate. Five replicates of each test sample were placed in the same row of the plate. Buffer was placed in the control wells for baseline correction. All empty wells were filled with water to prevent evaporation. The plate was sealed with an aluminum seal, placed in a fluorescent plate reader, and incubated at 37°C for 6 days with intermittent orbital shaking at 500-750 rpm. Fluorescence intensity was measured every 30 minutes using excitation at 444 nm and emission at 480 nm.

[0149] The fibril-forming potential of the test peptides was determined by measuring the mean time required to detect an increase in baseline-corrected fluorescence intensity: a time greater than 144 h indicates no increase in fluorescence intensity relative to baseline during the course of the experiment.

[0150] Binding of pramlintide to lipids (e.g., as in SEQ ID NOs: 3, 4, 5, 6, 112, 113) increases the tendency to form fibrils, as seen in Table 7.

[0151] [Table 25]

[0152] Example 4: Pharmacokinetic measurements in Sprague Dawley rats following IV and SC administration The objective of the pharmacokinetic (PK) study was to determine the plasma pharmacokinetic profile of lipidated pramlintide analog peptide in fasted male SD rats after single intravenous (IV) and subcutaneous (SC) administration. The PK study was performed to determine the half-life (T 1 / 2 ) was measured. 1 / 2 indicates the time required for the maximum plasma concentration (Cmax) of the test substance to decrease to half of its steady-state concentration in circulation.

[0153] Male SD rats were purchased from Si Bei Fu Laboratory Animal Technology Co., Ltd. (China). Animals were 6-8 weeks old and weighed 200-300 g on the day of dosing. Animals were housed under a 12-hour light / 12-hour dark cycle and fasted overnight before dosing. Animal weights were recorded before dosing and 24 and 48 h after dosing. Animals had free access to food and water, and food consumption was quantified daily.

[0154] The test substance was administered at 20 nmol / kg. Blood samples were collected from each animal via the jugular vein. Sampling times were as follows:

[0155] Blood samples per test article

[0156] [Table 26]

[0157] Blood samples were transferred to Eppendorf low-binding tubes containing K2EDTA. More than 0.150 mL of blood was collected at each time point. Blood samples were centrifuged at 4,000 g for 5 minutes at 4°C to obtain plasma. Plasma samples were stored frozen at -75±15°C until analysis.

[0158] The concentrations of the test substance in plasma samples were analyzed using an LC-MS / MS method. Data collection was performed using LabSolution version 5.89 software (Shimadzu, Kyoto, Japan). Data statistics were performed using Excel 97-2003 software. Pharmacokinetic parameters of the test substance were calculated using a non-compartmental approach in Phoenix™ WinNonlin® 6.1.

[0159] Whenever possible from the plasma concentration versus time data, the following pharmacokinetic parameters were calculated: IV administration: T 1 / 2 , C0, AUC last , AUC inf , MRT inf, Cl, Vss, Number of regression points. SC administration: T max , C max , AUC last , AUC inf , MRT inf ,F, Number of regression points.

[0160] [Table 27]

[0161] Pharmacokinetic studies have shown that the terminal half-life of amylin in rats is approximately 13 minutes, and the half-life for pramlintide in humans is approximately 20-45 minutes (Roth JD et.al. GLP-1R and amylin agonism in metabolic disease: complementary mechanisms and future opportunities. Br J Pharmacol. 2012;166(1):121-136). Lipidated polypeptides show marked improvement in prolonged circulatory T1 / 2 compared to pramlintide.

[0162] Example 6: Acute food intake study in rats Male Sprague Dawley rats were obtained from Taconic Denmark, ApS, at approximately 7 weeks of age. They were implanted with an identification microchip and housed four per cage with free access to enrichment, food, and water. They were allowed to acclimate for one week while undergoing noninvasive characterization. The rats were on a 12:12 light:dark cycle with a 1 pm:1 am switch. Food intake was monitored using an HM2 system (Lafayette Instrument), which allows for home-cage monitoring. As each rat entered the food access tunnel, the IR beam was interrupted and the implanted microchip was read. Any resulting changes in food weight were then assigned to a specific animal. Social order did not affect overall feeding patterns or food intake.

[0163] Rats were divided into groups based on day 1 body weight and 24-hour cumulative food intake (n=7 per group). On day 0, rats were weighed and then fasted for 6 hours. 30 minutes before food reintroduction, rats were subcutaneously dosed (5mL / kg) with 20nmol / kg of test compound or 60nmol / kg of peptide 1 (pramlintide) diluted in the appropriate vehicle, after which food was returned and lights were turned off. Automatic food intake was then monitored for 3 days, and rats were weighed once a day.

