Conjugated hepcidin mimetics

Abstract

To provide hepcidin analogs with improved in vivo half-lives, and related pharmaceutical compositions and methods of use thereof.SOLUTION: The present invention relates generally to hepcidin analog peptides and methods of making and using the same. In certain embodiments, hepcidin analogs exhibit one or more hepcidin activities. In certain embodiments, the present invention is directed to hepcidin peptide analogs comprising one or more peptide subunits, wherein the peptide subunits form a cyclized structure via an intramolecular bond, e.g., an intramolecular disulfide bond. In certain embodiments, cyclized structures have increased potency and selectivity compared to non-cyclized hepcidin peptides and analogs thereof. In certain embodiments, the hepcidin analog peptides of the present invention exhibit an extended half-life compared to hepcidin or conventional hepcidin analogs when delivered orally.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 057,582, filed July 28, 2020, U.S. Provisional Patent Application No. 63 / 057,577, filed July 28, 2020, U.S. Provisional Patent Application No. 63 / 169,527, filed April 1, 2021, U.S. Provisional Patent Application No. 63 / 169,533, filed April 1, 2021, U.S. Provisional Patent Application No. 63 / 169,515, filed April 1, 2021, U.S. Provisional Patent Application No. 63 / 057,583, filed July 28, 2020, and U.S. Provisional Patent Application No. 63 / 057,574, filed July 28, 2020, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated by reference in its entirety. The ASCII copy, created on July 28, 2021, is named PRTH_054_02WO_ST25.txt, and is 154KB in size.

[0003] The present invention particularly relates to certain hepcidin peptide analogs, including both peptide monomers and peptide dimers, as well as conjugates and derivatives thereof, and compositions comprising such peptide analogs, and the use of such peptide analogs in the treatment and / or prevention of various diseases, conditions, or disorders, including, for example, the treatment and / or prevention of polycythemia vera, iron overload diseases, such as hereditary hemochromatosis, iron overload anemia, and other conditions and disorders described herein. [Background technology]

[0004] Hepcidin (also known as LEAP-1), a peptide hormone produced by the liver, is a regulator of iron homeostasis in humans and other mammals. Hepcidin acts by binding to its receptor, the iron-transporting channel ferroportin, resulting in its internalization and degradation. Human hepcidin is a 25-amino acid peptide (Hep25). See Krause et al. (2000) FEBS Lett 480:147-150 and Park et al. (2001) J Biol Chem 276:7806-7810. The structure of the biologically active 25-amino acid form of hepcidin is a simple hairpin structure, with eight cysteines forming four disulfide bonds, as described by Jordan et al. J Biol Chem 284:24155-67. The N-terminal region is required for iron-regulatory function, and deletion of the five N-terminal amino acid residues results in loss of iron-regulatory function. See Nemeth et al. (2006) Blood 107:328-33.

[0005] Abnormal hepcidin activity is associated with iron overload disorders, including hereditary hemochromatosis (HH) and iron overload anemia. Hereditary hemochromatosis is a genetic iron overload disorder primarily caused by hepcidin deficiency or, in some cases, hepcidin resistance. This results in excessive dietary iron absorption, leading to iron overload. Clinical manifestations of HH include liver disease (e.g., cirrhosis, NASH, and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is regular phlebotomy, which places a significant burden on patients. Iron overload anemia is a genetic anemia associated with defective red blood cell production, such as β-thalassemia. Complications from iron overload are a major cause of morbidity and mortality in these patients. Hepcidin deficiency is a major cause of iron overload in non-transfused patients and also contributes to iron overload in transfused patients. The current treatment for iron overload in these patients involves iron chelation, which is extremely burdensome. Furthermore, in some cases they are ineffective and frequently cause side effects.

[0006] Hepcidin has several limitations that limit its use as a drug, including a difficult synthesis process, due in part to aggregation and precipitation during protein folding. Aggregation and precipitation during folding also lead to low bioavailability, injection site reactions, immunogenicity, and high cost of goods. What is needed in the art are compounds that have hepcidin activity and other beneficial physical properties, such as improved solubility, stability, and / or efficacy, that can be produced inexpensively and used to treat hepcidin-related diseases and disorders, such as those described herein. The present invention addresses such needs and provides novel peptide analogs, including both monomeric and dimeric peptide analogs, that possess hepcidin activity and other beneficial properties that make the peptides of the present invention suitable hepcidin substitutes. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Krause et al.(2000)FEBS Lett 480:147-150 [Non-patent document 2] Park et al. (2001) J Biol Chem 276:7806-7810 [Non-patent document 3] Jordan et al. J Biol Chem 284:24155-67 [Non-patent document 4] Nemeth et al.(2006)Blood 107:328-33 Summary of the Invention [Means for solving the problem]

[0008] The present invention relates generally to peptide analogs, including both monomers and dimers, that exhibit hepcidin activity and methods of use thereof.

[0009] In one embodiment, the present invention provides a compound of formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ia) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 is hydrogen, C1-C6 alkyl, C6-C 12 Aryl, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 is cycloalkanoyl, R 2 is NH, substituted amino, OH, or substituted hydroxy; X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, GluOMe, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X2 is Ala, Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, Gly, N-substituted Gly, His, or substituted His; X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X6 is absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys or Pen; X7 is absent, Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys or 123 triazole; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent, Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys or (D)Lys; and Each of X12 to X14 is absent or independently represents any amino acid, However, subject to the following conditions: i) the peptide may be further conjugated with any amino acid; ii) any of the amino acids of the peptide may be its corresponding (D)-amino acid or may be N-substituted; and iii) the peptide is a linear peptide or a cyclized lactam; and wherein Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, and 2-Nal is 2-naphthyl alanine, Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and the substituted Phe is phenyl is F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzoyl phenylalanine substituted with diaryloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine, and the substituted bhPhe is b-homophenylalanine in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine, and the substituted Trp is is N-methyl-L-tryptophan, a-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; the substituted bhTrp is N-methyl-Lb-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid; and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid; 123 Triazole is [ka] and Dab is [ka] is.

[0010] In one embodiment, the present invention provides a compound of formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ib) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 is hydrogen, C1-C6 alkyl, C6-C 12 Aryl, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 is cycloalkanoyl, R 2 is NH, substituted amino, OH, or substituted hydroxy; X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2; X2 is Ala, Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, Gly, N-substituted Gly, His, or substituted His; X4 is Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), or 2-pyrrolidinebutanoic acid (Pba); X6 is absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys or Pen; X7 is absent, Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent, Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys or (D)Lys; and Each of X12 to X14 is absent or independently represents any amino acid, However, subject to the following conditions: i) the peptide does not contain a disulfide bond or a thioether bond; ii) the peptide may be further conjugated with any amino acid; iii) any of the amino acids of the peptide may be its corresponding (D)-amino acid or may be N-substituted, wherein Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, and 2-Nal is 2-naphthylalanine. wherein Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, substituted Phe is phenylalanine in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine, and substituted bhPhe is phenyl in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine.is b-homophenylalanine substituted with 6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted Trp is N-methyl-L-tryptophan, a-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; the substituted bhTrp is N-methyl-Lb-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid; and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid.

[0011] In one embodiment, X1 is Glu, X2 is Thr, X4 is Dpa, or X5 is Pro.

[0012] In another aspect, the present invention provides a compound of formula (II): R 1 -Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (II) or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2 , X3, X6 to X14 are as defined in formula (I).

[0013] In another aspect, the present invention provides a compound of formula (IXa): R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2and X11 to X14 are as described in formula (I).

[0014] In another aspect, the present invention provides a compound of formula (XXI): R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (XXI), wherein R 1 , R 2 and X10 to X14 are as described in formula (I), X6 is absent, Ala, or a substituted Lys; X7 is absent, Ile, a substituted Lys, or a substituted (D)Lys; and X9 is absent or bhPhe; and X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety.

[0015] In one embodiment, R 1 is IVA or isovaleric acid.

[0016] In one embodiment, R 2 is NH. In one embodiment, R 2 is OH.

[0017] In certain embodiments of any of the hepcidin analogs of the invention, the substituted Lys or substituted (D)Lys is a Lys or (D)Lys substituted with an acid, or a residue thereof, selected from C12 (lauric acid), C14 (mysteric acid), C16 (palmitic acid), C18 (stearic acid), C20, C12 diacids, C14 diacids, C16 diacids, C18 diacids, C20 diacids, biotin, and isovaleric acid, either directly or via a linker. In one embodiment, the linker is Ahx, PEG, or PEG-Ahx.

[0018] In certain embodiments of any of the hepcidin analogs of the invention, X or X is Lys, or (D)Lys substituted with L1Z, where L is absent, Dapa, D-Dapa, or isoGlu, PEG, Ahx, isoGlu-PEG, PEG-isoGlu, PEG-Ahx, isoGlu-Ahx, or isoGlu-PEG-Ahx, where Ahx is an aminohexanoic acid moiety and PEG is -[C(O)-CH-(Peg) n -N(H)] m - or -[C(O)-CH2-CH2-(PEG) n -N(H)] m -, and Peg is -OCH2CH2-, m is 1, 2 or 3, and n is an integer between 1 and 100K, and Z is a half-life extending moiety. In one embodiment, the half-life extending moiety is C 10 -C 21 It is an alkanoyl.

[0019] In certain embodiments, a peptide analog or dimer of the present invention comprises an isovaleric acid moiety conjugated to the N-terminal X1 residue. In certain embodiments, a peptide analog or dimer of the present invention comprises an isovaleric acid moiety conjugated to the N-terminal Asp residue. In certain embodiments, a peptide analog or dimer of the present invention comprises an isovaleric acid moiety conjugated to the N-terminal Glu residue.

[0020] In certain embodiments, peptide analogs of the invention comprise an amidated C-terminal residue.

[0021] In a related embodiment, the invention includes polynucleotides encoding the hepcidin analog or dimeric (or monomeric subunit of a dimer) peptides of the invention.

[0022] In further related embodiments, the invention includes vectors comprising the polynucleotides of the invention. In certain embodiments, the vectors are expression vectors comprising a promoter operably linked to the polynucleotide, e.g., in a manner that promotes expression of the polynucleotide.

[0023] In another embodiment, the invention includes a pharmaceutical composition comprising a hepcidin analog, dimer, polynucleotide, or vector of the invention and a pharmaceutically acceptable carrier, excipient, or vehicle.

[0024] In another embodiment, the present invention provides a method of binding to or inducing the internalization and degradation of ferroportin, the method comprising contacting ferroportin with at least one hepcidin analog, dimer, or composition of the present invention.

[0025] In further embodiments, the present invention includes a method of treating a disorder of iron metabolism in a subject in need thereof, the method comprising providing the subject with an effective amount of a hepcidin analog or pharmaceutical composition of the present invention. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal, or topical administration. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral or subcutaneous administration. In certain embodiments, the disorder of iron metabolism is iron overload. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject at most or about twice a day, at most or about once a day, at most or about once every two days, at most or about once a week, or at most or about once a month. In certain embodiments, the hepcidin analog is provided to the subject at a dose of about 1 mg to about 100 mg, or about 1 mg to about 5 mg.

[0026] In another embodiment, the present invention provides a device comprising a hepcidin analog or pharmaceutical composition of the present invention for delivering the hepcidin analog or dimer of the present invention to a subject, optionally orally or subcutaneously.

[0027] In yet another embodiment, the invention includes a kit comprising a hepcidin analog or pharmaceutical composition of the invention packaged with reagents, devices, or reference materials, or a combination thereof. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention generally relates to hepcidin analog peptides, as well as methods for making and using the same. In certain embodiments, the hepcidin analog exhibits one or more hepcidin activities. In certain embodiments, the present invention relates to hepcidin peptide analogs comprising one or more peptide subunits, which form a cyclized structure via an intramolecular bond, such as an intramolecular disulfide bond. In certain embodiments, the cyclized structure has increased potency and selectivity compared to non-cyclized hepcidin peptides and analogs thereof. In certain embodiments, the hepcidin analog peptides of the present invention exhibit an extended half-life when delivered orally compared to hepcidin or conventional hepcidin analogs. In an embodiment of the present invention, for example, the following items are provided: (Item 1) Formula Ia: R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ia) or a pharmaceutically acceptable salt or solvate thereof, comprising a peptide according to During the ceremony, R 1 is hydrogen, C1-C6 alkyl, C6-C 12 Aryl, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C20 is cycloalkanoyl, R 2 is NH, substituted amino, OH, or substituted hydroxy; X1 is absent, Asp, isoAsp, Asp(OMe), Glu, Glu-OMe, bhGlu, bGlu, substituted Glu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X2 is Ala, Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, Gly, N-substituted Gly, His, or substituted His; X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X6 is absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys or Pen; X7 is absent, Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys or 123 triazole; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent, Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys or (D)Lys; and Each of X12 to X14 is absent or independently represents any amino acid, However, subject to the following conditions: i) the peptide may be further conjugated with any amino acid; ii) any of the amino acids of the peptide may be the corresponding (D)-amino acid or may be N-substituted; and iii) the peptide is a linear peptide or a cyclized lactam; and wherein Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, and Orn is ornithine. , Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, substituted Phe is phenylalanine in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; substituted bhPhe is b-homophenylalanine in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; Substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; Substituted bhTrp is N-methyl-Lb-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid, Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid, 123 Triazole is [ka] and Dab is [ka] or a pharmaceutically acceptable salt or solvate thereof. (Item 2) Formula Ib: R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ib) or a pharmaceutically acceptable salt or solvate thereof, comprising a peptide according to During the ceremony, R 1 is hydrogen, C1-C6 alkyl, C6-C 12 Aryl, C6-C 12 Aryl-C1-C6 alkyl, C1-C20 Alkanoyl, or C1-C 20 is cycloalkanoyl, R 2 is NH, substituted amino, OH, or substituted hydroxy; X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, Glu-OMe, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2; X2 is Ala, Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, Gly, N-substituted Gly, His, or substituted His; X4 is Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), or 2-pyrrolidinebutanoic acid (Pba); X6 is absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys or Pen; X7 is absent, Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent, Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys or (D)Lys; and Each of X12 to X14 is absent or independently represents any amino acid, However, subject to the following conditions: i) the peptide may be further conjugated with any amino acid; ii) any of the amino acids of the peptide may be the corresponding (D)-amino acid or may be N-substituted; and wherein Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, and Orn is orinithi ne), Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; substituted bhPhe is b-homophenylalanine in which the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; Substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; Substituted bhTrp is N-methyl-Lb-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; A hepcidin analogue, or a pharmaceutically acceptable salt or solvate thereof, wherein Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid. (Item 3) X1 is Asp, Glu, (D)Asp, Tet1, or Tet2; X2 is Thr or Ser, X3 is His or a substituted His, X7 is absent, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ile, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent or is Ala, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys, and 2. The hepcidin analog according to item 1, or a pharmaceutically acceptable salt or solvate thereof, wherein X11 is absent or is Pro, bhPhe, Lys, substituted Lys, or (D)Lys. (Item 4) X1 is Glu, Dab, Dap, Orn, Lys, or Tet1; X2 is Thr, X3 is His or 1MeHis, X4 is Dpa, The X5 is a Pro, X6 is absent, Ala, Glu, or substituted Lys; X7 is absent, Ile, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ile, Glu, Asp, 123 triazole, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent or is bhPhe; X10 is absent or is Ala, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys, and 2. The hepcidin analog according to item 1, or a pharmaceutically acceptable salt or solvate thereof, wherein X11 is absent or is Pro, bhPhe, Lys, substituted Lys, or (D)Lys. (Item 5) 5. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 4, wherein X1 is Glu. (Item 6) 5. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 4, wherein X2 is Thr. (Item 7) 5. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 4, wherein X4 is Dpa. (Item 8) 5. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 4, wherein X5 is Pro. (Item 9) The peptide has the formula II: R 1 -Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (II) In accordance with the formula, R 1 , R 2 9. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 8, wherein X3, X6 to X14 are the same as those in item 1 or item 2. (Item 10) 10. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 9, wherein X9 is absent, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 11) 10. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 9, wherein X9 is absent. (Item 12) 10. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 9, wherein X9 is bhPhe. (Item 13) The peptide has the formula III: R 1 -Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (III) In accordance with the formula, R 1 , R 2 13. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 12, wherein X, X3, X6 to X8, and X10 to X14 are the same as those in item 1 or item 2. (Item 14) 14. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 13, wherein X6 is Ala, Lys, or substituted Lys. (Item 15) 14. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 13, wherein X6 is Ala. (Item 16) The peptide has the formula IV: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-X7-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (IV) In accordance with the formula, R 1 , R 2 16. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 15, wherein X1, X2, X3, X7 to X8, and X10 to X14 are the same as those in item 1 or item 2. (Item 17) 17. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 16, wherein X7 is absent, Ile, Lys, or substituted Lys. (Item 18) 17. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 16, wherein X7 is absent. (Item 19) 17. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 16, wherein X7 is He. (Item 20) The peptide has the formula V: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (V) In accordance with the formula, R 1 , R 219. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 18, wherein X1, X2, X3, X8, and X10 to X14 are the same as those in item 1 or item 2. (Item 21) 21. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 20, wherein X8 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 22) 21. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 20, wherein X8 is (D)Lys or substituted (D)Lys. (Item 23) 21. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 20, wherein X8 is Lys, (D)Lys, Lys(Ac), or (D)Lys(Ac). (Item 24) The peptide has the formula VIa, VIb or VIc: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIa); R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIb); or R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIc); In accordance with the formula, R 1 , R 2 24. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 23, wherein X1, X2, and X3 are the same as those in item 1 or item 2. (Item 25) 25. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 24, wherein X8 is a conjugated amino acid. (Item 26) 25. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 24, wherein X8 is a conjugated Lys or (D)Lys. (Item 27) 25. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof of any one of items 1 to 24, wherein X8 is Lys(L1Z) or (D)Lys(L1Z), L1 is a linker, and Z is a half-life extending moiety. (Item 28) 25. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 24, wherein X3 is His. (Item 29) The peptide has the formula VIIa, VIIb or VIIc: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIa); R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIb); or R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIc); In accordance with the formula, R 1 , R 2 and X10 to X14 are the same as those in item 1 or 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 30) 30. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 29, wherein X3 is (1-Me)His. (Item 31) The peptide has the formula VIIIa or VIIIb: R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIa); or R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIb) In accordance with the formula, R 1 , R 2 and X10 to X14 are the same as those in item 1 or item 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 32) 32. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 31, wherein X10 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 33) 32. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 31, wherein X10 is (D)Lys or substituted (D)Lys. (Item 34) The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of Items 1 to 31, wherein X10 is (D)Lys or (D)Lys(Ac). (Item 35) 32. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 31, wherein X10 is Lys(Ahx_Palm). (Item 36) 36. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 35, wherein X10 is a conjugated amino acid. (Item 37) 36. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 35, wherein X10 is a conjugated Lys or (D)Lys. (Item 38) 36. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof of any one of items 1 to 35, wherein X10 is Lys(L1Z) or (D)Lys(L1Z), L1 is a linker, and Z is a half-life extending moiety. (Item 39) 39. The hepcidin analog of either item 30 or 38, wherein L1 is a single bond. (Item 40) 37. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to either of items 30 or 36, wherein L1 is iso-Glu. (Item 41) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein L1 is Ahx. (Item 42) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein L1 is iso-Glu-Ahx. (Item 43) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein L1 is PEG. (Item 44) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein L1 is PEG-Ahx. (Item 45) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein L1 is iso-Glu-PEG-Ahx. (Item 46) 42. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 41, wherein PEG is -[C(O)-CH-(Peg)N(H)]- or -[C(O)-CH-CH-(Peg)N(H)]-, Peg is -OCHCH-, m is 1, 2, or 3, and n is an integer between 1 and 100, or 10K, 20K, or 30K. (Item 47) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein m is 1. (Item 48) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein m is 2. (Item 49) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein n is 2. (Item 50) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein n is 4. (Item 51) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein n is 8. (Item 52) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein n is 11. (Item 53) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein n is 12. (Item 54) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein n is 20K. (Item 55) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 1Peg2, and 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-. (Item 56) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 2Peg2, and 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-. (Item 57) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 1Peg2-1Peg2, and each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-. (Item 58) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 1Peg2-1Peg2, and 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-. (Item 59) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 2Peg4, and 2Peg4 is —C(O)—CH—CH—(Peg)—N(H)— or —[C(O)—CH—CH—(OCHCH)—NH]—. (Item 60) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 1Peg8, and 1Peg8 is —C(O)—CH—(Peg)—N(H)— or —[C(O)—CH—(OCHCH)—NH]—. (Item 61) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 2Peg8, and 2Peg8 is —C(O)—CH—CH—(Peg)—N(H)— or —[C(O)—CH—CH—(OCHCH)—NH]—. (Item 62) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-. (Item 63) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 2Peg11, and 2Peg11 is -C(O)-CH2-CH2-(Peg)11-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)11-NH]-. (Item 64) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein PEG is 2Peg11' or 2Peg12, and 2Peg11' or 2Peg12 is -C(O)-CH2-CH2-(Peg)12-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)12-NH]-. (Item 65) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein when PEG is bound to Lys, -C(O)- of PEG is bound to Ne of Lys. (Item 66) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein when PEG is bound to isoGlu, —N(H)— of PEG is bound to —C(O)— of isoGlu. (Item 67) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein, when PEG is bound to Ahx, —N(H)— of PEG is bound to —C(O)— of Ahx. (Item 68) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein, when PEG is bound to Palm, -N(H)- of PEG is bound to -C(O)- of Palm. (Item 69) 39. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 38, wherein Z is Palm. (Item 70) The peptide has formula IXa or IXb: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); or R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXb) In accordance with the formula, R 1 , R 2 and X11 to X14 are the same as those in item 1 or item 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 71) The peptide has the formula Xa or Xb: R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (Xa); or R 1-Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (Xb) In accordance with the formula, R 1 , R 2 and X11 to X14 are the same as those in item 1 or item 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 72) 72. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 71, wherein the peptide is a linear peptide. (Item 73) 72. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 71, wherein the peptide is a lactam. (Item 74) 72. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 71, wherein the peptide is a lactam and any free -NH2 is cyclized with any free -C(O)2H. (Item 75) The peptide has the formula XXI: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (XXI), In accordance with the formula, R 1 , R 2 , and X10 to X14 are the same as item 1 or item 2, X6 is absent, Ala, or a substituted Lys; X7 is absent, Ile, a substituted Lys, or a substituted (D)Lys; and X9 is absent or bhPhe; and X8 is Lys(L1Z) or (D)Lys(L1Z), wherein L1 is a linker and Z is a half-life extending moiety. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 1. (Item 76) 76. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 75, wherein X8 is Lys(L1Z). (Item 77) 76. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 75, wherein X8 is (D)Lys(L1Z). (Item 78) The peptide has the formula XXII: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXII), In accordance with the formula, R 1 , R 2 , and X10 to X14 are the same as item 1 or item 2, 2. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 1, wherein X6 is absent, Ala, or substituted Lys; X7 is absent, Ile, substituted Lys, or substituted (D)Lys; and X9 is absent or bhPhe. (Item 79) 79. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 75 to 78, wherein X6 is absent. (Item 80) 79. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 75 to 78, wherein X6 is a substituted Lys. (Item 81) 79. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 75 to 78, wherein X6 is Ala. (Item 82) The peptide has the formula XXIIIa or XXIIIb: R 1 -Glu-Thr-His-[Dpa]-Pro-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIIIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIIIb), In accordance with the formula, R 1 , R 2 , and X10 to X14 are the same as item 1 or item 2, 2. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 1, wherein X7 is absent, Ile, substituted Lys, or substituted (D)Lys, and X9 is absent or bhPhe. (Item 83) 83. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 82, wherein X7 is absent. (Item 84) 83. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 82, wherein X7 is a substituted (D)Lys. (Item 85) 83. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 82, wherein X7 is a substituted Lys. (Item 86) 83. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 82, wherein X7 is He. (Item 87) The peptide has the formula XXIVa, XXIVb, XXIVc or XXIVd: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVb), R 1-Glu-Thr-His-[Dpa]-Pro- [Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVd), In accordance with the formula, R 1 , R 2 , and X10 to X14 are the same as item 1 or item 2, 2. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 1, wherein X9 is absent or bhPhe. (Item 88) 88. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 87, wherein X9 is absent. (Item 89) The peptide has the formula XXVa, XXVb, XXVc or XXVd: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVb), R 1 -Glu-Thr-His-[Dpa]-Pro- [Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVd), In accordance with the formula, R 1 , R 2and X10 to X14 are the same as those in item 1 or 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 90) 88. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 87, wherein X9 is bhPhe. (Item 91) The peptide is XXVIa, XXVIb, XXVIc or XXVId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIb), R 1 -Glu-Thr-His-[Dpa]-Pro- [Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVId), In accordance with the formula, R 1 , R 2 and X10 to X14 are the same as those in item 1 or 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 92) 92. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 91, wherein X10 is Lys or (D)Lys. (Item 93) 92. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 91, wherein X10 is (D)Lys. (Item 94) The peptide is XXVIIa, XXVIIb, XXVIIc or XXVIId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-[(D)Lys]-X11-X12-X13-X14-R 2 (XXVIIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIIb), R 1 -Glu-Thr-His-[Dpa]-Pro- [Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIIc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIId), In accordance with the formula, R 1 , R 2 and X11 to X14 are the same as those in item 1 or item 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 95) 92. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 91, wherein X10 is absent. (Item 96) The peptide is XXVIIIa, XXVIIIb, XXVIIIc or XXVIIId: R 1-Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIb), R 1 -Glu-Thr-His-[Dpa]-Pro- [Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIId), In accordance with the formula, R 1 , R 2 and X11 to X14 are the same as those in item 1 or item 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 97) 97. The hepcidin analogue of any one of items 78 to 96, wherein L1 is a single bond. (Item 98) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1 is iso-Glu. (Item 99) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1 is Ahx. (Item 100) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1 is iso-Glu-Ahx. (Item 101) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1 is PEG. (Item 102) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1 is PEG-Ahx. (Item 103) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1 is iso-Glu-PEG-Ahx. (Item 104) 42. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to item 41, wherein PEG is -[C(O)-CH-(Peg)N(H)]- or -[C(O)-CH-CH-(Peg)N(H)]-, Peg is -OCHCH-, m is 1, 2, or 3, and n is an integer between 1 and 100, or 10K, 20K, or 30K. (Item 105) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein m is 1. (Item 106) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein m is 2. (Item 107) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein n is 2. (Item 108) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein n is 4. (Item 109) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein n is 8. (Item 110) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein n is 11. (Item 111) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein n is 12. (Item 112) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein n is 20K. (Item 113) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 1Peg2, and 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-. (Item 114) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 2Peg2, and 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-. (Item 115) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 1Peg2-1Peg2, and each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-. (Item 116) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 1Peg2-1Peg2, and 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-. (Item 117) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 2Peg4, and 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-. (Item 118) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 1Peg8, and 1Peg8 is —C(O)—CH—(Peg)—N(H)— or —[C(O)—CH—(OCHCH)—NH]—. (Item 119) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 2Peg8, and 2Peg8 is —C(O)—CH—CH—(Peg)—N(H)— or —[C(O)—CH—CH—(OCHCH)—NH]—. (Item 120) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-. (Item 121) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein PEG is 2Peg11, and 2Peg11 is -C(O)-CH2-CH2-(Peg)11-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)11-NH]-. (Item 122) The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of Items 78 to 96, wherein PEG is 2Peg11' or 2Peg12, and 2Peg11' or 2Peg12 is -C(O)-CH2-CH2-(Peg)12-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)12-NH]-. (Item 123) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein when PEG is bound to Lys, -C(O)- of PEG is bound to Ne of Lys. (Item 124) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein when PEG is bound to isoGlu, -N(H)- of PEG is bound to -C(O)- of isoGlu. (Item 125) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein, when PEG is bound to Ahx, -N(H)- of PEG is bound to -C(O)- of Ahx. (Item 126) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein, when PEG is bound to Palm, -N(H)- of PEG is bound to -C(O)- of Palm. (Item 127) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein Z is Palm. (Item 128) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1Z is -Ahx_Palm. (Item 129) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1Z is -bAla_Palm. (Item 130) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1Z is -IsoGlu_Palm. (Item 131) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1Z is PEG12_Palm. (Item 132) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein L1Z is -1PEG2_1PEG2_Ahx_C18_diacid. (Item 133) 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 96, wherein X11, X12, X13, and X14 are each absent. (Item 134) The peptide has the formula XXI: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXI), In accordance with the formula, R 1 , R 2 , and X10 to X14 are the same as item 1 or item 2, 79. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 78 to 78, wherein X6 is absent or a substituted Lys, X7 is absent or a substituted Lys, and X9 is absent or a bhPhe. (Item 135) -L1Z are independent of each other. PEG11_OMe; PEG12_C18 acid; 1PEG2_1PEG2_Ahx_Palm; 1PEG2_Ahx_Palm; Ado_Palm; Ahx_Palm; Ahx_PEG20K; PEG12_Ahx_IsoGlu_Behenic; PEG12_Ahx_Palm; PEG12_DEKHKS_Palm; PEG12_IsoGlu_C18 acid; PEG12_Ahx_C18 acid; PEG12_IsoGlu_Palm; PEG12_KKK_Palm; PEG12_KKKG_Palm; PEG12_DEKHKS_Palm; PEG12_Palm; PEG12_PEG12_Palm; PEG20K; PEG4_Ahx_Palm; PEG4_Palm; PEG8_Ahx_Palm; or IsoGlu_Palm; -1PEG2_1PEG2_Dap_C18_Diacid; -1PEG2_1PEG2_IsoGlu_C10_Diacid; -1PEG2_1PEG2_IsoGlu_C12_diacid; -1PEG2_1PEG2_IsoGlu_C14_diacid; -1PEG2_1PEG2_IsoGlu_C16_diacid; -1PEG2_1PEG2_IsoGlu_C18_diacid; -1PEG2_1PEG2_IsoGlu_C22_diacid; -1PEG2_1PEG2_Ahx_C18_diacid; -1PEG2_1PEG2_C18_dioic acid; -1PEG8_IsoGlu_C18_Diacid; -IsoGlu_C18_diacid; -PEG12_Ahx_C18_diacid; -PEG12_C16_dioic acid; -PEG12_C18_dioic acid; -1PEG2_1PEG2_1PEG2_C18_dioic acid; -1PEG2_1PEG2_1PEG2_IsoGlu_C18_diacid; -PEG12_IsoGlu_C18_Diacid; -PEG4_IsoGlu_C18_Diacid; or PEG4 PEG4 IsoGlu C18 diacid, During the ceremony, PEG11_OMe is -[C(O)-CH2-CH2-(OCH2CH2) 11 -OMe], 1PEG2 is -C(O)-CH2-(OCH2CH2)2-NH-, PEG4 is -C(O)-CH2-CH2-(OCH2CH2)4-NH-, PEG8 is —[C(O)—CH—CH—(OCHCH)—NH—; 1PEG8 is -[C(O)-CH2-(OCH2CH2)8-NH-, PEG12 is -[C(O)-CH2-CH2-(OCH2CH2) 12 -NH-, Ado is -[C(O)-(CH2) 11 -NH]-, Cn acid is -C(O)(CH2) n-2 -CH3, and C18 acids are -C(O)-(CH2) 16 -Me, Palm is -C(O)-(CH2) 14 -Me, isoGlu is isoglutamic acid isoGlu_Palm is [ka] and Ahx is -[C(O)-(CH2)5-NH]-, Cn_ diacids are -C(O)-(CH2) n-2 135. The hepcidin analogue according to any one of items 1 to 134, or a pharmaceutically acceptable salt or solvate thereof, wherein n is —COOH, wherein n is 10, 12, 14, 16, 18, or 22. (Item 136) X8 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid), Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 137) X8 or X10 is (D) Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid), (D) Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 138) X8 or X10 is Lys(1PEG8_IsoGlu_C n _ diacid), Lys(1PEG8_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 139) X8 or X10 is (D)Lys(1PEG8_IsoGlu_C n _ diacid), (D) Lys(1PEG8_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 140) X8 or X10 is Lys(1PEG2_1PEG2_Dap_C n _ diacid), Lys(1PEG2_1PEG2_Dap_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 141) X8 or X10 is Lys(IsoGlu_C n _ diacid), Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 142) X8 or X10 is (D)Lys(IsoGlu_C n _ diacid), (D) Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 143) X8 or X10 is Lys(PEG12_IsoGlu_C n _ diacid), Lys(PEG12_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 144) X8 or X10 is (D)Lys(PEG12_IsoGlu_C n_ diacid), (D) Lys(PEG12_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 145) X8 or X10 is Lys(PEG4_IsoGlu_C n _ diacid), Lys(PEG4_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 146) X8 or X10 is (D)Lys(PEG4_IsoGlu_C n _ diacid), (D) Lys(PEG4_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 147) X8 or X10 is Lys(PEG4_PEG4_IsoGlu_C n _ diacid), Lys(PEG4_PEG4_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 148) X8 or X10 is (D) Lys(PEG4_PEG4_IsoGlu_C n _ diacid), (D) Lys(PEG4_PEG4_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 149) X8 or X10 is Lys(IsoGlu_C n _ diacid), Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 150) X8 or X10 is (D)Lys(IsoGlu_C n _ diacid), (D) Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 151) X8 or X10 is Lys(PEG12_Ahx_C n_ diacid), Lys(PEG12_Ahx_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18. (Item 152) X8 or X10 is Lys(PEG12_Ahx_C n _ diacid), Lys(PEG12_Ahx_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 153) X8 or X10 is (D)Lys(PEG12_Ahx_C n _ diacid), (D) Lys(PEG12_Ahx_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 154) X8 or X10 is Lys(PEG12_C n _ diacid), Lys(PEG12_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 155) X8 or X10 is (D)Lys(PEG12_C n _ diacid), (D) Lys(PEG12_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18. (Item 156) 135. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 134, wherein X8 or X10 is 123 triazole. (Item 157) 157. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 156, wherein X11 is absent, Ala, (D)Lys, or substituted Lys. (Item 158) 157. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 156, wherein X11 is absent. (Item 159) 157. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 156, wherein X11 is Ala. (Item 160) 157. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 156, wherein X11 is (D)Lys. (Item 161) 157. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 156, wherein X11 is Lys(Ahx_Palm). (Item 162) 162. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 161, wherein X12 is absent or Ala. (Item 163) 162. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 161, wherein X12 is absent. (Item 164) 162. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 161, wherein X12 is Ala. (Item 165) 165. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 164, wherein X13 is absent. (Item 166) 166. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 165, wherein X14 is absent. (Item 167) R 2 167. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 166, wherein is NH2. (Item 168) R 2 167. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 166, wherein is a substituted amino. (Item 169) R 2 167. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 166, wherein is N-alkylamino. (Item 170) R 2 is N-alkylamino, wherein the alkyl is further substituted or unsubstituted. (Item 171) R 2167. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 166, wherein is N-alkylamino, wherein the alkyl is further substituted aryl or heteroaryl. (Item 172) R 2 is alkylamino, wherein alkyl is unsubstituted or substituted with aryl, and alkyl is ethyl, propyl, butyl, or pentyl. (Item 173) R 2 is alkylamino, wherein alkyl is unsubstituted or substituted with phenyl, and alkyl is ethyl, propyl, butyl, or pentyl. (Item 174) R 2 167. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 166, wherein is OH. (Item 175) R 1 But C1-C 20 175. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 174, which is alkanoyl. (Item 176) R 1 175. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 174, wherein is IVA or isovaleric acid. (Item 177) 177. The hepcidin analogue or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 176, wherein the peptide is a linear peptide. (Item 178) 177. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 176, wherein the peptide is a lactam. (Item 179) 177. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 176, wherein the peptide is a lactam and any free -NH2 is cyclized with any free -C(O)2H. (Item 180) A hepcidin analog, or a pharmaceutically acceptable salt or solvate thereof, comprising or consisting of a peptide, wherein the peptide is any one of the peptides listed in Tables 6A-C. (Item 181) 1. A hepcidin analogue comprising or consisting of a peptide, or a pharmaceutically acceptable salt or solvate thereof, wherein said peptide is: ID number 321 [ka] ; ID number 319 [ka] ; ID number 322 [ka] ; ID number 318 [ka] ; ID number 320 [ka] ; ID number 56 [ka] ; ID number 286 [ka] ; ID number 58 [ka] ; ID number 287 [ka] ; ID number 156 [ka] ,or ID number 292 [ka] ; or a pharmaceutically acceptable salt or solvate thereof. (Item 182) 182. A polynucleotide encoding a peptide present in the hepcidin analogue or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 181. (Item 183) Item 183. A vector comprising the polynucleotide of Item 182. (Item 184) A pharmaceutical composition comprising the hepcidin analogue according to any one of items 1 to 181 or a pharmaceutically acceptable salt or solvate thereof, the polynucleotide according to item 182, or the vector according to item 86, and a pharmaceutically acceptable carrier, excipient, or vehicle. (Item 185) 184. A method for binding to or inducing internalization and degradation of ferroportin, comprising contacting the ferroportin with at least one hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of items 1 to 181, or the pharmaceutical composition according to item 184. (Item 186) 184. A method for treating a disease of iron metabolism in a subject in need thereof, comprising providing to the subject an effective amount of a hepcidin analog according to any one of items 1 to 181 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to item 184. (Item 187) 183. A method for treating a disease or disorder associated with dysregulation of hepcidin signaling in a subject in need thereof, comprising providing to the subject an effective amount of a hepcidin analogue according to any one of items 1 to 181 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to item 184. (Item 188) 188. The method of claim 186 or 187, wherein the pharmaceutical composition is provided to the subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal, or topical route of administration. (Item 189) 189. The method of claim 188, wherein the pharmaceutical composition is provided to the subject by oral or subcutaneous administration. (Item 190) 189. The method according to any one of items 186 to 189, wherein the disease or disorder is a disease of iron metabolism. (Item 191) Item 191. The method of item 190, wherein the disease of iron metabolism is iron overload. (Item 192) 189. The method of any one of items 186 to 189, wherein the disease or disorder is hemochromatosis, thalassemia, or polycythemia vera. (Item 193) 193. The method of any one of items 186 to 192, wherein the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition is provided to the subject at most twice a day, at most once a day, at most every two days, at most once a week, or at most once a month. (Item 194) 193. The method of any one of items 186 to 192, wherein the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition is provided to the subject in a dose of about 1 mg to about 100 mg. (Item 195) 185. A device comprising the pharmaceutical composition of item 184 for delivering the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof to a subject, optionally orally or subcutaneously. (Item 196) 185. A kit comprising the pharmaceutical composition of item 184 packaged together with a reagent, device, or instruction manual, or a combination thereof.