[0164] Food intake per rat was summarized into one bout per hour and imported into Gubra's GubraView data management system. Individual food intake data was exported to MS Excel, which provided cumulative food intake data. The cumulative food intake data was then transferred to GraphPad Prism (v8.0.1) for analysis of dark-period feeding.

[0165] The lipidated polypeptides show a marked suppression of food intake compared to pramlintide.

[0166] [Table 28]

Claims

1. Amino acid sequence: Xaa(-4)-Xaa(-3)-Xaa(-2)-Xaa(-1)-Xaa1-Cys2-Asn3-Xaa4-Ala5-Thr6-C ys7-Ala8-Thr9-Gln10-Arg11-Leu12-Ala13-Xaa14-Xaa15-Xaa16-Xaa17-Hi s18-Ser19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Thr30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-amide [SEQ ID NO: 2] (In the formula, Xaa(-4) is either Lys (albumin binding site) or absent; Xaa(-3) is either Gly or absent; Xaa(-2) is either Gly or absent; Xaa(-1) is either Gly (albumin binding site), Lys (albumin binding site), or absent; Xaa1 is either Lys, Lys(albumin binding site), (albumin binding site), or absent; Xaa4 is Thr, Ile, or Ala; Xaa14 is Asn, His, Glu, 2,4-diaminobutanoic acid (Dab), or alpha-methylamino acid; Xaa15 is either Phe or Trp; Xaa16 is Leu or D-Leu(dL); Xaa17 is Val, Ser, Glu, Arg, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp), Dab, or alpha-methyl amino acid (e.g., 2-amino-2-methylpropanoic acid [Aib]); Xaa20 is Ser, Ile, Pro, or alpha-methyl amino acid (e.g., (S)-2-amino-3-hydroxy-2-methylpropanoic acid [αMeSer]); Xaa21 is Asn, Dab, His, Pro, Ser, Arg, Lys, Gly or Glu, Ala, Hyp or alpha-methyl amino acid (e.g., Aib); Xaa22 is Asn, His, Hyp, Dab, or an alpha-methylamino acid (e.g., Aib); Xaa23 is Phe, Hyp, or alpha-methyl amino acid (e.g., (S)-2-amino-2-methyl-3-phenylpropanoic acid [αMePhe]); Xaa24 is Gly, Pro, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa25 is Pro, Ala, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa26 is Ile, D-Ile (dI), Arg, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa27 is Leu, dL, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa28 is Pro, D-Pro(dP), Ser, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa29 is Pro, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa31 is Asn, Glu, His, Arg, Pro, Dab, or alpha-methylamino acid (e.g., Aib); Xaa32 is Val, Hyp, Dab, or an alpha-methyl amino acid (e.g., Aib); Xaa33 is Gly, Pro, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa34 is Ser, Pro, His, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa35 is Asn, Pro, Arg, Glu, Dab, Hyp, or alpha-methylamino acid (e.g., Aib); Xaa36 is Thr, Hyp or alpha-methyl amino acid (e.g., Aib); and Xaa37 is Tyr, Pro, Hyp, or alpha-methylamino acid (e.g., Aib). A polypeptide comprising or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least one albumin-binding site, the at least one albumin-binding site comprises a lipid, and the polypeptide is subject to the following amino acid modifications or combinations of amino acid modifications: -1G, -2G, 17Aib 4A, 15W, 21P, 24P, 25A, 28S 4I, 20I, 21A, 35R 4I, 21Dab, 35R 14Dab 14Dab, 17Aib, 31E 14Dab, 23αMePhe, 31E 14Dab, 31E 14E, 17Aib 14E, 17Aib, 21H 14E, 17R 14E, 17R, 23αMePhe 14E, 21Aib 14H, 17Aib 14H, 17Aib, 21Aib, 31E 14H, 21Aib 14H, 21Aib, 35E 16dL, 21Aib 17Aib 17Aib, 37P 17Aib, 21Aib 17Aib, 21Aib, 37P 17Aib, 21G 17Aib, 21H 17Aib, 21K 17Aib, 21P 17Aib, 21P, 31E 17Aib, 21P, 35E 17Aib, 21R 17Aib, 21S 17Aib, 21Dab 17Aib, 21Dab, 31E 17Aib, 22H 17Aib, 22H, 35E 17Aib, 23αMePhe 17Aib, 26Aib 17Aib, 26R 17Aib, 27Aib 17Aib, 27dL 17Aib, 28Aib 17Aib, 29Aib 17Aib, 31Aib 17Aib, 31E 17Aib, 31H, 35E 17Aib, 31P 17Aib, 31R 17Aib, 32Aib 17Aib, 33Aib 17Aib, 34Aib 17Aib, 34H 17Aib, 34P 17Aib, 35Aib 17Aib, 35E 17Aib, 35R 17E, 21Aib 17R, 21Aib 17R, 21Aib, 31Aib 17R, 21Aib, 31E 17R, 21Aib, 31R 17R, 21Aib, 35Aib 17R, 23αMePhe 17R, 23αMePhe, 31E 17R, 26Aib 17S, 21Aib 17S, 21Aib, 31H 17S, 21Aib, 31P 17S, 21Aib, 31R 17S, 21Aib, 33P 17S, 21Aib, 35P 20αMeSeer 20P, 21P, 24P, 25A, 28S 21Aib 21Aib, 24P, 25A, 28S, 31Dab 21Aib, 24P, 25A, 28S, 35Dab 21Aib, 26dI 21Aib, 26Aib 21Aib, 27Aib 21Aib, 27dL 21Aib, 28Aib 21Aib, 28dP 21Aib, 31Aib 21Aib, 31E 21Aib, 31H 21Aib, 31R 21Aib, 33Aib 21Aib, 34Aib 21Aib, 35Aib 21Aib, 35E 21Aib, 35R 21 Aib, 36 Aib 21Aib, 37Aib 21Aib, 37P 21Aib, 24Hyp, 25A, 28S 21Dab, 25Aib 22Aib 22H, 35E 23αMePhe, 31E 23αMePhe, 31R 23αMePhe, 35R 24Aib 26Aib 27Aib Δ1K, 4I, 21Dab, 35R or Δ1K, 14E, 17R, 23αMePhe A polypeptide or a pharmaceutically acceptable salt thereof containing any one of the following.