[0029] Definitions and Nomenclature Unless otherwise defined herein, scientific and technical terms used herein shall have the same meaning as commonly understood by those skilled in the art. Generally, the nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein chemistry and nucleic acid chemistry described herein are known and commonly used in the art.

[0030] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0031] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a specified integer (or component) or group of integers (components) but not the exclusion of any other integer (or component) or group of integers (or components).

[0032] The singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0033] The term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0034] The terms "patient," "subject," and "individual" may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as, for example, humans, primates, livestock animals (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats). The term "mammal" refers to any mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, etc.

[0035] As used herein, the term "peptide" refers broadly to a sequence of two or more amino acids that are linked together by peptide bonds. It is understood that the term does not connote a specific length of an amino acid polymer, nor is it intended to suggest or distinguish whether the polypeptide is produced using recombinant technology, chemical synthesis, enzymatic synthesis, or is naturally occurring.

[0036] As used herein, the term "peptide analog" or "hepcidin analog" broadly refers to peptide monomers and peptide dimers that share one or more structural features and / or functional activities with hepcidin or a functional region thereof. In certain embodiments, peptide analogs include peptides that share substantial amino acid sequence identity with hepcidin, including peptides that contain one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence of wild-type hepcidin, such as human hepcidin. In certain embodiments, peptide analogs contain one or more additional modifications, such as, for example, conjugation to another compound. The term "peptide analog" encompasses any peptide monomer or peptide dimer of the present invention. In certain examples, "peptide analog" as used herein may alternatively be referred to as a "hepcidin analog," a "hepcidin peptide analog," or a "hepcidin analog peptide."

[0037] As used herein, terms such as "sequence identity," "percent identity," "percent homology," or, for example, "a sequence that is 50% identical to" refer to the degree to which sequences are identical at each nucleotide or amino acid level over a comparison window. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0038] Calculation of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0039] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences, and the length of each gap.

[0040] Comparison of sequences and determination of percent identity between two sequences may be performed using a mathematical algorithm. In some embodiments, percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch, (1970, J. Mol. Biol. 48:444-453), incorporated into the GAP program of the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package, using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. Another exemplary setting of parameters includes a scoring matrix of Blossum 62, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences may be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4:11-17), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0041] The peptide sequences described herein may be used as "query sequences" to search public databases, for example, to identify other family members or to identify related sequences. Such searches may be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol. 215:403-10). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches may be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be used as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0042] As used herein, the term "conservative substitution" refers to the replacement of one or more amino acids with another, biologically similar residue. Examples include the replacement of amino acid residues with similar properties, such as small, acidic, polar, basic, hydrophobic, and aromatic amino acids. See, for example, the table below. In some embodiments of the invention, one or more Met residues are replaced with norleucine (Nle). Nle is a biological equivalent of Met, but, in contrast to Met, is not readily oxidized. In some embodiments, one or more Trp residues are replaced with Phe, or one or more Phe residues are replaced with Trp. Meanwhile, in some embodiments, one or more Pro residues are replaced with Npc, or one or more Npc residues are replaced with Pro. Another example of a conservative substitution using a residue not normally present in endogenous mammalian peptides or proteins is the conservative substitution of Arg or Lys with, for example, ornithine, canavanine, aminoethylcysteine, or another basic amino acid. In some embodiments, another conservative substitution is the substitution of one or more Pro residues with bhPro or Leu or D-Npc (isonipecotic acid). For details regarding phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et al. Science 247, 1306-1310, 1990. In the following scheme, conservative amino acid substitutions are grouped by physicochemical properties: I: neutral, hydrophilic; II: acid and amide; III: basic; IV: hydrophobic; V: aromatic, bulky amino acids. [Table 1]

[0043] In the following scheme, conservative substitutions of amino acids are grouped by physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. [Table 2]

[0044] As used herein, the term "amino acid" or "any amino acid" refers to any amino acid, including natural amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and non-naturally occurring amino acids. This includes both D-amino acids and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that are incorporated into peptide chains to form the building blocks of countless proteins. These are primarily L-stereoisomers, although some D-amino acids occur in bacterial envelopes and some antibiotics. The 20 "standard" natural amino acids are listed in the table above. "Non-standard" natural amino acids are pyrrolysine (present in methanogens and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Non-natural" or "unnatural" amino acids are non-proteinogenic amino acids (i.e., not naturally encoded or present in the genetic code) that do not occur in nature or are chemically synthesized. There are over 140 known naturally occurring amino acids, with thousands of possible combinations. Examples of "unnatural" amino acids include β-amino acids (β 3 and β 2 ), homo-amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not found on amino acids in nature.

[0045] As will be apparent to those skilled in the art, the peptide sequences disclosed herein are presented proceeding from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C-terminus of the peptide. Some sequences disclosed herein incorporate a "Hy-" moiety at the amino terminus (N-terminus) of the sequence and either an "-OH" or "-NH2" moiety at the carboxy terminus (C-terminus) of the sequence. In such cases, and unless otherwise indicated, the "Hy-" moiety at the N-terminus of the subject sequence represents a hydrogen atom, which corresponds to the presence of a free primary or secondary amino group at the N-terminus, and the "-OH" or "-NH2" moiety at the C-terminus of the sequence represents a hydroxy or amino group, which corresponds to the presence of an amide (CONH2) group at the C-terminus. In each of the sequences of the present invention, the C-terminal "-OH" moiety may be replaced with a C-terminal "-NH2" moiety, or vice versa. It is further understood that the amino- or carboxy-terminal moiety can be linked, such as covalently, particularly when the amino- or carboxy-terminal moiety is attached to a linker, or another chemical moiety, such as a PEG moiety.

[0046] As used herein, the term "NH" refers to the free amino group present at the amino terminus of a polypeptide. As used herein, the term "OH" refers to the free carboxy group present at the carboxy terminus of a peptide. Additionally, as used herein, the term "Ac" refers to acetyl protection via acylation of the C-terminus or N-terminus of a polypeptide.

[0047] As used herein, the term "carboxy" refers to -CO2H.

[0048] For the most part, the names of natural and unnatural aminoacyl residues used herein follow the naming conventions proposed by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature, as set forth in "Nomenclature of α-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and the appended claims differ from these proposals, they will be made clear to the reader. Some abbreviations useful in describing this invention are defined below in Tables 1A and 1B. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 4]

[0049] Throughout this specification, unless the naturally occurring amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they are designated 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 less common or unnatural amino acids, the frequently adopted three-letter or four-letter codes for those residues are adopted, unless they are referred to by their full names (e.g., sarcosine, ornithine, etc.). Examples include Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Daba (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), pGlu (pyroglutamic acid), Gaba (γ-aminobutanoic acid), β-Pro (pyrrolidine-3-carboxylic acid), 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (4-aminobutyric acid), bhPro (β-homo-proline), bhPhe (β-homo-L-phenylalanine), bhAsp (β-homo-aspartic acid), Dpa (β,β-diphenylalanine), Ida (iminodiacetic acid), hCys (homocysteine), and bhDpa (β-homo-β,β-diphenylalanine).

[0050] Furthermore, R 1 can be substituted with isovaleric acid or an equivalent in all sequences. In some embodiments, when a peptide of the present invention is conjugated to an acidic compound, such as isovaleric acid, isobutyric acid, valeric acid, etc., the presence of such a conjugate is referred to in its acidic form. Thus, for example and without limitation, instead of referring to a conjugate of isovaleric acid to a peptide as isovaleroyl, in some embodiments, the present application may refer to such a conjugate as isovaleric acid.

[0051] For each of the hepcidin analog formulas provided herein, it is understood that bonds may be indicated by a "-" or may be implied based on the formula and components. For example, "B7(L1Z)" is understood to include a bond between B7 and L1 if L1 is present, and a bond between B7 and Z if L1 is absent. Similarly, "B5(L1Z)" is understood to include a bond between B5 and L1 if L1 is present, and a bond between B5 and Z if L1 is absent. Furthermore, it is understood that when both L1 and Z are present, a bond also exists between L1 and Z. Thus, for example, the definitions of certain substituents, such as B7, L1, and J, may include a "-" before and / or after the defined substituent. However, it is understood that in each case, the substituent is connected to the other substituent via a single bond. For example, if "J" is defined as Lys, D-Lys, Arg, Pro, -Pro-Arg-, etc., it is understood that J is connected to Xaa2 and Y1 via a single bond. Thus, a substituent definition may or may not include a "-" but is understood to be attached to the adjacent substituent in either case.

[0052] As used herein, the term "L-amino acid" refers to the "L" isomeric form of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomeric form of a peptide. In certain embodiments, the amino acid residues described herein are in the L-isomer form, but residues in the "D" isomeric form can be substituted with any L-amino acid residue, so long as the desired functionality of the peptide is retained.

[0053] Unless otherwise indicated, reference is made to the L-isomer forms of natural and unnatural amino acids of interest, which have chiral centers. Where appropriate, the D-isomer forms of amino acids are designated in the conventional manner by prefixing "D" before the conventional three-letter code (e.g., Dasp, (D)Asp, or D-Asp; Dphe, (D)Phe, or D-Phe).

[0054] As used herein, a "lower homolog of Lys" refers to an amino acid that has the structure of lysine but has one or more fewer carbon atoms in the side chain than lysine.

[0055] As used herein, a "higher homolog of Lys" refers to an amino acid that has the structure of lysine but has one or more additional carbon atoms in the side chain compared to lysine.

[0056] As used herein, the term "DRP" refers to a disulfide-rich peptide.

[0057] As used herein, the term "dimer" broadly refers to a peptide comprising two or more monomer subunits. A particular dimer comprises two DRPs. Dimers of the present invention include homodimers and heterodimers. The monomer subunits of a dimer may be linked at their C-terminus or N-terminus, or may be linked via internal amino acid residues. Each monomer subunit of a dimer may be linked via the same site, or each may be linked via a different site (e.g., C-terminus, N-terminus, or internal site).

[0058] The terms "isostere replacement" or "isostere substitution" are used interchangeably herein and refer to any amino acid or other analog moiety that has similar chemical and / or structural properties to the specified amino acid. In certain embodiments, an isostere replacement is a conservative substitution with a natural or unnatural amino acid.

[0059] As used herein, the term "cyclization" refers to a reaction in which one portion of a polypeptide molecule is linked to another portion of the polypeptide molecule to form a closed circle by forming a disulfide bridge or other similar bond.

[0060] As used herein, the term "subunit" refers to one of a pair of polypeptide monomers that are combined to form a dimeric peptide composition.

[0061] As used herein, the term "linker moiety" refers broadly to a chemical structure that can link or connect two peptide monomer subunits to form a dimer.

[0062] In the context of the present invention, the term "solvate" refers to a complex formed with a defined stoichiometry between a solute (e.g., a hepcidin analog according to the present invention or a pharmaceutically acceptable salt thereof) and a solvent. In this context, the solvent may be, for example, water, ethanol, or another pharmaceutically acceptable, typically small, organic substance, such as, but not limited to, acetic acid or lactic acid. When the solvent of interest is water, such a solvate is typically referred to as a hydrate.

[0063] As used herein, "diseases of iron metabolism" include diseases in which abnormal iron metabolism is a direct cause of the disease, or diseases that result from dysregulated blood levels of iron, or diseases in which dysregulated iron is the result of another disease, or diseases that can be treated by regulating iron levels. More specifically, diseases of iron metabolism according to the present disclosure include iron overload, iron deficiency, disorders of iron biodistribution, other disorders of iron metabolism, and other disorders that may be related to iron metabolism. Disorders of iron metabolism include hemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, sideroblastic anemia, porphyria, porphyria cutanea tarda, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital dyserythropoiesis anemia, hypochromic microcytic anemia, sickle cell disease, and polycythemia vera (primary Hepcidin overproduction is a rare condition that can occur in a variety of conditions, including: hepatitis, urinary tract infections (including urinary tract infections, urinary tract infections, and urinary tract infections), hepatitis, liver cirrhosis, hepatitis B, liver cirrhosis, hepatitis C, hepatitis B, hepatitis C, hepatitis C (hepatic and secondary), secondary erythrocytosis, e.g., chronic obstructive pulmonary disease (COPD), post-renal transplant, Chuvash, HIF and PHD mutations, and idiopathic myelodysplasia, pyruvate kinase deficiency, iron deficiency in obesity, other anemias, benign or malignant tumors that overproduce or induce the overproduction of hepcidin, hepcidin excess states, Friedreich's ataxia, Greisl's syndrome, Hallervorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer, hepatitis, liver cirrhosis, pica, chronic renal failure, insulin resistance, diabetes, atherosclerosis, neurodegenerative disorders, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0064] In some embodiments, the disease and disorder is associated with iron overload disorders, such as, for example, ferrohemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0065] In some embodiments, the hepcidin analogs of the present invention are used to treat diseases and disorders that are not usually identified as iron-related.For example, hepcidin is highly expressed in the pancreas of mice, suggesting that diabetes (type I or type II), insulin resistance, glucose intolerance, and other disorders can be improved by treating the underlying iron metabolic disorder.Ilyin, G. et al.(2003)FEBS Lett.542 22-26, which are incorporated herein by reference. Thus, the peptides of the present invention can be used to treat these diseases and conditions. One skilled in the art can readily determine whether a given disease can be treated with a peptide according to the present invention using methods known in the art, including the assays described in WO 2004092405, which is incorporated herein by reference. The assays monitor the levels and expression of hepcidin, hemojuvelin, or iron and are known in the art, such as those described in U.S. Pat. No. 7,534,764, which is incorporated herein by reference.

[0066] In certain embodiments of the invention, the disorder of iron metabolism is an iron overload disorder, which includes hereditary hemochromatosis, iron overload anemia, alcoholic liver disease, and chronic hepatitis C.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to a salt or zwitterionic form of a peptide or compound of the present invention that is soluble or dispersible in water or oil, suitable for treating disease without undue toxicity, irritation, or allergic reaction, commensurate with a reasonable benefit / risk ratio, and effective for its intended use. Salts can be prepared during the final isolation and purification of the compound, or can be prepared separately by reacting an amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, and mesitylenesulfonate. , methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Amino groups in the compounds of the present invention may also be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. The pharmaceutically acceptable salt may preferably be a salt selected from acid addition salts and base salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts.Examples of basic salts include salts in which the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, and substituted ammonium ions, such as ions of the N(R1)(R2)(R3)(R4) type, where R1, R2, R3, and R4 independently typically represent hydrogen, optionally substituted C1-6-alkyl, or optionally substituted C2-6-alkenyl. Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl, and 2-propyl groups. Examples of potentially relevant C2-6-alkenyl groups include ethenyl, 1-propenyl, and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its latest edition), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66:2 (1977). Also, a summary of suitable salts can be found in Handbook of See Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Other suitable base salts are formed from bases that form non-toxic salts. Representative examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemi-salts of acids and bases may also be formed, including, for example, hemisulfate and hemicalcium salts.

[0068] As used herein, the term "N(alpha) methylation" describes the methylation of the alpha amine of an amino acid, also commonly referred to as N-methylation.

[0069] As used herein, the terms "sym methylation" or "Arg-Me-sym" describe symmetric methylation of both nitrogens of the guanidine group of arginine, and "asym methylation" or "Arg-Me-asym" describe methylation of one nitrogen of the guanidine group of arginine.

[0070] As used herein, the term "acylating organic compound" refers to various compounds having a carboxylic acid functional group that are used to acylate the N-terminus of an amino acid subunit prior to forming a C-terminal dimer. Non-limiting examples of acylating organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3,3,3-trifluoropropionic acid, 3-fluoromethylbutyric acid, and tetrahydro-2H-pyran-4-carboxylic acid.

[0071] The term "alkyl" includes straight-chain or branched-chain, acyclic or cyclic saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; saturated branched-chain alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

[0072] As used herein, a "therapeutically effective amount" of a peptide agonist of the invention is intended to describe a sufficient amount of peptide agonist to treat a hepcidin-related disease, including, but not limited to, any of the diseases and disorders described herein (e.g., diseases of iron metabolism). In certain embodiments, a therapeutically effective amount achieves a desirable benefit / risk ratio applicable to any medical treatment.

[0073] Peptide analogues of hepcidin The present invention provides peptide analogs of hepcidin, which may be monomeric or dimeric (collectively "hepcidin analogs").

[0074] In some embodiments, the hepcidin analogs of the present invention bind to ferroportin, such as human ferroportin. In certain embodiments, the hepcidin analogs of the present invention specifically bind to human ferroportin. As used herein, "specifically bind" refers to the preferential interaction of a specific binding substance with a given ligand over other substances in a sample. For example, a specific binding substance that specifically binds to a given ligand binds to the given ligand in an amount or degree that is observed above any nonspecific interactions with other components in the sample under appropriate conditions. Suitable conditions are those that allow interaction between a given specific binding substance and a given ligand. These conditions include pH, temperature, concentration, solvent, incubation time, etc., and may vary depending on the specific binding substance and ligand pair, but can be easily determined by one of skill in the art. In some embodiments, the hepcidin analogs of the present invention bind to ferroportin with greater specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the invention exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1000%, or 10,000% greater ferroportin specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the invention exhibit at least about 5-fold, or at least about 10-fold, 20-fold, 50-fold, or 100-fold greater ferroportin specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0075] In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity. In some embodiments, the activity is an in vitro activity or an in vivo activity, such as an in vitro activity or an in vivo activity described herein. In some embodiments, the hepcidin analogs of the present invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99% of the activity exhibited by a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0076] In some embodiments, the hepcidin analogs of the invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99% of the ferroportin binding ability exhibited by the hepcidin reference compound. In some embodiments, the hepcidin analogs of the invention exhibit a lower EC50 or IC for binding to ferroportin (e.g., human ferroportin) compared to the hepcidin reference compound. 50 In some embodiments, the hepcidin analogs of the invention have an EC50 or IC that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than a hepcidin reference compound in a ferroportin competitive binding assay. 50 It has.

[0077] In certain embodiments, the hepcidin analogue of the present invention exhibits increased hepcidin activity compared with hepcidin reference compound.In some embodiments, activity is in vitro activity or in vivo activity, for example the in vitro activity or in vivo activity described herein.In certain embodiments, the hepcidin analogue of the present invention exhibits 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 times higher hepcidin activity than hepcidin reference compound. In certain embodiments, hepcidin analogs of the invention exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 700%, or greater than 1000% greater activity than a hepcidin reference compound.