2. Amino acid sequence: Xaa(-4)-Xaa(-3)-Xaa(-2)-Xaa(-1)-Xaa1-Cys2-Asn3-Xaa4-Ala5-Thr6-C ys7-Ala8-Thr9-Gln10-Arg11-Leu12-Ala13-Xaa14-Xaa15-Xaa16-Xaa17-Hi s18-Ser19-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Thr30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-amide [SEQ ID NO: 2] (In the formula, Xaa(-4) is either Lys (albumin binding site) or absent; Xaa(-3) is either Gly or absent; Xaa(-2) is either Gly or absent; Xaa(-1) is either (albumin binding site), Lys(albumin binding site), or absent; Xaa1 is either Lys, Lys(albumin binding site), (albumin binding site), or absent; Xaa4 is Thr; Xaa14 is Asn, Glu, or Dab; Xaa15 is Phe; Xaa16 is Leu; Xaa17 is Val, Arg, or Aib; Xaa20 is Ser; Xaa21 is Asn; Xaa22 is Asn; Xaa23 is Phe or αMePhe; Xaa24 is Gly; Xaa25 is Pro; Xaa26 is Ile; Xaa27 is Leu; Xaa28 is Pro; Xaa29 is Pro; Xaa31 is Asn, Glu, or Arg; Xaa32 is Val; Xaa33 is Gly; Xaa34 is Ser; Xaa35 is either Asn or Arg; Xaa36 is Thr; and Xaa37 is Tyr. A polypeptide comprising or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least one albumin-binding site, and the at least one albumin-binding site comprises a lipid, optionally, (i) Xaa23 is αMePhe; (ii) Xaa14 is either Glu or Dab; (iii) Xaa17 is Arg or Aib; (iv) Xaa31 is Glu or Arg; (v) Xaa35 is Arg; and / or (vi) The polypeptide or pharmaceutically acceptable salt according to claim 1, wherein Xaa(1) is absent.

3. The lipid is bound to the N-terminus of the polypeptide, for example, the lipid is bound to lysine at the N-terminus of the polypeptide; optionally, (i) The lipid is attached to an amino acid residue at Xaa-4, Xaa-3, Xaa-2, Xaa-1 or Xaa1; (ii) The lipid is bound to Xaa-4, Xaa-1 or Xaa1; optionally, the lipid is bound to the N-terminus or side chain of Xaa-4, Xaa-1 or Xaa1; or (iii) The polypeptide or pharmaceutically acceptable salt according to claim 1 or 2, wherein Xaa(-4) is Lys(albumin binding site), Xaa(-1) is Lys(albumin binding site) or (albumin binding site), or Xaa1 is Lys(albumin binding site).

4. The polypeptide or pharmaceutically acceptable salt according to any one of claims 1 to 3, wherein the lipid comprises a hydrocarbon chain having 10 to 26 carbon atoms, for example 14 to 24 carbon atoms, for example 16 to 22 carbon atoms, or comprises 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms; optionally, the lipid comprises a dicarboxylic acid; and further optionally, the lipid is selected from C12 diacid, C14 diacid, C16 diacid, C17 diacid, C18 diacid, C19 diacid or C20 diacid.

5. The albumin-binding site is bound to the amino acid residue by a linker; optionally, the linker comprises one or more residues of any naturally occurring or non-naturally occurring amino acids; further optionally, (i) The linker comprises a combination of residues as a single unit or repeating unit, each of which independently comprises Glu, γ-Glu, Lys, ε-Lys, Asp, β-Asp, Gaba, β-Ala (3-aminopropanoyl), O2Oc (2-(2-(2-aminoethoxy)ethoxy)acetic acid), PEG2 (3-(2-(2-aminoethoxy)ethoxy)propanoic acid), It may be a residue selected from PEG4 (1-amino-3,6,9,12-tetraoxapentadecane-15-acid), PEG8 (1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-acid), or PEG12 (1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoquinonatricontane-39-acid); or (ii) The polypeptide or pharmaceutically acceptable salt according to any one of claims 1 to 4, wherein the linker comprises a residue of γ-Glu, and further optionally the linker comprises γGlu, γGlu-γGlu, γGlu-(O2Oc)-(O2Oc), or γGlu-(PEG2)-(PEG2).

6. A pharmaceutical composition comprising a polypeptide or pharmaceutically acceptable salt according to any one of claims 1 to 5, and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6 for treating and / or preventing a disease or disorder in a subject.

8. The pharmaceutical composition according to claim 7, wherein the disease or disorder is obesity, metabolic disease, symptoms associated with obesity, eating disorders, Alzheimer's disease, fatty liver ("fatty liver"), renal failure, arteriosclerosis (e.g., atherosclerosis), cardiovascular disease, macrovascular disease, microvascular disease, diabetic heart (including diabetic cardiomyopathy and heart failure as diabetic complications), coronary heart disease, peripheral artery disease or seizure, cancer, dumping syndrome, hypertension (e.g., pulmonary hypertension) or dyslipidemia (e.g., atherosclerotic dyslipidemia), cholecystitis or short bowel syndrome.

9. (i) The symptoms related to obesity are obesity or obesity-related reproductive health complications such as being overweight, morbid obesity, preoperative obesity, obesity-related inflammation, obesity-related gallbladder disease, sleep apnea and respiratory disorders, dyslipidemia, cartilage degeneration, osteoarthritis or infertility; or (ii) The pharmaceutical composition according to claim 8, wherein the metabolic disease is a metabolic disease including diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, prediabetes, insulin resistance, impaired glucose tolerance (IG1), conditions associated with elevated blood glucose levels, metabolic syndrome, or hyperglycemia, such as abnormal postprandial hyperglycemia.

10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the pharmaceutical composition is administered to the subject by subcutaneous injection.

11. The pharmaceutical composition according to any one of claims 7 to 9, which is administered to the subject by self-administration.

12. A polypeptide or pharmaceutically acceptable salt according to any one of claims 1 to 5 for treating and / or preventing a disease or disorder in a subject.

13. The polypeptide or pharmaceutically acceptable salt according to claim 12, wherein the disease or disorder is obesity, metabolic disease, symptoms associated with obesity, eating disorders, Alzheimer's disease, fatty liver ("fatty liver"), renal failure, arteriosclerosis (e.g., atherosclerosis), cardiovascular disease, macrovascular disease, microvascular disease, diabetic heart (including diabetic cardiomyopathy and heart failure as diabetic complications), coronary heart disease, peripheral artery disease or seizure, cancer, dumping syndrome, hypertension (e.g., pulmonary hypertension) or dyslipidemia (e.g., atherosclerotic dyslipidemia), cholecystitis or short bowel syndrome.

14. (i) The symptoms related to obesity are obesity or obesity-related reproductive health complications such as being overweight, morbid obesity, preoperative obesity, obesity-related inflammation, obesity-related gallbladder disease, sleep apnea and respiratory disorders, dyslipidemia, cartilage degeneration, osteoarthritis or infertility; or (ii) The polypeptide or pharmaceutically acceptable salt according to claim 13, wherein the metabolic disease is a metabolic disease including diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, prediabetes, insulin resistance, impaired glucose tolerance (IG1), conditions associated with elevated blood glucose levels, metabolic syndrome, or hyperglycemia, such as abnormal postprandial hyperglycemia.

15. The polypeptide or pharmaceutically acceptable salt according to any one of claims 12 to 14, wherein the polypeptide or pharmaceutically acceptable salt is administered to the subject by subcutaneous injection.

16. The polypeptide or pharmaceutically acceptable salt according to any one of claims 12 to 14, wherein the polypeptide or pharmaceutically acceptable salt is administered to the subject by self-administration.

17. A method for producing a polypeptide according to any one of claims 1 to 5, comprising optionally synthesizing the polypeptide by a solid-phase or liquid-phase method, and optionally isolating and purifying the final product.

18. A product comprising a polypeptide or pharmaceutically acceptable salt according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 6.

19. A kit comprising a polypeptide or pharmaceutically acceptable salt according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6, further optionally comprising instructions for use.