[0078] In some embodiments, the peptide analogs of the invention exhibit in vitro activity with respect to inducing degradation of human ferroportin protein that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 700%, or greater than 1000% greater than the activity of a hepcidin reference compound, wherein the activity is measured according to the methods described herein.

[0079] In some embodiments, the peptides or peptide dimers of the invention exhibit in vivo activity with respect to inducing a reduction in free plasma iron that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 700%, or greater than 1000% greater than the activity of a hepcidin reference compound, where the activity is measured according to the methods described herein.

[0080] In some embodiments, the activity is an in vitro activity or an in vivo activity, such as an in vitro activity or an in vivo activity described herein. In certain embodiments, the hepcidin analogs of the invention exhibit an activity that is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times greater than, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% greater than, a hepcidin reference compound. , 700% or 1000% greater activity, where the activity is in vitro activity of inducing ferroportin degradation, e.g., as measured according to the Examples herein, or the activity is in vivo activity of reducing free plasma iron, e.g., as measured according to the Examples herein.

[0081] In some embodiments, the hepcidin analog of the present invention mimics the hepcidin activity of Hep25, the biologically active human 25-amino acid form, and is herein referred to as "mini-hepcidin." As used herein, in certain embodiments, a compound (e.g., a hepcidin analog) with "hepcidin activity" means that when administered to a subject (e.g., by parenteral injection or oral administration), the compound has the ability to dose-dependently and time-dependently reduce plasma iron concentration in a subject (e.g., a mouse or human). See, for example, Rivera et al. (2005), Blood 106:2196-9. In some embodiments, the peptides of the invention reduce plasma iron concentrations in a subject by at least about 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

[0082] In some embodiments, hepcidin analogs of the present invention have in vitro activity, as analyzed by their ability to cause ferroportin internalization and degradation in ferroportin-expressing cell lines, as taught in Nemeth et al. (2006) Blood 107:328-33. In some embodiments, in vitro activity is measured by the dose-dependent fluorescence loss of cells engineered to display ferroportin fused to green fluorescent protein, as taught in Nemeth et al. (2006) Blood 107:328-33. Cell aliquots are incubated with graded concentrations of a reference preparation of Hep25 or mini-hepcidin for 24 hours. As provided herein, EC 50 Values ​​are provided as the concentration of a given compound (e.g., a hepcidin analog peptide or peptide dimer of the invention) at which 50% of the maximum fluorescence produced by the reference compound is extinguished. EC2 values ​​for Hep25 preparations in the assay are shown in Table 1. 50 In certain embodiments, preferred hepcidin analogs of the present invention have an EC of about 1,000 nM or less in an in vitro activity assay. 50 In certain embodiments, the hepcidin analogs of the invention have an EC value of less than about any one of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM in an in vitro activity assay (as described in Nemeth et al. (2006) Blood 107:328-33 or in the Examples herein). 50 In some embodiments, the hepcidin analog or biopharmaceutical composition (e.g., any one of the pharmaceutical compositions described herein) has an EC 50 Value or IC 50 It has a value.

[0083] Other methods known in the art for calculating the hepcidin activity and in vitro activity of hepcidin analogs of the present invention may also be used. For example, in certain embodiments, the in vitro activity of a hepcidin analog or reference peptide is measured by its ability to internalize cellular ferroportin, as determined by immunohistochemistry or flow cytometry using an antibody that recognizes an extracellular epitope of ferroportin. Alternatively, in certain embodiments, the in vitro activity of a hepcidin analog or reference peptide is determined by its ability to dose-dependently inhibit iron efflux from ferroportin-expressing cells preloaded with a radioisotope or stable isotope, as described in Nemeth et al. (2006) Blood 107:328-33.

[0084] In some embodiments, the hepcidin analogue of the present invention exhibits increased stability (for example, measured by half-life, proteolysis rate) compared with hepcidin reference compound.In certain embodiments, the stability of the hepcidin analogue of the present invention is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 times higher than hepcidin reference compound, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500% higher than hepcidin reference compound.In some embodiments, stability is the stability described herein. In some embodiments, the stability is plasma stability, e.g., stability optionally measured according to the methods described herein, hi some embodiments, stability is stability when delivered orally.

[0085] In certain embodiments, the hepcidin analogs of the present invention exhibit a longer half-life than a hepcidin reference compound. In certain embodiments, the hepcidin analogs of the present invention exhibit a half-life of at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 4 days, at least about 7 days, at least about 1 hour, at least about 1 day, at least about 2 days, at least about 4 days, at least about 7 days, at least about 1 hour, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 1 day, at least about 12 hours, at least about 18 ... The hepcidin analogs of the present invention have a half-life of about 0 days, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months or more, or any half-life or range therebetween, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 4 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months or more, or any half-life or range therebetween. In some embodiments, the half-life of the hepcidin analogs of the present invention is extended by conjugation to one or more lipophilic substituents or half-life extending moieties, such as any of the lipophilic substituents or half-life extending moieties disclosed herein. In some embodiments, the half-life of the hepcidin analogs of the present invention is extended by conjugation to one or more polymer moieties, such as any of the polymer moieties or half-life extending moieties disclosed herein. In certain embodiments, the hepcidin analogs of the present invention have the above-mentioned half-lives under a given set of conditions, where the temperature is about 25°C, about 4°C, or about 37°C, and the pH is physiological pH or a pH of about 7.4.

[0086] In certain embodiments, hepcidin analogs of the invention comprising a conjugated half-life extending moiety have increased serum half-life following oral, intravenous, or subcutaneous administration compared to the analogs without the conjugated half-life extending moiety. In certain embodiments, the serum half-life of a hepcidin analog of the invention following oral, intravenous, or subcutaneous administration is at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 72 hours, or at least 168 hours. In certain embodiments, the serum half-life is 12-168 hours, 24-168 hours, 36-168 hours, or 48-168 hours.

[0087] In certain embodiments, hepcidin analogs of the invention, e.g., hepcidin analogs comprising a conjugated half-life extending moiety, reduce serum iron concentrations following oral, intravenous, or subcutaneous administration to a subject. In certain embodiments, the subject's serum iron concentration is reduced by less than 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the serum iron concentration that would be present if the subject were not administered the hepcidin analog. In certain embodiments, the reduction in serum iron concentration is maintained for at least 1 hour, at least 4 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours after administration to the subject. In certain embodiments, the reduction in serum iron concentration is maintained for 12 to 168 hours, 24 to 168 hours, 36 to 168 hours, or 48 to 168 hours. In one embodiment, the subject's serum iron concentration decreases by less than 20% about 4 hours or about 10 hours after administration, e.g., intravenous, oral, or subcutaneous, to the subject. In one embodiment, the subject's serum iron concentration decreases by less than 50% or less than 60% about 24 hours to about 30 hours after administration, e.g., intravenous, oral, or subcutaneous.

[0088] In some embodiments, half-life is measured in vitro using any suitable method known in the art.For example, in some embodiments, the stability of the hepcidin analogue of the present invention is determined by incubating it with pre-warmed human serum (Sigma) at 37 ℃.Typically, samples are taken at various time points up to 24 hours, and the stability of the samples is analyzed by separating the hepcidin analogue from serum proteins, and then using LC-MS to analyze the presence of the hepcidin analogue of interest.

[0089] In some embodiments, the stability of hepcidin analogues is measured in vivo by using any suitable method known in the art.For example, in some embodiments, the stability of hepcidin analogues is measured in vivo by administering peptide or peptide dimer to a subject, such as a human or any mammal (e.g., a mouse), and then collecting samples from the subject by drawing blood at various time points, typically up to 24 hours.The samples are then analyzed as described above for the in vitro method of measuring half-life.In some embodiments, the in vivo stability of the hepcidin analogues of the present invention is measured by the method disclosed in the examples herein.

[0090] In some embodiments, the present invention provides a hepcidin analog described herein, which exhibits improved solubility or improved aggregation properties compared to a hepcidin reference compound. Solubility can be determined by any suitable method known in the art. In some embodiments, suitable methods known in the art for determining solubility include incubating a peptide (e.g., a hepcidin analog of the invention) in various buffers (e.g., acetate pH 4.0, acetate pH 5.0, Phos / citrate pH 5.0, Phos citrate pH 6.0, Phos pH 6.0, Phos pH 7.0, Phos pH 7.5, strong PBS pH 7.5, Tris pH 7.5, Tris pH 8.0, glycine pH 9.0, water, acetate (pH 5.0, and other buffers known in the art) and examining for aggregation or solubility using standard methods. These methods include, but are not limited to, visual precipitation, dynamic light scattering, circular dichroism, and fluorescent dyes to measure surface hydrophobicity and detect aggregation or fibrillation. In some embodiments, improved solubility means that the peptide (e.g., a hepcidin analog of the invention) is more soluble in a given liquid than a hepcidin reference compound.

[0091] In certain embodiments, the invention provides hepcidin analogs described herein that exhibit increased solubility in a particular solution or buffer, such as, for example, water or a buffer known in the art or disclosed herein, that is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times greater, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater, than a hepcidin reference compound.

[0092] In certain embodiments, the invention provides hepcidin analogs described herein, which exhibit reduced aggregation, such that aggregation of the peptide in solution is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times less, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% less than a hepcidin reference compound in a particular solution or buffer, e.g., water, or a buffer known in the art or disclosed herein.

[0093] In some embodiments, the present invention provides hepcidin analogs described herein, wherein the hepcidin analogs exhibit reduced degradation (i.e., high degradation stability) compared to hepcidin reference compounds, for example, about 10% higher or lower, about 20% higher or lower, about 30% higher or lower, about 40% higher or lower, or about 50% higher or lower. In some embodiments, degradation stability is determined by any suitable method known in the art. In some embodiments, suitable methods known in the art for determining degradation stability include Hawe et al. J Pharm Sci, VOL.101, NO.3, 2012, p895-913, which is incorporated herein in its entirety. In some embodiments, this method is used to select potential sequences with long shelf life.

[0094] In some embodiments, the hepcidin analogs of the present invention are synthetically produced, hi other embodiments, the hepcidin analogs of the present invention are recombinantly produced.

[0095] The various hepcidin analog monomeric and dimeric peptides of the present invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may contain unnatural or non-natural amino acids, including, but not limited to, modified amino acids. In certain embodiments, modified amino acids include natural amino acids that have been chemically modified to include a group, groups, or chemical moiety on the amino acid that is not naturally present on the amino acid. The hepcidin analogs of the present invention may additionally contain D-amino acids. Furthermore, the hepcidin analog peptide monomers and dimers of the present invention may contain amino acid analogs. In certain embodiments, peptide analogs of the present invention include any of those described herein, where one or more natural amino acid residues of the peptide analog are replaced with an unnatural or non-natural amino acid, or a D-amino acid.

[0096] In certain embodiments, hepcidin analogs of the invention include one or more modified or unnatural amino acids. For example, in certain embodiments, hepcidin analogs include one or more of Daba, Dapa, Pen, Sar, Cit, Pba, Cav, HLeu, 2-Nal, 1-Nal, d-1-Nal, d-2-Nal, Bip, Phe(4-OMe), Tyr(4-OMe), βhTrp, βhPhe, Phe(4-CF3), 2-2-indan, 1-1-indan, cyclobutyl, βhPhe, hLeu, Gla, Phe(4-NH2), hPhe, 1-Nal, Nle, 3-3-diPhe, cyclobutyl-Ala, Cha, Bip, β-Glu, Phe(4-Guan), homoamino acids, D-amino acids, and various N-methylated amino acids. Those skilled in the art will recognize that modified or unnatural amino acids and various other substitutions of natural amino acids with other modified or unnatural amino acids may be made to achieve similar desirable results, and that such substitutions are within the teachings and spirit of the present invention.

[0097] The present invention includes any of the hepcidin analogs described herein in free or salt form.

[0098] The compounds described herein include isotopically labeled compounds that are identical to those listed in the various formulas and structures presented herein, but differ by the fact that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as: 2 H, 3 H, 1 3C, 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Cl, etc. 3 H and 14 Certain isotopically labeled compounds described herein that incorporate a radioactive isotope, such as C, are useful in drug and / or substrate tissue distribution assays. 2 Substitution with an isotope such as H provides certain therapeutic benefits resulting from increased metabolic stability, such as increased in vivo half-life or reduced dosage requirements. In certain embodiments, the compound is isotopically substituted with deuterium. In even more particular embodiments, the most labile hydrogen is substituted with deuterium.

[0099] The hepcidin analogs of the present invention include any of the peptide monomers or dimers described herein linked to a linker moiety, including any of the specific linker moieties described herein.

[0100] Hepcidin analogs of the present invention include, for example, monomeric or dimeric peptides comprising peptide monomer subunits having at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to a hepcidin analog peptide sequence described herein (e.g., any one of the peptides disclosed herein), including, but not limited to, any of the amino acid sequences shown in Tables 2 and 3.

[0101] In certain embodiments, a peptide analog of the invention, or a monomeric subunit of a dimeric peptide analog of the invention, comprises or consists of 7-35 amino acid residues, 8-35 amino acid residues, 9-35 amino acid residues, 10-35 amino acid residues, 7-25 amino acid residues, 8-25 amino acid residues, 9-25 amino acid residues, 10-25 amino acid residues, 7-18 amino acid residues, 8-18 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues, and optionally one or more additional non-amino acid moieties, e.g., a half-life extending moiety, a conjugation chemical moiety such as a PEG or a linker moiety. In certain embodiments, a monomeric subunit of a hepcidin analog comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues. In certain embodiments, a monomeric subunit of a hepcidin analog of the invention comprises or consists of 10-18 amino acid residues and, optionally, one or more additional non-amino acid moieties, such as, for example, a conjugation chemical moiety, e.g., a PEG or a linker moiety. In various embodiments, a monomeric subunit comprises or consists of 7-35 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues. In certain embodiments of any of the various formulas described herein, X comprises or consists of 7 to 35 amino acid residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues, 10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25 amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues.

[0102] In certain embodiments, hepcidin analogs or dimers of the invention do not include any of the compounds described in PCT / US2014 / 030352 or PCT / US2015 / 038370.

[0103] Peptide Hepcidin Analogues In certain embodiments, the hepcidin analogs of the present invention comprise a single peptide subunit, which is optionally conjugated to an acid moiety, hi certain embodiments, the acid moiety is conjugated directly or via a linker.

[0104] In one embodiment, the present invention provides a compound of formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ia) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 is hydrogen, C1-C6 alkyl, C6-C 12 Aryl, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 is cycloalkanoyl, R 2 is NH, substituted amino, OH, or substituted hydroxy; X1 is absent, Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X2 is Ala, Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, Gly, N-substituted Gly, His, or substituted His; X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X6 is absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys or Pen; X7 is absent, Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys or 123 triazole; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent, Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys or (D)Lys; and Each of X12 to X14 is absent or independently represents any amino acid, However, subject to the following conditions: i) the peptide may be further conjugated with any amino acid; ii) any of the amino acids of the peptide may be its corresponding (D)-amino acid or may be N-substituted; and iii) the peptide is a linear peptide or a cyclized lactam; and wherein Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, and bhTrp is b-homotricarboxylic acid. 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and the substituted Phe is phenyl is F, Cl, Br, I, OH, methoxy, dimethoxy. phenylalanine substituted with thio, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; substituted bhPhe is b-homophenylalanine substituted with phenyl by F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; substituted bhTrp is N-methyl-Lb-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid; and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid; 123 Triazole is [ka] and Dab is [ka] is.

[0105] In one embodiment, the present invention provides a compound of formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ib) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 is hydrogen, C1-C6 alkyl, C6-C 12 Aryl, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 is cycloalkanoyl, R 2 is -NH2 or -OH, X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2; X2 is Ala, Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, His, or substituted His; X4 is Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), or 2-pyrrolidinebutanoic acid (Pba); X6 is absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys or Pen; X7 is absent, Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent, Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys or (D)Lys; and Each of X12 to X14 is absent or independently represents any amino acid, However, subject to the following conditions: i) the peptide does not consist of disulfide or thioether bonds; ii) the peptide may be further conjugated with any amino acid; iii) any of the amino acids of the peptide may be its corresponding (D)-amino acid or may be further N-substituted; Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, and 2-Nal is 2- naphthylalanine, Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Pba is 2-(R)-pyrrolidinebutanoic acid, and the substituted Phe is phenyl is F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t phenylalanine substituted with -Bu, carboxyl, CN or guanidine, the substitution bhPhe is b-homophenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, the substitution Trp is N-methyl-L -tryptophan, a-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; the substituted bhTrp is N-methyl-Lb-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid; and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid.

[0106] In certain embodiments of any of the hepcidin analogs of the invention, X or X is Lys, or (D)Lys substituted with L1Z, where L is absent, Dapa, D-Dapa, or isoGlu, PEG, Ahx, isoGlu-PEG, PEG-isoGlu, PEG-Ahx, isoGlu-Ahx, or isoGlu-PEG-Ahx, where Ahx is an aminohexanoic acid moiety and PEG is -[C(O)-CH-(Peg) n -N(H)] m - or -[C(O)-CH2-CH2-(PEG) n -N(H)] m -, and Peg is -OCH2CH2-, m is 1, 2 or 3, and n is an integer between 1 and 100K, and Z is a half-life extending moiety. In one embodiment, the half-life extending moiety is C 10 -C 21 It is an alkanoyl.

[0107] In one embodiment of any of the peptides, including but not limited to those of formula (Ia) or (Ib), X1 is Asp, Glu, (D)Asp, Tet1, or Tet2; X2 is Thr or Ser, X3 is His or a substituted His, X7 is absent, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X8 is absent, Ile, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent, Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X10 is absent or is Ala, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys, and X11 is absent or is Pro, bhPhe, Lys, substituted Lys or (D)Lys.

[0108] In one embodiment of any of the peptides, including but not limited to those of formula (Ia) or (Ib), X1 is Glu, Dab, Dap, Orn, Lys, or Tet1; X2 is Thr, X3 is His or 1MeHis, X4 is Dpa, The X5 is a Pro, X6 is absent, Ala, Glu, or substituted Lys; X7 is absent, Ile, Lys, substituted Lys, (D)Lys or substituted (D)Lys; X8 is absent, Ile, Glu, Asp, 123 triazole, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent or is bhPhe; X10 is absent or is Ala, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys or substituted (D)Lys, and X11 is absent or is Pro, bhPhe, Lys, substituted Lys or (D)Lys.

[0109] In one embodiment, X1 is Glu.

[0110] In one embodiment, X2 is Thr.

[0111] In one embodiment, X4 is Dpa.

[0112] In one embodiment, X5 is Pro.

[0113] In one embodiment, the peptide has the formula II: R 1-Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (II) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , X3, X6 to X14 are as described in formula (Ia) or formula (Ib).

[0114] In one embodiment, X9 is absent, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0115] In one embodiment, X9 is absent.

[0116] In one embodiment, X9 is bhPhe.

[0117] In one embodiment, the peptide has the formula III: R 1 -Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (III) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , X3, X6 to X8, and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0118] In one embodiment, X6 is Ala, Lys, or a substituted Lys.

[0119] In one embodiment, X6 is Ala.

[0120] In one embodiment, the peptide has Formula IV: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-X7-X8-[bhPhe]-X10-X11-X12-X13-X14-R2 (IV) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , X3, X7 to X8, and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0121] In one embodiment, X7 is absent, Ile, Lys, or a substituted Lys.

[0122] In one embodiment, X7 is absent.

[0123] In one embodiment, X7 is Ile.

[0124] In one embodiment, the peptide has the formula V: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (V) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , X3, X8, and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0125] In one embodiment, X8 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0126] In one embodiment, X8 is (D)Lys or a substituted (D)Lys.

[0127] In one embodiment, X8 is Lys or Lys(Ac).

[0128] In one embodiment, X8 is (D)Lys or (D)Lys(Ac).

[0129] In one embodiment, X8 is a conjugated amino acid.

[0130] In one embodiment, X8 is a conjugated Lys or (D)Lys.

[0131] In one embodiment, X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety.

[0132] In one embodiment, the peptide has the formula VIa or VIb: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIa); or R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIb) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , X3 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0133] In one embodiment, the peptide has the formula VIc: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIc); or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , X3 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0134] In one embodiment, X3 is His.

[0135] In one embodiment, the peptide has the formula VIIa or VIIb: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIa); or R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIb) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0136] In one embodiment, the peptide has the formula VIIc: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIc); or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0137] In one embodiment, X3 is (1-Me)His.

[0138] In one embodiment, the peptide has the formula VIIIa or VIIIb: R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIa); or R1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIb) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0139] In one embodiment, X10 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0140] In one embodiment, X10 is (D)Lys or a substituted (D)Lys.

[0141] In one embodiment, X10 is (D)Lys or (D)Lys(Ac).

[0142] In one embodiment, X10 is Lys(Ahx_Palm).

[0143] In one embodiment, X10 is a conjugated amino acid.

[0144] In one embodiment, X10 is a conjugated Lys or (D)Lys.

[0145] In one embodiment, X10 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety.

[0146] In one embodiment, PEG is —[C(O)—CH—(Peg) n -N(H)] m - or -[C(O)-CH2-CH2-(Peg) n -N(H)] m-, Peg is -OCH2CH2-, m is 1, 2 or 3, and n is an integer between 1 and 100, or 10K, 20K or 30K.

[0147] In one embodiment, m is 1. In another embodiment, m is 2.

[0148] In one embodiment, n is 2. In another embodiment, n is 4. In another embodiment, n is 8. In another embodiment, n is 11. In another embodiment, n is 12. In another embodiment, n is 20K.

[0149] In one embodiment, PEG is 1Peg2, where 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0150] In another embodiment, PEG is 2Peg2, wherein 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0151] In another embodiment, PEG is 1Peg2-1Peg2, where each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0152] In another embodiment, PEG is 1Peg2-1Peg2, where 1Peg2-1Peg2 is —[(C(O)—CH2—(OCH2CH2)2—NH—C(O)—CH2—(OCH2CH2)2—NH—]—.

[0153] In another embodiment, PEG is 2Peg4, wherein 2Peg4 is —C(O)—CH—CH—(Peg)—N(H)—, or —[C(O)—CH—CH—(OCH—CH)—NH]—.

[0154] In another embodiment, PEG is 1Peg8, wherein 1Peg8 is —C(O)—CH2—(Peg)8-N(H)—, or —[C(O)—CH2—(OCH2CH2)8-NH]—.

[0155] In another embodiment, PEG is 2Peg8, wherein 2Peg8 is —C(O)—CH—CH—(Peg)—N(H)—, or —[C(O)—CH—CH—(OCHCH)—NH]—.

[0156] In another embodiment, PEG is 1Peg11, which is -C(O)-CH2-(Peg) 11 -N(H)-, or -[C(O)-CH2-(OCH2CH2) 11 -NH]-.

[0157] In another embodiment, PEG is 2Peg11, and 2Peg11 is -C(O)-CH2-CH2-(Peg) 11 -N(H)-, or -[C(O)-CH2-CH2-(OCH2CH2) 11 -NH]-.

[0158] In another embodiment, PEG is 2Peg11′ or 2Peg12, wherein 2Peg11′ or 2Peg12 is —C(O)—CH2—CH2—(Peg) 12 -N(H)-, or -[C(O)-CH2-CH2-(OCH2CH2) 12 -NH]-.

[0159] In one embodiment, when PEG is attached to Lys, the —C(O)— of PEG is the N- ε Combine with.

[0160] In one embodiment, when PEG is attached to isoGlu, the -N(H)- of PEG is attached to the -C(O)- of isoGlu.

[0161] In one embodiment, when PEG is attached to Ahx, the -N(H)- of PEG is attached to the -C(O)- of Ahx.

[0162] In one embodiment, when PEG is bound to Palm, the -N(H)- of PEG is bound to the -C(O)- of Palm.

[0163] In one embodiment, the peptide has the formula IX: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2 , X6, X7 and X11 to X14 are as described in formula (Ia) or formula (Ib).

[0164] In one embodiment, the peptide has the formula IXa or IXb: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); or R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXb) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 and X11 to X14 are as described in formula (Ia) or formula (Ib).

[0165] In one embodiment, the peptide has the formula Xa or Xb: R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (Xa); or R 1-Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (Xb) or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 and X11 to X14 are as described in formula (Ia) or formula (Ib).

[0166] In one embodiment, the peptide is a linear peptide.

[0167] In one embodiment, the peptide is a lactam.

[0168] In one embodiment, the peptide is a lactam, in which any free -NH2 is cyclized with any free -C(O)2H.

[0169] In one embodiment, the peptide has the formula XXI: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (XXI), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib), X6 is absent, Ala, or a substituted Lys; X7 is absent, Ile, a substituted Lys, or a substituted (D)Lys; and X9 is absent or bhPhe; and X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety.

[0170] In one embodiment, X8 is Lys(L1Z).

[0171] In one embodiment, X8 is (D)Lys(L1Z).

[0172] In one embodiment, the peptide has the formula XXII: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXII), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib), X6 is absent, Ala, or a substituted Lys; X7 is absent, Ile, a substituted Lys, or a substituted (D)Lys; and X9 is absent or bhPhe.

[0173] In one embodiment, X6 is absent.

[0174] In one embodiment, X6 is a substituted Lys.

[0175] In one embodiment, X6 is Ala.

[0176] In one embodiment, the peptide has the formula XXIIIa or XXIIIb: R 1 -Glu-Thr-His-[Dpa]-Pro-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIIIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIIIb), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib), X7 is absent, Ile, substituted Lys, or substituted (D)Lys, and X9 is absent or bhPhe.

[0177] In one embodiment, X7 is absent.

[0178] In one embodiment, X7 is a substituted (D)Lys.

[0179] In one embodiment, X7 is a substituted Lys.

[0180] In one embodiment, X7 is Ile.

[0181] In one embodiment, the peptide has the formula XXIVa, XXIVb, XXIVc, or XXIVd: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVb), R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVd), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib), X9 is absent or is bhPhe.

[0182] In one embodiment, X9 is absent.

[0183] In one embodiment, the peptide has the formula XXVa, XXVb, XXVc, or XXVd: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVb), R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVd), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0184] In one embodiment, X9 is bhPhe.

[0185] In one embodiment, the peptide has the formula XXVIa, XXVIb, XXVIc, or XXVId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIb), R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVId), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib).

[0186] In one embodiment, X10 is Lys or (D)Lys.

[0187] In one embodiment, X10 is (D)Lys.

[0188] In one embodiment, the peptide has the formula XXVIIa, XXVIIb, XXVIIc, or XXVIId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-[(D)Lys]-X11-X12-X13-X14-R 2 (XXVIIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIIb), R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIIc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIId), In accordance with the formula, R 1 , R 2 and X11 to X14 are as described in formula (Ia) or formula (Ib).

[0189] In one embodiment, X10 is absent.

[0190] In one embodiment, the peptide has the formula XXVIIIa, XXVIIIb, XXVIIIc, or XXVIIId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIa), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIb), R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIc), R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIId), In accordance with the formula, R 1 , R 2 and X11 to X14 are as described in formula (Ia) or formula (Ib).

[0191] In one embodiment, L1 is a single bond.

[0192] In one embodiment, L1 is iso-Glu.

[0193] In one embodiment, L1 is Ahx.

[0194] In one embodiment, L1 is iso-Glu-Ahx.

[0195] In one embodiment, L1 is PEG.

[0196] 97. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 78 to 96, wherein L1 is PEG-Ahx.

[0197] In one embodiment, L1 is iso-Glu-PEG-Ahx.

[0198] In one embodiment, PEG is -[C(O)-CH-(Peg)N(H)]- or -[C(O)-CH-CH-(Peg)N(H)]-, where Peg is -OCHCH-, m is 1, 2, or 3, and n is an integer between 1 and 100, or 10K, 20K, or 30K.

[0199] In one embodiment, m is 1.

[0200] In one embodiment, m is 2.

[0201] In one embodiment, n is 2.

[0202] In one embodiment, n is 4.

[0203] In one embodiment, n is 8.

[0204] In one embodiment, n is 11.

[0205] In one embodiment, n is 12.

[0206] In one embodiment, n is 20K.

[0207] In one embodiment, PEG is 1Peg2, where 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0208] In one embodiment, PEG is 2Peg2, where 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0209] In one embodiment, PEG is 1Peg2-1Peg2, where each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0210] In one embodiment, PEG is 1Peg2-1Peg2, where 1Peg2-1Peg2 is —[(C(O)—CH2—(OCH2CH2)2—NH—C(O)—CH2—(OCH2CH2)2—NH—]—.

[0211] In one embodiment, PEG is 2Peg4, and 2Peg4 is —C(O)—CH—CH—(Peg)—N(H)—, or —[C(O)—CH—CH—(OCH—CH)—NH]—.

[0212] In one embodiment, PEG is 1Peg8, where 1Peg8 is —C(O)—CH2—(Peg)8-N(H)— or —[C(O)—CH2—(OCH2CH2)8-NH]—.

[0213] In one embodiment, PEG is 2Peg8, where 2Peg8 is —C(O)—CH—CH—(Peg)—N(H)—, or —[C(O)—CH—CH—(OCH—CH)—NH]—.

[0214] In one embodiment, PEG is 1Peg11, where 1Peg11 is —C(O)—CH2—(Peg)11-N(H)— or —[C(O)—CH2—(OCH2CH2)11-NH]—.

[0215] In one embodiment, PEG is 2Peg11, where 2Peg11 is —C(O)—CH2—CH2—(Peg)11-N(H)—, or —[C(O)—CH2—CH2—(OCH2CH2)11-NH]—.

[0216] In one embodiment, PEG is 2Peg11′ or 2Peg12, and 2Peg11′ or 2Peg12 is —C(O)—CH2—CH2—(Peg)12-N(H)—, or —[C(O)—CH2—CH2—(OCH2CH2)12-NH]—.

[0217] In one embodiment, when PEG is attached to Lys, the -C(O)- of PEG is attached to the Ne of Lys.

[0218] In one embodiment, when PEG is attached to isoGlu, the -N(H)- of PEG is attached to the -C(O)- of isoGlu.

[0219] In one embodiment, when PEG is attached to Ahx, the -N(H)- of PEG is attached to the -C(O)- of Ahx.

[0220] In one embodiment, when PEG is bound to Palm, the -N(H)- of PEG is bound to the -C(O)- of Palm.

[0221] In one embodiment, Z is Palm.

[0222] In one embodiment, L1Z is -Ahx_Palm.

[0223] In one embodiment, L1Z is -bAla_Palm.

[0224] In one embodiment, L1Z is -IsoGlu_Palm.

[0225] In one embodiment, L1Z is PEG12_Palm.

[0226] In one embodiment, L1Z is -1PEG2_1PEG2_Ahx_C18_diacid.

[0227] In one embodiment, each of X11, X12, X13 and X14 is absent.

[0228] In one embodiment, the peptide has the formula XXI: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXI), In accordance with the formula, R 1 , R 2 and X10 to X14 are as described in formula (Ia) or formula (Ib), X6 is absent or a substituted Lys, X7 is absent or a substituted Lys, and X9 is absent or bhPhe.

[0229] In one embodiment, each -L1Z is independently: PEG11_OMe; PEG12_C18 acid; 1PEG2_1PEG2_Ahx_Palm; 1PEG2_Ahx_Palm; Ado_Palm; Ahx_Palm; Ahx_PEG20K; PEG12_Ahx_IsoGlu_Behenic; PEG12_Ahx_Palm; PEG12_DEKHKS_Palm; PEG12_IsoGlu_C18 acid; PEG12_Ahx_C18 acid; PEG12_IsoGlu_Palm; PEG12_KKK_Palm; PEG12_KKKG_Palm; PEG12_DEKHKS_Palm; PEG12_Palm; PEG12_PEG12_Palm; PEG20K; PEG4_Ahx_Palm; PEG4_Palm; PEG8_Ahx_Palm; or IsoGlu_Palm; -1PEG2_1PEG2_Dap_C18_Diacid; -1PEG2_1PEG2_IsoGlu_C10_Diacid; -1PEG2_1PEG2_IsoGlu_C12_diacid; -1PEG2_1PEG2_IsoGlu_C14_diacid; -1PEG2_1PEG2_IsoGlu_C16_diacid; -1PEG2_1PEG2_IsoGlu_C18_diacid; -1PEG2_1PEG2_IsoGlu_C22_diacid; -1PEG2_1PEG2_Ahx_C18_diacid; -1PEG2_1PEG2_C18_dioic acid; -1PEG8_IsoGlu_C18_Diacid; -IsoGlu_C18_diacid; -PEG12_Ahx_C18_diacid; -PEG12_C16_dioic acid; -PEG12_C18_dioic acid; -1PEG2_1PEG2_1PEG2_C18_dioic acid; -1PEG2_1PEG2_1PEG2_IsoGlu_C18_diacid; -PEG12_IsoGlu_C18_Diacid; -PEG4_IsoGlu_C18_Diacid; or PEG4 PEG4 IsoGlu C18 diacid, During the ceremony, PEG11_OMe is -[C(O)-CH2-CH2-(OCH2CH2) 11 -OMe], 1PEG2 is -C(O)-CH2-(OCH2CH2)2-NH-, PEG4 is -C(O)-CH2-CH2-(OCH2CH2)4-NH-, PEG8 is —[C(O)—CH—CH—(OCHCH)—NH—; 1PEG8 is -[C(O)-CH2-(OCH2CH2)8-NH-, PEG12 is -[C(O)-CH2-CH2-(OCH2CH2) 12 -NH-, Ado is -[C(O)-(CH2) 11 -NH]-, Cn acid is -C(O)(CH2) n-2 -CH3, and C18 acids are -C(O)-(CH2) 16 -Me, Palm is -C(O)-(CH2) 14 -Me, isoGlu is isoglutamic acid isoGlu_Palm is [ka] and Ahx is -[C(O)-(CH2)5-NH]-, Cn_ diacids are -C(O)-(CH2) n-2 -COOH, where n is 10, 12, 14, 16, 18, or 22.

[0230] In one embodiment, X8 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid), and (1PEG2_1PEG2_IsoGlu_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0231] In one embodiment, X8 or X10 is (D)Lys(1PEG2_1PEG2_IsoGlu_C n_ diacid), and (D) Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0232] In one embodiment, X8 or X10 is Lys(1PEG8_IsoGlu_C n _ diacid), and Lys(1PEG8_IsoGlu_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0233] In one embodiment, X8 or X10 is (D)Lys(1PEG8_IsoGlu_C n _ diacid), and (D) Lys(1PEG8_IsoGlu_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0234] In one embodiment, X8 or X10 is Lys(1PEG2_1PEG2_Dap_C n _ diacid), and Lys(1PEG2_1PEG2_Dap_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0235] In one embodiment, X8 or X10 is Lys(IsoGlu_C n _ diacid), and Lys(IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0236] In one embodiment, X8 or X10 is (D)Lys(IsoGlu_C n _ diacid), and (D) Lys(IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0237] In one embodiment, X8 or X10 is Lys(PEG12_IsoGlu_C n _ diacid), and Lys(PEG12_IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0238] In one embodiment, X8 or X10 is (D)Lys(PEG12_IsoGlu_C n _ diacid), and (D) Lys(PEG12_IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0239] In one embodiment, X8 or X10 is (PEG4_IsoGlu_C n _ diacid), and Lys(PEG4_IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0240] In one embodiment, X8 or X10 is (D)Lys(PEG4_IsoGlu_C n _ diacid), and (D) Lys(PEG4_IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0241] In one embodiment, X8 or X10 is Lys(PEG4_PEG4_IsoGlu_C n _ diacid), and Lys(PEG4_PEG4_IsoGlu_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0242] In one embodiment, X8 or X10 is (D)Lys(PEG4_PEG4_IsoGlu_C n _ diacid), and (D) Lys(PEG4_PEG4_IsoGlu_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0243] In one embodiment, X8 or X10 is Lys(IsoGlu_C n _ diacid), and Lys(IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0244] In one embodiment, X8 or X10 is (D)Lys(IsoGlu_C n _ diacid), and (D) Lys(IsoGlu_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0245] In one embodiment, X8 or X10 is Lys(PEG12_Ahx_C n _ diacid), and Lys(PEG12_Ahx_C n _ diacid) is [ka] and n is 10, 12, 14, 16, or 18.

[0246] In one embodiment, X8 or X10 is Lys(PEG12_Ahx_C n _ diacid), and Lys(PEG12_Ahx_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0247] In one embodiment, X8 or X10 is (D)Lys(PEG_Ahx_C n _ diacid), and (D) Lys(PEG12_Ahx_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0248] In one embodiment, X8 or X10 is Lys(PEG_C n _ diacid), and Lys(PEG12_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0249] In one embodiment, X8 or X10 is (D)Lys(PEG_C n _ diacid), and (D) Lys(PEG12_C n _ diacid) is [ka] ; and n is 10, 12, 14, 16, or 18.

[0250] In one embodiment, X8 or X10 is 123 triazole.

[0251] In one embodiment, X11 is absent, Ala, (D)Lys, or substituted Lys.

[0252] In one embodiment, X11 is absent.

[0253] 157. The hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 156, wherein X11 is Ala.

[0254] In one embodiment, X11 is (D)Lys.

[0255] In one embodiment, X11 is Lys(Ahx_Palm).

[0256] In one embodiment, X12 is absent or Ala.

[0257] In one embodiment, X12 is absent.

[0258] In one embodiment, X12 is Ala.

[0259] In one embodiment, X13 is absent.

[0260] In one embodiment, X14 is absent.

[0261] In one embodiment, R 2 is NH2.

[0262] In one embodiment, R 2 is a substituted amino.

[0263] In one embodiment, R 2 is N-alkylamino.

[0264] In one embodiment, R 2 is N-alkylamino, where the alkyl is further substituted or unsubstituted.

[0265] In one embodiment, R 2 is N-alkylamino, where alkyl is a further substituted aryl or heteroaryl.

[0266] In one embodiment, R 2 is alkylamino, where alkyl is unsubstituted or substituted with aryl, and alkyl is ethyl, propyl, butyl, or pentyl.

[0267] In one embodiment, R 2is alkylamino, where alkyl is unsubstituted or substituted with phenyl, and alkyl is ethyl, propyl, butyl, or pentyl.

[0268] In one embodiment, R 2 is OH.

[0269] In one embodiment, R 1 is C1-C 20 It is an alkanoyl.

[0270] In one embodiment, R 1 is IVA or isovaleric acid.

[0271] In one embodiment, the peptide is a linear peptide.

[0272] In one embodiment, the peptide is a lactam.

[0273] In one embodiment, the peptide is a lactam, in which any free -NH2 is cyclized with any free -C(O)2H.

[0274] In one embodiment, X11 is absent, Ala, (D)Lys, or substituted Lys.

[0275] In one embodiment, X11 is absent.

[0276] In one embodiment, X11 is Ala.

[0277] In one embodiment, X11 is (D)Lys.

[0278] In one embodiment, X11 is Lys(Ahx_Palm).

[0279] In one embodiment, X12 is absent or Ala.

[0280] In one embodiment, X12 is absent.

[0281] In one embodiment, X12 is Ala.

[0282] In one embodiment, X13 is absent.

[0283] In one embodiment, X14 is absent.

[0284] In one embodiment, R 2 is NH. In another embodiment, R 2 is a substituted amino. In another embodiment, R 2 is alkylamino, or (substituted alkyl)amino. In another embodiment, R 2 is methylamino, ethylamino, propylamino, benzylamino, or phenethylamino.

[0285] In one embodiment, R 2 is OH.

[0286] In one embodiment, R 1 is C1-C 20 It is an alkanoyl.

[0287] In one embodiment, R 1 is IVA or isovaleric acid.

[0288] In certain embodiments of any of the peptide analogs having any of the various formulas described herein, R 1 is selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and conjugated amides of lauric acid, hexadecanoic acid, and γ-Glu-hexadecanoic acid.

[0289] In certain embodiments, the substituted Lys is selected from Ac, PEG, Ahx, isoGlu, C 10 -C 20Alkanoyl, PEG-Ahx, PEG-isoGlu, Ahx-C 10 -C 20 Alkanoyl, isoGlu-C 10 -C 20 Alkanoyl, PEG-Ahx-C 10 -C 20 Alkanoyl, PEG-isoGlu-C 10 -C 20 In one embodiment, Lys is substituted with alkanoyl, or any other group described herein. ε is substituted for.

[0290] In certain embodiments, the substituted (D)Lys is selected from Ac, PEG, Ahx, isoGlu, C 10 -C 20 Alkanoyl, PEG-Ahx, PEG-isoGlu, Ahx-C 10 -C 20 Alkanoyl, isoGlu-C 10 -C 20 Alkanoyl, PEG-Ahx-C 10 -C 20 Alkanoyl, PEG-isoGlu-C 10 -C 20 In one embodiment, (D)Lys is (D)Lys substituted with alkanoyl, or any other group described herein. In one embodiment, (D)Lys is (D)Lys substituted with N of (D)Lys. ε is substituted for.

[0291] In certain embodiments, C 10 -C 20 The alkanoyl is Palm.

[0292] In certain embodiments, the invention includes polypeptides comprising an amino acid sequence set forth in Tables 6A-C, or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity to any of these amino acid sequences.

[0293] In certain embodiments, the present invention includes hepcidin analogs having the structure set forth below or comprising the amino acid sequence set forth below: Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PA-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-P-[Lys(Ahx_Palm)]-NH2;Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ahx_Palm)]-[bhPhe]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PA-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-P-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-NH2; Iso-glycine-ETH-[Dpa]-PLI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-L-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid)]-[bhPhe]-[(D)Lys]-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid)]-NH2; Iso-glycine-ETH-[Dpa]-PI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PSI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PII-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PFI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PEI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-P-[(D)Lys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-P-[Lys(Ahx_Palm)]-I-[(D)Lys]-[bhPhe]-[Lys(Ac)]-NH2; Iso-glycine-ATH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid-[(D)Lys]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid)]-[(D)Lys]-[bhPhe]-NH2; Iso-glycine-ETH-[Dpa]-PAI-[(D)Lys]-A-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ETH-[Dpa]-PAI-[(D)Ala]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ETH-[Dpa]-PAA-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ATH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-EAH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ETA-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Iso-glycine-ET-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-[Tet1]-TH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-[Tet2]-TH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-[Tet1]-T-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Isovaleric acid-[Tet2]-T-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Isovaleric acid-[Tet2]-T-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[bhPhe]-[(D)Lys]-A-NH2; Isovaleric acid-[Tet1]-T-[(1-Me)His]-[Dpa]-PAI-[bhPhe]-[(D)Lys]-A-NH2, or Isovaleric acid-[Tet1]-T-[(1-Me)His]-[Dpa]-PAI-[bhPhe]-[(D)Lys]-NH2.

[0294] In certain embodiments, the present invention includes hepcidin analogs having the structure set forth below or comprising the amino acid sequence set forth below: ID number 321 [ka] ; ID number 319 [ka] ; ID number 322 [ka] ; ID number 318 [ka] ; ID number 320 [ka] ; ID number 56 [ka] ; ID number 286 [ka] ; ID number 58 [ka] ; ID number 287 [ka] ; ID number 156 [ka] ,or ID number 292 [ka] ;

[0295] In a specific embodiment, the peptide is any one of the peptides having an FPN activity of less than 100 nM. In another specific embodiment, the peptide is any one of the peptides having an FPN activity of less than 50 nM. In another specific embodiment, the peptide is any one of the peptides having an FPN activity of less than 20 nM. In another specific embodiment, the peptide is any one of the peptides having an FPN activity of less than 10 nM. In an even more specific embodiment, the peptide is any one of the peptides having an FPN activity of less than 5 nM.

[0296] Peptide Analog Conjugates In certain embodiments, the hepcidin analogs of the present invention, including both monomers and dimers, contain one or more conjugated chemical substituents, such as lipophilic substituents and polymer moieties, collectively referred to herein as half-life extending moieties. Without wishing to be bound by any particular theory, it is believed that the lipophilic substituents bind to albumin in the bloodstream, thereby shielding the hepcidin analog from enzymatic degradation and extending its half-life. Furthermore, it is believed that the polymer moieties extend the half-life, reducing clearance in the bloodstream, and in some cases increasing epithelial permeability and enhancing retention in the lamina propria. It is also speculated that in some cases, these substituents may increase epithelial permeability and enhance retention in the lamina propria. Those skilled in the art will be well aware of suitable compound preparation methods to be employed in the context of the present invention. Non-limiting examples of suitable chemical methods are described, for example, in WO98 / 08871, WO00 / 55184, WO00 / 55119, Madsen, et al. See, e.g., Knudsen et al. (J. Med. Chem. 2007, 50, 6126-32), and Knudsen et al. 2000 (J. Med. Chem. 43, 1664-1669).

[0297] In one embodiment, the side chain of one or more amino acid residues (e.g., Lys residues) in the hepcidin analog of the present invention is further conjugated (e.g., covalently bonded) to a lipophilic substituent or other half-life extending moiety. The lipophilic substituent may be covalently bonded to an atom in the amino acid side chain or may be conjugated to the amino acid side chain via one or more spacer or linker moieties. The spacer or linker moiety, if present, may provide spacing between the hepcidin analog and the lipophilic substituent.

[0298] In certain embodiments, the lipophilic substituent or half-life extending moiety comprises a hydrocarbon chain having 4 to 30 C atoms, e.g., at least 8 or 12 C atoms, preferably 24 or fewer C atoms, or 20 or fewer C atoms. The hydrocarbon chain may be linear or branched, saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted with a moiety that forms the linkage to the amino acid side chain or spacer, such as an acyl group, a sulfonyl group, an N atom, an O atom, or an S atom. In some embodiments, the hydrocarbon chain is substituted with an acyl group, whereby the hydrocarbon chain may form part of an alkanoyl group, e.g., palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0299] Lipophilic substituents can be conjugated to any amino acid side chain in the hepcidin analog of the present invention.In certain embodiments, the amino acid side chain comprises a carboxyl group, a hydroxyl group, a thiol group, an amide group, or an amine group to form an ester, a sulfonyl ester, a thioester, an amide, or a sulfonamide with a spacer or a lipophilic substituent.For example, lipophilic substituents can be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys, or Dbu, Dpr, or Orn.In certain embodiments, lipophilic substituents are conjugated to Lys.The amino acid shown as Lys in any of the formulas provided herein can be replaced with, for example, Dbu, Dpr, or Orn, and lipophilic substituents are added thereto.

[0300] Alternatively, in further embodiments of the invention, the side chains of one or more amino acid residues in the hepcidin analogs of the invention may be conjugated to polymeric or other half-life extending moieties, e.g., to increase solubility, and / or extend in vivo half-life (e.g., plasma half-life), and / or increase bioavailability. Such modifications are also known to decrease clearance (e.g., renal clearance) of therapeutic proteins and peptides.

[0301] As used herein, "polyethylene glycol" or "PEG" refers to a compound of the general formula H-(O-CH-CH) nPEG is a polyether compound with -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE). As used herein, PEO, PEE, or POE refer to oligomers or polymers of ethylene oxide, depending on their molecular weight. While the three terms are chemically synonymous, PEG tends to refer to oligomers or polymers with molecular weights less than 20,000 g / mol, PEO tends to refer to polymers with molecular weights greater than 20,000 g / mol, and POE tends to refer to polymers of any molecular weight. PEG and PEO are liquids or low-melting solids, depending on their molecular weight. The three terms are used interchangeably throughout this disclosure. PEG is prepared by polymerization of ethylene oxide and is commercially available over a wide range of molecular weights, from 300 g / mol to 10,000,000 g / mol. While PEG and PEO of different molecular weights are used in different applications and have different physical properties (e.g., viscosity) due to chain length effects, their chemical properties are largely the same. The polymer moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymer moieties include polyethylene glycol (PEG), PEG homopolymers or copolymers, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See, e.g., Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992). Also included are PEGs prepared for half-life extension, such as mono-activated alkoxy-terminated polyalkylene oxides (POA), e.g., mono-methoxy-terminated polyethylene glycol (mPEG); bis-activated polyethylene glycol oxide (glycol), or other PEG derivatives. Suitable polymers vary considerably, with weights typically selected for the purposes of the present invention ranging from about 200 to about 40,000 kJ. In certain embodiments, PEG having a molecular weight of 200 to 2,000 daltons, or 200 to 500 daltons, is used.Various forms of PEG can also be used, depending on the initiator used in the polymerization process. For example, common initiators are monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), truncated mPEG. Other suitable initiators are known in the art and are suitable for use in the present invention.

[0302] Low molecular weight PEGs are also available as pure oligomers, also referred to as monodisperse, homogeneous, or discontinuous, and are used in certain embodiments of the present invention.

[0303] PEGs are available in different structures. Branched PEGs have 3-10 PEG chains extending from a central core group. Star PEGs have 10-100 PEG chains extending from a central core group. Comb PEGs usually have multiple PEG chains attached to the polymer backbone. PEGs can also be linear. A number often included in the name of PEGs indicates their average molecular weight (e.g., PEG with n=9 has an average molecular weight of approximately 400 daltons and is labeled PEG400).

[0304] As used herein, "PEGylation" refers to the act of attaching (e.g., covalently linking) a PEG structure to a hepcidin analog of the invention, and in certain embodiments, is referred to as a "PEGylated hepcidin analog." In certain embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG portion of the conjugated half-life extending moiety is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, it is PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8. In some embodiments, the spacer is PEGylated. In certain embodiments, the PEG of the PEGylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8.

[0305] In some embodiments, the present invention includes a hepcidin analog peptide (or a dimer thereof) conjugated with PEG, where the PEG is covalently attached, for example, via amide, thiol, click chemistry, or any other suitable means known in the art. In certain embodiments, the PEG is attached via an amide bond. Accordingly, the particular PEG derivative used is appropriately functionalized. For example, in certain embodiments, PEG11, O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol, has both an amine and a carboxylic acid and is attached to the peptide of the present invention. In certain embodiments, PEG25 contains a diacid moiety and 25 glycol moieties.

[0306] Other suitable polymer moieties include poly-amino acids such as poly-lysine, poly-aspartic acid, and poly-glutamic acid (see, e.g., Gombotz, et al. (1995), Bioconjugate Chem., vol. 6:332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57, and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73, :721-729). The polymer moiety may be linear or branched. In some embodiments, the molecular weight is between 500 and 40,000 Da, e.g., between 500 and 10,000 Da, between 1000 and 5000 Da, between 10,000 and 20,000 Da, or between 20,000 and 40,000 Da.

[0307] In some embodiments, a hepcidin analog of the invention may include two or more such polymer moieties, in which case the total molecular weight of all of the moieties generally falls within the ranges described above.

[0308] In some embodiments, the polymer moiety may be attached (covalently) to the amino, carboxyl, or thiol groups of amino acid side chains. Particular examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, and may also include the carboxyl groups of Asp and Glu residues.

[0309] Those skilled in the art will recognize the appropriate techniques that can be used to carry out the conjugation reaction.For example, PEG moieties carrying methoxy groups can be conjugated to Cys thiol groups via maleimide linkages using commercially available reagents from Nektar Therapeutics AL.For details of suitable chemical methods, see WO 2008 / 101017 and the references cited above.Maleimide-functionalized PEG can also be conjugated to the side chain sulfhydryl group of Cys residues.

[0310] As used herein, oxidation of disulfide bonds can occur in one step or is a two-step process. As used herein, one oxidation step often employs a trityl protecting group during assembly, allowing deprotection during cleavage, followed by solution oxidation. If a second disulfide bond is required, there are options for natural oxidation or selective oxidation. For selective oxidation, which requires orthogonal protecting groups, Acm and trityl are used as protecting groups for cysteines. Cleavage removes the protecting groups for the cysteine ​​pair, allowing oxidation of the pair. A second oxidative deprotection step of the Acm-protected cysteines then occurs. For natural oxidation, trityl protecting groups are used for all cysteines, allowing the peptide to fold natively.

[0311] Those skilled in the art will recognize suitable techniques that can be used to carry out the oxidation step.

[0312] In certain embodiments, the hepcidin analogs of the present invention comprise a half-life extending moiety, which may be selected from, but is not limited to, the following: Ahx-Palm, PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-lauric acid, isoGlu-mysteric acid, and isoGlu-isovaleric acid.

[0313] In certain embodiments, the hepcidin analog comprises a half-life extending moiety having the structure shown below, where n=0-24 or n=14-24. [ka]

[0314] In certain embodiments, the hepcidin analogs of the invention comprise a conjugated half-life extending moiety as shown in Table 2. [Table 5-1] [Table 5-2]

[0315] In some embodiments, the half-life extending moiety is conjugated directly to the hepcidin analog, while in other embodiments, the half-life extending moiety is conjugated to the hepcidin analog peptide via a linker moiety, such as, for example, any of the linkers shown in Table 3. [Table 6-1] [Table 6-2]

[0316] For the linker structures shown in Table 3, the notation for n=1-24 or n=1-25, etc. (e.g., L4 or L5) indicates that n can be any integer within the recited range. Additional linker moieties that can be used are shown in the "Abbreviations" table.

[0317] In certain embodiments, the hepcidin analogs of the invention comprise any of the linker moieties shown in Table 3 and any of the half-life extending moieties shown in Table 2, including any of the following combinations shown in Table 4: [Table 7] [Table 8] [Table 9] [Table 10]

[0318] In certain embodiments, the hepcidin analog comprises two or more linkers, hi certain embodiments, the two or more linkers are concatamerized, i.e., linked to one another.

[0319] In related embodiments, the invention includes polynucleotides that encode polypeptides having peptide sequences present in any of the hepcidin analogs described herein.

[0320] Furthermore, the present invention includes vectors, such as expression vectors, which comprise the polynucleotides of the present invention.

[0321] Treatment method In some embodiments, the present invention provides a method for treating a subject suffering from a disease or disorder associated with dysregulated hepcidin signaling, comprising administering a hepcidin analog of the present invention to the subject. In some embodiments, the hepcidin analog administered to the subject is present in a composition (e.g., a pharmaceutical composition). In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by increased ferroportin activity or expression, comprising administering to the individual a hepcidin analog or composition of the present invention in an amount sufficient to bind (partially or fully) to, agonize, or mimic hepcidin in the subject. In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by dysregulated iron metabolism, comprising administering to the subject a hepcidin analog or composition of the present invention.

[0322] In some embodiments, the methods of the present invention include providing a hepcidin analog or composition of the present invention to a subject in need thereof. In certain embodiments, the subject in need thereof has been diagnosed with or has been determined to be at risk of developing a disease or disorder characterized by dysregulated iron levels (e.g., a disease or disorder of iron metabolism, a disease or disorder associated with iron overload, and a disease or disorder associated with abnormal hepcidin activity or expression). In certain embodiments, the subject is a mammal (e.g., a human).

[0323] In certain embodiments, the disease or disorder is a disease of iron metabolism, such as, for example, an iron overload disease, an iron deficiency disorder, a disorder of iron biodistribution, or another disorder of iron metabolism, and other disorders potentially related to iron metabolism. In certain embodiments, the disease of iron metabolism is hemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, beta thalassemia, sideroblastic anemia, porphyria, porphyria cutanea tarda, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital dyserythropoiesis anemia, hypochromic microcytic anemia, sickle cell disease, polycythemia vera (primary and secondary), secondary and idiopathic myelodysplasia, pyruvate kinase deficiency, hypochromic microcytic anemia, transfusion-dependent anemia, hemolytic anemia, iron deficiency in obesity, other anemias, benign or malignant tumors that overproduce or induce the overproduction of hepcidin, hepcidin excess states, Friedreich's ataxia, Greisl's syndrome, Hallervorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer (e.g., liver cancer), hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes, atherosclerosis, neurodegenerative disorders, dementia, multiple sclerosis, Parkinson's disease, Huntington's disease, or Alzheimer's disease.

[0324] In certain embodiments, the disease or disorder is associated with an iron overload disorder, such as, for example, ferrohemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0325] In certain embodiments, the disease or disorder is one that is not typically identified as iron-related. For example, hepcidin is highly expressed in the mouse pancreas, suggesting that diabetes (type I or type II), insulin resistance, glucose intolerance, and other disorders may be improved by treating underlying iron metabolic disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, which is incorporated herein by reference. Thus, the peptides of the present invention can be used to treat these diseases and conditions. Those skilled in the art can easily determine whether a given disease can be treated with the peptides of the present invention using methods known in the art, including the assays described in WO2004092405. This patent application is incorporated herein by reference. The assays, which monitor the levels and expression of hepcidin, hemojuvelin, or iron, are known in the art, such as those described in U.S. Pat. No. 7,534,764. That patent is incorporated herein by reference.

[0326] In certain embodiments, the disease or disorder is postmenopausal osteoporosis.

[0327] In certain embodiments of the invention, the disorder of iron metabolism is an iron overload disorder, including hereditary hemochromatosis, iron overload anemia, alcoholic liver disease, heart disease and / or heart failure, cardiomyopathies, and chronic hepatitis C.

[0328] In certain embodiments, any of these diseases, disorders, or indications is caused by or associated with a hepcidin deficiency or iron overload.

[0329] In some embodiments, the methods of the present invention comprise providing a hepcidin analog of the present invention (i.e., a first therapeutic agent) in combination with a second therapeutic agent to a subject in need thereof. In certain embodiments, the second therapeutic agent is provided to the subject before, simultaneously with, and / or after the pharmaceutical composition is administered to the subject. In certain embodiments, the second therapeutic agent is an iron chelator. In certain embodiments, the second therapeutic agent is selected from an iron chelator, deferoxamine, and deferasirox (Exjade™). In another embodiment, the method comprises administering a third therapeutic agent to the subject.

[0330] The present invention provides compositions (e.g., pharmaceutical compositions) comprising one or more hepcidin analogs of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent. A pharmaceutically acceptable carrier, excipient, or diluent refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. Prevention of microbial influence may be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like.

[0331] The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in "Remington's Pharmaceutical Sciences," 17th The method is described in "The American Journal of Pharmacology, Vol. 1, No. 1, pp. 1985-1985," Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, sterile saline and phosphate-buffered saline at weakly acidic or physiological pH may be used. Suitable pH buffers may be, for example, phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetate (e.g., sodium acetate), or mixtures thereof. The term also encompasses any carrier agent listed in the United States Pharmacopoeia for use in animals, including humans.

[0332] In certain embodiments, the composition comprises two or more hepcidin analogs disclosed herein, in which the combination is selected from one of the following: (i) any two or more of the hepcidin analog peptide monomers disclosed herein, (ii) any two or more of the hepcidin analog peptide dimers disclosed herein, or (iii) any one or more of the hepcidin analog peptide monomers disclosed herein and any one or more of the hepcidin analog peptide dimers disclosed herein.

[0333] It should be understood that containing a hepcidin analog of the present invention (i.e., one or more hepcidin analog peptide monomers of the present invention, or one or more hepcidin analog peptide dimers of the present invention) in a pharmaceutical composition also encompasses containing a pharmaceutically acceptable salt or solvate of the hepcidin analog of the present invention. In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, excipients, or vehicles.

[0334] In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog, or a pharmaceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders disclosed herein or elsewhere (see, e.g., methods of treatment herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide monomer, or a pharmaceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders otherwise disclosed herein (see, e.g., methods of treatment herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide dimer, or a pharmaceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders disclosed herein.

[0335] The hepcidin analogs of the present invention may be formulated as pharmaceutical compositions, which are suitable for administration, with or without storage, and typically comprise a therapeutically effective amount of at least one hepcidin analog of the present invention together with a pharmaceutically acceptable carrier, excipient, or vehicle.

[0336] In some embodiments, the hepcidin analog pharmaceutical composition of the present invention is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form may be presented as a packaged preparation, for example, a package containing individual quantities of the preparation, such as packaged tablets, capsules, or powders in vials or ampoules. The unit dosage form may be, for example, a capsule, cachet, or tablet per se, or an appropriate number of any of these packaged forms. The unit dosage form may also be provided in a single-dose injectable form, for example, in the form of a pen device containing a liquid phase (typically aqueous) composition. The composition may be formulated for any suitable route and means of administration, for example, any one of the routes and means of administration disclosed herein.

[0337] In certain embodiments, the hepcidin analog or a pharmaceutical composition comprising the hepcidin analog is suspended in a sustained-release matrix. A sustained-release matrix, as used herein, is a matrix made of a material, usually a polymer, that is enzymatically degradable, degradable by acid-base hydrolysis, or degradable by dissolution. Once inserted into the body, the matrix is ​​activated by enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from biodegradable materials, such as liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. One embodiment of a biodegradable matrix is ​​a matrix of either polylactide, polyglycolide, or polylactide-co-glycolide (a copolymer of lactic acid and glycolic acid).

[0338] In certain embodiments, the composition is administered parenterally, subcutaneously, or orally.In certain embodiments, the composition is administered orally, intracisternally, intravaginally, intraperitoneally, intrarectally, topically (including intravitreal delivery, intranasal delivery, and inhalation delivery as powder, ointment, drop, suppository, or transdermal patch), or buccally.As used herein, the term "parenteral" refers to administration modes including intravenous, intramuscular, intraperitoneal, substernal, subcutaneous, intradermal, and intraarticular injection and infusion.Therefore, in certain embodiments, the composition is formulated for delivery by any of these administration routes.

[0339] In certain embodiments, pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders, which are reconstituted into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose, and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained by using coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prolonged absorption of injectable pharmaceuticals can be achieved by including agents that delay absorption, such as aluminum monostearate or gelatin.

[0340] Injectable depot formulations include those prepared by forming microencapsulated matrices of a hepcidin analog in one or more biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), poly(anhydrides), and (poly)glycols, e.g., PEG. The release rate of the hepcidin analog can be controlled depending on the ratio of peptide to polymer and the nature of the particular polymer employed. Depot-type injectable formulations can also be prepared by entrapping the hepcidin analog in liposomes or microemulsions that are compatible with body tissues.

[0341] Injectable preparations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0342] The hepcidin analogs of the present invention may be administered in liposomes or other lipid-based carriers. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes may be used. In addition to the hepcidin analogs of the present invention, the liposome-formed compositions of the present invention may contain stabilizers, preservatives, excipients, etc. In certain embodiments, the lipids include both natural and synthetic phosphatidylcholines (lecithins) and serine-containing phospholipids. Methods for forming liposomes are known in the art.

[0343] Pharmaceutical compositions used in the present invention suitable for parenteral administration may comprise sterile aqueous solutions and / or suspensions of the peptide inhibitors made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, or the like.

[0344] In some embodiments, the present invention provides pharmaceutical compositions for oral delivery.The compositions and hepcidin analogs of the present invention can be prepared for oral administration according to any of the methods, techniques, and / or delivery vehicles described herein.Furthermore, those skilled in the art will recognize that the hepcidin analogs of the present invention can be modified or integrated into systems or delivery vehicles that are not disclosed herein but are known in the art and are suitable for use in oral delivery of peptides.

[0345] In certain embodiments, formulations for oral administration may include adjuvants to artificially increase the permeability of the intestinal wall (e.g., resorcinol and / or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether), and / or enzyme inhibitors to inhibit enzymatic degradation (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF), or trasylol). In certain embodiments, a hepcidin analog in a solid dosage form for oral administration may be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, a synthetic or semi-synthetic polymer, or a glyceride. These dosage forms may also contain other types of additives, such as inert diluents, lubricants such as, for example, magnesium stearate, preservatives such as parabens, e.g., sorbic acid, antioxidants such as ascorbic acid, alpha-tocopherol, e.g., cysteine, disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavorings, or perfuming agents.

[0346] In certain embodiments, oral dosage forms or unit doses compatible with the hepcidin analogs of the present invention may include a mixture of a hepcidin analog and non-drug ingredients or excipients, as well as other non-recyclable materials that may be considered either ingredients or packaging materials. Oral compositions may include at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, oral dosage forms containing an effective amount of a hepcidin analog are provided, where the dosage form includes at least one of a pill, tablet, capsule, gel, paste, beverage, syrup, ointment, and suppository. In some examples, oral dosage forms are provided that are designed and configured to provide delayed release of the hepcidin analog in the small intestine and / or colon of a subject.

[0347] In one embodiment, oral pharmaceutical compositions containing a hepcidin analog of the present invention include an enteric coating designed to delay release of the hepcidin analog in the small intestine. In at least some embodiments, pharmaceutical compositions are provided that include a hepcidin analog of the present invention and a protease inhibitor, such as aprotinin, in a delayed-release pharmaceutical formulation. In some examples, pharmaceutical compositions of the present invention include an enteric coating that is soluble in gastric juices at a pH of about 5.0 or higher. In at least one embodiment, pharmaceutical compositions are provided that include an enteric coating that includes a polymer with a dissociable carboxylic acid group, such as a cellulose derivative, including, for example, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate, as well as similar derivatives of cellulose and other carbohydrate polymers.

[0348] In one embodiment, a pharmaceutical composition comprising a hepcidin analog of the present invention is provided in an enteric coating, which is designed to protect and release the pharmaceutical composition in a controlled manner within a subject's lower gastrointestinal system to avoid systemic side effects. In addition to enteric coatings, the hepcidin analog of the present invention may be encapsulated, coated, engaged, or otherwise associated with any compatible oral drug delivery system or component. For example, in some embodiments, the hepcidin analog of the present invention is provided in a lipid carrier system, including at least one of polymer hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0349] To overcome peptide degradation in the small intestine, some embodiments of the present invention include a hydrogel polymer carrier system containing the hepcidin analog of the present invention, whereby the hydrogel polymer protects the hepcidin analog from proteolytic degradation in the small intestine and / or colon. The hepcidin analog of the present invention may also be formulated to be compatible for use with carrier systems designed to increase dissolution kinetics and enhance intestinal absorption of the peptide. These methods include the use of liposomes, micelles, and nanoparticles to increase GI tract penetration of the peptide.

[0350] Various bioresponsive systems may be combined with one or more hepcidin analogs of the present invention to provide a therapeutic agent for oral delivery. In some embodiments, the hepcidin analogs of the present invention are used in combination with bioresponsive systems, such as hydrogels and mucoadhesive polymers having hydrogen-bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan, and alginate), to provide a therapeutic agent for oral administration. Other embodiments include methods for optimizing or extending the drug residence time of the hepcidin analogs disclosed herein, in which the surface of the hepcidin analog is modified to provide mucoadhesion through hydrogen bonds, polymers with linked mucins, or / and hydrophobic interactions. These modified peptide molecules may exhibit extended drug residence time within a subject, according to a desirable feature of the present invention. Furthermore, targeted mucoadhesive systems may specifically bind to receptors on the surface of enterocytes and M cells, thereby further increasing the uptake of particles containing the hepcidin analog.

[0351] Another embodiment includes a method for oral delivery of the hepcidin analog of the present invention, in which the hepcidin analog is provided to a subject in combination with a permeation enhancer that promotes peptide transport across the intestinal mucosa by increasing paracellular or paracellular permeation. For example, in one embodiment, a permeation enhancer is combined with the hepcidin analog, in which the permeation enhancer includes at least one of a long-chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent. In one embodiment, a permeation enhancer including sodium N-[hydroxybenzoyl)amino]caprylate is used to form a weak covalent bond with the hepcidin analog of the present invention, in which the permeation enhancer favors membrane transport and further dissociation upon reaching the blood circulation. In another embodiment, the hepcidin analog of the present invention is conjugated to oligoarginine, thereby increasing the cellular penetration of the peptide into various cell types. Additionally, in at least one embodiment, a non-covalent bond is formed between a peptide inhibitor of the present invention and a permeation enhancer selected from the group consisting of cyclodextrins (CDs) and dendrimers, wherein the permeation enhancer reduces peptide aggregation and increases the stability and solubility of the hepcidin analog molecule.

[0352] Another embodiment of the present invention provides a method of treating a subject using a hepcidin analog of the present invention with an extended half-life. In one aspect, the present invention provides a hepcidin analog having a half-life of at least several hours to a day, sufficient for once-daily (qd) or twice-daily (bid) therapeutically effective dose administration in vitro or in vivo (e.g., when administered to a human subject). In another embodiment, the hepcidin analog has a half-life of three days or more, sufficient for once-weekly (qw) therapeutically effective dose administration. In yet another embodiment, the hepcidin analog has a half-life of eight days or more, sufficient for every-other-weekly (biw) or monthly therapeutically effective dose administration. In another embodiment, the hepcidin analog is derivatized or modified to have a longer half-life compared to the underivatized or unmodified hepcidin analog. In another embodiment, the hepcidin analog contains one or more chemical modifications that extend its serum half-life.

[0353] When used in at least one of the treatments or delivery systems described herein, the hepcidin analogs of the present invention may be employed in pure form or, if such forms exist, in the form of a pharmaceutically acceptable salt.

[0354] dose The total daily use amount of the hepcidin analogue and composition of the present invention can be determined by the attending physician within the scope of sound medical judgment.The specific therapeutically effective dosage level for any specific subject depends on various factors, including: a) the disorder to be treated and the severity of the disorder, b) the activity of the specific composition used, c) the specific composition used, the patient's age, weight, general health, sex and diet, d) the time of administration, the route of administration, the excretion rate of the specific hepcidin analogue used, e) the duration of treatment, f) the drugs used in combination with or simultaneously with the specific hepcidin analogue used, and similar factors known in the pharmaceutical field.

[0355] In certain embodiments, the total daily dose of the hepcidin analogs of the present invention administered to a human or other mammalian host, in single or divided doses, may be, for example, 0.0001 to 300 mg / kg body weight per day, or 1 to 300 mg / kg body weight per day. In certain embodiments, the dose of the hepcidin analogs of the present invention is in the range of about 0.0001 to about 100 mg / kg body weight per day, e.g., about 0.0005 to about 50 mg / kg body weight per day, e.g., about 0.001 to about 10 mg / kg body weight per day, e.g., about 0.01 to about 1 mg / kg body weight per day, administered in one or more doses, e.g., 1 to 3 doses. In certain embodiments, the total dose is, for example, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg for a human patient, approximately once or twice per week. In certain embodiments, the total dose ranges from about 1 mg to about 5 mg, or from about 1 mg to about 3 mg, or from about 2 mg to about 3 mg per human patient, for example, once a week.

[0356] In various embodiments, the hepcidin analogs of the invention may be administered continuously (e.g., by intravenous administration or another continuous drug administration method) or may be administered to a subject periodically, typically at regular intervals, such as once daily, twice daily, every other day, every third day, every fourth day, every fifth day, or every sixth day, weekly or twice weekly, monthly or twice monthly, etc., depending on the desired dose and pharmaceutical composition selected by one of skill in the art for a particular subject.

[0357] Under certain circumstances, such as during chronic long-term administration, it may be beneficial to interrupt the regular dosing regimen of the hepcidin analog of the present invention for a period during which the medicated subject reduces the level of the drug or stops taking the drug. This is often referred to as taking a "drug holiday." Drug holidays are useful, for example, to maintain or restore drug sensitivity, particularly during long-term chronic treatment, or to reduce undesirable side effects of long-term chronic treatment of a subject with a drug. The timing of the drug holiday depends on the timing of the regular dosing regimen and the purpose of the drug holiday (e.g., to restore drug sensitivity and / or reduce undesirable side effects of continuous, long-term administration). In some embodiments, the drug holiday may be a reduction in the drug dose (e.g., reducing the dose to below the therapeutically effective amount for a specific period of time). In other embodiments, drug administration is stopped for a specific period of time, and then administration resumes using the same or a different dosing regimen (lower or higher doses, and / or more or less frequent dosing). Thus, the drug holiday of the present invention can be selected from a wide range of durations and dosing regimens. Exemplary drug holiday periods are those of two or more days, one or more weeks, or one or more months, up to a maximum of about 24 months. Thus, for example, a regular daily dosing regimen using the peptide, peptide analog, or dimer of the present invention may be interrupted by, for example, a one-week, two-week, or four-week drug holiday, after which the previous regular dosing regimen (e.g., a daily or weekly dosing regimen) is resumed. It is contemplated that various other drug holiday regimens are useful for administering the hepcidin analogs of the present invention.

[0358] Thus, the hepcidin analog may be delivered by a dosing regimen that includes two or more dosing phases, each separated by a washout phase.

[0359] During each administration phase, the hepcidin analog is administered to the recipient subject in a therapeutically effective amount according to a predetermined administration pattern. The administration pattern may include continuously administering the agent to the recipient subject during the administration phase. Alternatively, the administration pattern may include administering multiple doses of the hepcidin analog to the recipient subject, wherein the doses are separated by administration intervals.

[0360] The dosing pattern may include at least two doses per dosing phase, at least five doses per dosing phase, at least 10 doses per dosing phase, at least 20 doses per dosing phase, at least 30 doses per dosing phase, or more.

[0361] The dosing intervals may be regular dosing intervals, which may be set as described above, including once daily, twice daily, every other day, every third day, every fifth day, or every sixth day, once or twice weekly, once or twice monthly, or regular less frequent dosing intervals, depending on the particular dosage formulation, bioavailability, and pharmacokinetic profile of the hepcidin analogs of the present invention.

[0362] The administration phase may be at least two days, at least one week, at least two weeks, at least four weeks, at least one month, at least two months, at least three months, at least six months, or longer.

[0363] If a dosing pattern includes multiple doses, the length of the subsequent drug holiday phase is longer than the dosing interval used in that dosing pattern. If the dosing intervals are irregular, the length of the drug holiday phase may be longer than the average interval between doses during the dosing phase. Alternatively, the length of the drug holiday may be longer than the longest interval between consecutive doses during the dosing phase.

[0364] The length of the drug holiday may be at least twice the length of the relevant dosing interval (or the average thereof), at least three times, at least four times, at least five times, at least ten times, or at least 20 times the length of the relevant dosing interval or the average thereof.

[0365] Within these constraints, the washout phase may be at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or longer, depending on the dosing pattern during the previous dosing phase.

[0366] The dosing regimen comprises at least two dosing phases, each successive dosing phase being separated by a washout phase. Thus, the dosing regimen may comprise at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 dosing phases, or more, each separated by a washout phase.

[0367] Sequential administration phases may utilize the same administration pattern, although this is not necessarily desirable or necessary. However, when other drugs or active agents are administered in combination with the hepcidin analogs of the present invention, the same combination of drugs or active agents are typically administered in sequential administration phases. In certain embodiments, the recipient subject is a human.

[0368] In some embodiments, the present invention provides compositions and pharmaceutical products comprising at least one hepcidin analog disclosed herein. In some embodiments, the present invention provides methods for producing pharmaceutical products comprising at least one hepcidin analog disclosed herein for treating disorders of iron metabolism, such as iron overload disorders. In some embodiments, the present invention provides methods for producing pharmaceutical products comprising at least one hepcidin analog disclosed herein for treating diabetes (type I or type II), insulin resistance, or glucose intolerance. Also provided are methods for treating disorders of iron metabolism in a subject, such as a mammalian subject, and preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount. Also provided are methods for treating diabetes (type I or type II), insulin resistance, or glucose intolerance in a subject, such as a mammalian subject, and preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount. Also provided are methods for treating diabetes (type I or type II), insulin resistance, or glucose intolerance in a subject, such as a mammalian subject, and preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount.

[0369] In some embodiments, the present invention provides processes for producing the hepcidin analogs or hepcidin analog compositions (eg, pharmaceutical compositions) disclosed herein.

[0370] In some embodiments, the present invention provides a device comprising at least one hepcidin analog of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for delivering the hepcidin analog to a subject.

[0371] In some embodiments, the present invention provides methods of binding to or inducing the internalization and degradation of ferroportin, the methods comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0372] In some embodiments, the present invention provides methods for binding to ferroportin, blocking the pore, and blocking the external transport function without causing internalization of ferroportin, comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0373] In some embodiments, the present invention provides kits comprising at least one hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) disclosed herein packaged together with reagents, devices, instructions, or a combination thereof.

[0374] In some embodiments, the invention provides methods of administering a hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) of the invention to a subject via an implant or osmotic pump, by a cartridge or micropump, as known to those of skill in the art, or by other means recognized by those of skill in the art. In some embodiments, the present invention provides complexes comprising at least one hepcidin analog disclosed herein bound to ferroportin, preferably human ferroportin, or bound to an antibody, e.g., an antibody that specifically binds to a hepcidin analog disclosed herein, Hep25, or a combination thereof.

[0375] In some embodiments, the hepcidin analogs of the invention have a measured EC value (e.g., EC ) of less than 500 nM in an FPN internalization assay. 50). As will be appreciated by those skilled in the art, the function of a hepcidin analog depends on the tertiary structure and the binding interface presented by the hepcidin analog. Thus, minor changes can be made to the sequence encoding the hepcidin analog that do not affect folding or are not on the binding interface and still maintain function. In other embodiments, the present invention provides hepcidin analogs that have 85% or greater (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity or homology to the amino acid sequence of any hepcidin analog described herein that exhibits activity (e.g., hepcidin activity) or alleviates symptoms of a disease or indication in which hepcidin is implicated.

[0376] In other embodiments, the present invention provides hepcidin analogs that have 85% or greater (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity or homology to any hepcidin analog presented herein or to the amino acid sequence of a peptide according to any one of the formulas or hepcidin analogs described herein.

[0377] In some embodiments, the hepcidin analogs of the present invention may include functional fragments or variants thereof having up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to one or more of the specific peptide analog sequences listed herein.

[0378] In addition to the methods described in the examples herein, the hepcidin analogue of the present invention can be produced by methods known in the art, including chemical synthesis, biosynthesis, or in vitro synthesis using recombinant DNA methods, and solid-phase synthesis.For example, see Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol.12, JK Setlow ed., Plenum Press, NY, pp.1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce, Rockford, IL.These documents are incorporated herein by reference in their entirety. The hepcidin analogs of the present invention can be purified using protein purification techniques known in the art, such as reverse-phase high-performance liquid chromatography (HPLC), ion exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem. 12(16):3121-3126; and Scopes (1982) Protein Purification, Springer-Verlag, NY. These documents are incorporated herein by reference in their entirety. Alternatively, the hepcidin analogs of the present invention can be produced by recombinant DNA techniques known in the art. Thus, polynucleotides encoding the polypeptides of the present invention are contemplated herein. In certain preferred embodiments, the polynucleotides are isolated. As used herein, "isolated polynucleotide" refers to a polynucleotide that is in an environment different from the environment in which the polynucleotide naturally occurs. [Example]

[0379] The following examples illustrate specific embodiments of the present invention. The following examples were carried out using standard techniques known and routine to those skilled in the art, unless otherwise specified in detail. It should be understood that these examples are for illustrative purposes only and are not intended to be completely definitive regarding the conditions or scope of the present invention. Therefore, they should not be interpreted as limiting the scope of the present invention in any way. Abbreviation DCM: dichloromethane DMF: N,N-dimethylformamide NMP: N-methylpyrrolidone HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU: 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate DCC: dicyclohexylcarbodiimide NHS: N-hydroxysuccinimide DIPEA: Diisopropylethylamine EtOH: ethanol Et2O: Diethyl ether Hy: Hydrogen TFA: Trifluoroacetic acid TIS: Triisopropylsilane ACN: acetonitrile HPLC: High-performance liquid chromatography ESI-MS: Electrospray ionization mass spectrometry PBS: phosphate buffered saline Boc: t-butoxycarbonyl Fmoc: fluorenylmethyloxycarbonyl Acm: acetamidomethyl IVA: Isovaleric acid (or isovaleryl)

[0380] K(): In the peptide sequences provided herein, when a compound or chemical group is presented in parentheses immediately following a lysine residue, the compound or chemical group within the parentheses is understood to be the side chain conjugated to the lysine residue. Thus, for example, but in no way limiting, K-[(PEG8)]- indicates that a PEG8 moiety is conjugated to the side chain of the lysine.

[0381] Palm: indicates the conjugation of palmitic acid (palmitoyl).

[0382] Synthesis Protocol-1 Synthesis of peptide monomers The peptide monomers of the present invention were synthesized using Merrifield solid-phase synthesis on a Protein Technology Symphony multichannel synthesizer. Peptides were assembled using HBTU (O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate) and diisopropylethylamine (DIEA) coupling conditions. For some amino acid couplings, PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) and DIEA conditions were used. Rink Amide MBHA resin (100-200 mesh, 0.57 mmol / g) was used for peptides with C-terminal amides, while Wang resin preloaded with N-α-Fmoc-protected amino acids was used for peptides with C-terminal acids. Coupling reagents (HBTU and DIEA premixed) were prepared at a concentration of 100 mmol. Similarly, amino acid solutions were prepared at a concentration of 100 mmol. The peptide inhibitors of the present invention are identified based on medicinal chemistry optimization and / or phage display and screened to identify those with superior binding and / or inhibitory properties.

[0383] assembly Peptides were assembled using the standard Symphony protocol. The peptide sequence was assembled as follows: In each reaction vial, resin (250 mg, 0.14 mmol) was washed twice with 4 ml of DMF, followed by treatment with 2.5 ml of 20% 4-methylpiperidine (Fmoc deprotection) for 10 minutes. The resin was then filtered, washed twice with 4 ml of DMF, and re-treated with piperidine for another 30 minutes. The resin was again washed three times with 4 ml of DMF, followed by the addition of 2.5 ml of amino acid and 2.5 ml of HBTU-DIEA mixture. After 45 minutes of frequent agitation, the resin was filtered and washed three times with 4 ml of DMF. For a typical peptide of the present invention, a double coupling was performed. After the coupling reaction was complete, the resin was washed three times with 4 ml of DMF before proceeding to the next amino acid coupling.

[0384] Disconnect After completion of peptide assembly, the peptide was cleaved from the resin by treatment with a cleavage reagent such as Reagent K (82.5% trifluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, 2.5% 1,2-ethanedithiol), which cleaved the peptide from the resin as well as any remaining side chain protecting groups.

[0385] The cleaved peptide was precipitated in cold diethyl ether and subsequently washed twice with ethyl ether. The filtrate was decanted, a second aliquot of cold ether was added, and the procedure was repeated. The crude peptide was dissolved in a solution of acetonitrile:water (7:3, containing 1% TFA) and filtered. The quality of the linear peptide was then verified using electrospray ionization mass spectrometry (ESI-MS) (Micromass / Waters ZQ) and subsequently purified.

[0386] purification Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed on a Gemini C18 column (4.6 mm × 250 mm) (Phenomenex). Semi-preparative reversed-phase HPLC was performed on a Gemini 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of B buffer in A (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at flow rates of 1 mL / min (analytical) and 20 mL / min (preparative). Separation was performed using a linear gradient of B buffer in A (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at flow rates of 1 mL / min (analytical) and 15 mL / min (preparative).

[0387] Synthesis Protocol-1 Synthesis of peptide monomers Peptide monomers of the present invention were synthesized using standard Fmoc solid-phase synthesis techniques on a CEM Liberty Blue™ microwave peptide synthesizer. Peptides were assembled using Oxyma / DIC (ethyl cyanohydroxyiminoacetate / diisopropylcarbodiimide) with microwave heating. Rink Amide-MBHA resin (100-200 mesh, 0.66 mmol / g) was used for peptides with C-terminal amides, while Wang resin preloaded with N-α-Fmoc-protected amino acids was used for peptides with C-terminal acids. Oxyma was prepared as a 1 M solution in DMF containing 0.1 M DIEA. DIC was prepared as a 0.5 M solution in DMF. Amino acids were prepared at 200 mM. Peptide inhibitors of the present invention were identified based on medicinal chemistry optimization and / or phage display, and screened to identify those with superior binding and / or inhibitory properties.

[0388] assembly Peptides were prepared using the standard CEM Liberty Blue™ protocol. The peptide sequences were assembled as follows: resin (400 mg, 0.25 mmol) was suspended in 10 ml of 50 / 50 DMF / DCM. The resin was then transferred to a reaction vessel in a microwave cavity. Peptides were assembled using repeated cycles of Fmoc deprotection and Oxyma / DIC coupling. For deprotection, a 20% solution of 4-methylpiperidine in DMF was added to the reaction vessel and heated to 90°C for 65 seconds. The deprotection solution was drained, and the resin was washed three times with DMF. For most amino acids, 5 equivalents of Oxyma and DIC were then added to the reaction vessel, and the reaction mixture was rapidly heated to 90°C for 4 minutes by microwave irradiation. For arginine and histidine residues, moderate conditions were used, using temperatures of 75°C and 50°C for 10 minutes, respectively, to prevent racemization. Rare and expensive amino acids were often coupled manually at room temperature overnight using only 1.5–2 equivalents of reagent. Difficult couplings were often performed with 2 × 4 min double couplings at 90 °C. After coupling, the resin was washed with DMF, and the entire cycle was repeated until the desired peptide assembly was complete.

[0389] Disconnect After peptide assembly was complete, the peptide was then cleaved from the resin by treatment with a standard cleavage cocktail of 91:5:2:2 TFA / HO / TIPS / DODT for 2 hours. If multiple Arg(Pbf) residues were present, cleavage was carried out for an additional hour.

[0390] The cleaved peptide was precipitated in cold diethyl ether. The filtrate was decanted, a second aliquot of cold ether was added, and the procedure was repeated. The quality of the linear peptide was then verified using electrospray ionization mass spectrometry (ESI-MS) (Waters® Micromass® ZQ™) and subsequently purified.

[0391] purification Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed on a Gemini® C18 column (4.6 mm × 250 mm) (Phenomenex). Semi-preparative reversed-phase HPLC was performed on a Gemini 10 μm C18 column (22 mm × 250 mm) (Phenomenex) or a Jupiter® 10 μm, 300A column. O Separation was performed on a C18 column (21.2 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of B buffer in A (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at a flow rate of 1 mL / min (analytical) and 20 mL / min (preparative).

[0392] Example 1A Synthesis of peptide analogues Unless otherwise specified, the reagents and solvents employed below were commercially available at standard laboratory reagent or analytical grade and were used without further purification.

[0393] Procedure for solid phase peptide synthesis Method A The peptide analogs of the present invention were chemically synthesized using an optimized 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol. For C-terminal amides, rink-amide resin was used, while wang and trityl resins were also used to generate C-terminal acids. The side chain protecting groups were as follows: Glu, Thr, and Tyr: 0t-butyl; Trp and Lys: t-Boc (t-butyloxycarbonyl); Arg: N-gamma-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; His, Gln, Asn, and Cys: trityl. For selective disulfide bridge formation, Acm (acetamidomethyl) was also used as a Cys protecting group. For coupling, a 4- to 10-fold excess of a solution containing Fmoc amino acid, HBTU, and DIEA (1:1:1.1) was added to the swollen resin [HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA: diisopropylethylamine; DMF: dimethylformamide]. HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) was used instead of HBTU to improve coupling efficiency in difficult regions. Removal of the Fmoc protecting group was achieved by treatment with a DMF, piperidine (2:1) solution.

[0394] Method B : Alternatively, peptides were synthesized using a CEM Liberty Blue Microwave-assisted peptide synthesizer. FMOC deprotection was performed using the Liberty Blue by adding a 20% solution of 4-methylpiperidine in DMF containing 0.1M Oxyma in DMF, followed by heating to 90°C for 4 minutes using microwave irradiation. After a DMF wash, FMOC-amino acids were coupled by adding 0.2M amino acid (4-6 equivalents), 0.5M DIC (4-6 equivalents), and 4-6 equivalents of 1M Oxyma (containing 0.1M DIEA) (all in DMF). The coupling solution was heated to 90°C for 4 minutes using microwave irradiation. When coupling Arg or other sterically hindered amino acids, a second coupling was performed. When coupling with histidine, the reaction was heated to 50°C for 10 minutes. The cycle was repeated until the full-length peptide was obtained.

[0395] Procedure for cleaving peptide from resin Deprotection and cleavage of the side chains of peptide analogs of the present invention (e.g., Compound No. 2) were carried out by stirring the dried resin in a solution containing trifluoroacetic acid, water, ethanedithiol, and triisopropylsilane (90:5:2.5:2.5) for 2-4 hours. After removing the TFA, the peptide was precipitated using ice-cold diethyl ether. The solution was centrifuged, and the ether was decanted, followed by a second diethyl ether wash. The peptide was dissolved in a solution of acetonitrile and water (1:1) containing 0.1% TFA (trifluoroacetic acid), and the resulting solution was filtered. The quality of the linear peptide was assessed using electrospray ionization mass spectrometry (ESI-MS).

[0396] Peptide purification procedure Purification of peptides of the present invention (e.g., Compound No. 2) was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Analysis was performed using a C18 column (3 μm, 50 × 2 mm) at a flow rate of 1 mL / min. Purification of linear peptides was performed using preparative RP-HPLC with a C18 column (5 μm, 250 × 21.2 mm) at a flow rate of 20 mL / min. Separation was performed using a linear gradient of buffer B in A (Buffer A: 0.05% TFA in water, Buffer B: 0.043% TFA, 90% acetonitrile in water).

[0397] Those skilled in the art will appreciate that standard methods of peptide synthesis may be used to produce the compounds of the present invention.

[0398] Half-life extending moiety conjugates Peptide conjugation was performed on the resin. Lys(ivDde) ​​was used as the primary amino acid. After on-resin peptide assembly, selective deprotection of the ivDde group was performed using 2% hydrazine in DMF for 5 min (3 times 5 min). Linker activation and acylation were performed using HBTU and 1-2 equivalents of DIEA for 3 h. Fmoc removal followed by a second acylation with a lipid acid afforded the conjugated peptide. Example 1B Synthesis of peptide: Isovaleric acid-Glu-Thr-His-DIP-Pro-Ala-Ile-Lys(Ahx-Palm)-bhF-NH2 (Peptide No. 9)

[0399] The TFA salt of Peptide No. 9 was synthesized on a 0.13 mmol scale. Upon completion, 45.31 mg of >95% pure Peptide No. 9 was isolated as a white powder for an overall yield of 21.5%.

[0400] Peptide No. 9 was synthesized using Merrifield solid-phase synthesis on a Protein Technology Symphony multichannel synthesizer and assembled onto Rink Amide MBHA (100-200 mesh, 0.66 mmol / g) resin using standard Fmoc-protection synthesis conditions. The assembled peptide was isolated from the resin and protecting groups by cleavage with strong acid followed by precipitation. The crude precipitate was then purified by RP-HPLC. Lyophilization of the pure fractions afforded the final product, Peptide No. 9.

[0401] Peptide assembly Swollen resin: 200 mg of Rink Amide MBHA solid phase resin (0.66 mmol / g loading) was transferred to a 25 mL reaction vessel (for Symphony peptide synthesizers). The resin was swelled with 3.75 mL of DMF (3 x 10 min).

[0402] Step 1: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-βhomo-L-Phe-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min) before the next deprotection / coupling cycle was initiated.

[0403] Step 2: Coupling of FMOC-L-Lys(IvDde)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 mL of 20% piperidine in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Lys(IvDde)-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0404] Step 3: Coupling of FMOC-L-Dpa-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 mL of 20% piperidine in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Ile-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0405] Step 4: Coupling of FMOC-L-Ala-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Ala-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0406] Step 5: Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of the amino acid FMOC-Pro-OH in DMF and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0407] Step 6: Coupling of FMOC-L-DIP-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-DIP-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0408] Step 7: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-His(Trt)-OH and 2.5 mL of a DMF mixture of the coupling reagents HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0409] Step 8: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Thr(tBu)-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0410] Step 9: Coupling of FMOC-L-Glu(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 mL of 20% piperidine in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Glu(tBu)-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0411] Step 10: Coupling of isovaleric acid: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin with 2.5 mL of a 20% piperidine solution in DMF twice for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of isovaleric acid in DMF and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0412] Step 11: Removal of IvDde and coupling of Fmoc-Ahx-OH: 2-5% The IvDde was removed from the Lys C-terminus of the resin-bound peptide using hydrazine in DMF (4 × 30 min), followed by a DMF wash. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid Fmoc-Ahx-OH and 2.0 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min) before the next deprotection / coupling cycle was initiated.

[0413] Step 12: Coupling of palmitic acid: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of isovaleric acid in DMF and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0414] Step 13: TFA cleavage and ether precipitation: 10 ml of cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT)] was added to the protected resin-bound peptide and shaken for 2 hours. Cold diethyl ether was added to form a white precipitate, followed by centrifugation. The ether was decanted and discarded, and the precipitate was washed twice more with ether. The resulting white precipitate cake was dissolved in acetonitrile / water (7:3), filtered, and subsequently purified.

[0415] Step 14: RP-HPLC purification: Semi-preparative reverse-phase HPLC was performed on a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of B buffer in A (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at a flow rate of 20 mL / min (preparative).

[0416] Step 15: Final lyophilization and analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions with a purity greater than 95% were combined. The combined fractions were lyophilized to obtain peptide No. 9 as a white powder with a purity of 97%. Low-resolution LC / MS of the purified peptide No. 9 gave a single charge state for the peptide, an M+2 / 2 of 807.70, and a molecular ion [M+1] of 1613.80. The experimental mass is consistent with the theoretical mass of 1614.0 Da [M+1]. Example 1C Synthesis of peptide: Isovaleric acid-Glu-Thr-His-Dpa-Pro-Ala-Ile-(D)Lys-bhF-Lys(Ahx-Palm)-NH2 (Peptide No. 4)

[0417] The TFA salt of Peptide No. 4 was synthesized on a 0.13 mmol scale. Upon completion, 27.74 mg of >95% pure Peptide No. 4 was isolated as a white powder for an overall yield of 12.2%.

[0418] Peptide No. 4 was synthesized using Merrifield solid-phase synthesis. The peptide was synthesized on a Rink Amide MBHA (100-200 mesh, 0.66 mmol / g) resin using standard Fmoc-protection synthesis conditions on a Sigma-Aldrich Technology Symphony multichannel synthesizer. The assembled peptide was isolated from the resin and protecting groups by cleavage with strong acid followed by precipitation. The crude precipitate was then purified by RP-HPLC. Lyophilization of the pure fractions afforded the final product, peptide number 4.

[0419] Peptide assembly Swollen resin: 200 mg of Rink Amide MBHA solid phase resin (0.66 mmol / g loading) was transferred to a 25 mL reaction vessel (for Symphony peptide synthesizers). The resin was swelled with 3.75 mL of DMF (3 x 10 min).

[0420] Step 1: Coupling of FMOC-L-Lys(IvDde)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Lys(IvDde)-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0421] Step 2: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-βhomo-L-Phe-OH and 2.5 mL of a DMF mixture of the coupling reagents HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min) before the next deprotection / coupling cycle was initiated.

[0422] Step 3: Coupling of FMOC-D-Lys(Boc)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-D-Lys(Boc)-OH and 2.5 mL of a DMF mixture of the coupling reagents HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0423] Step 4: Coupling of FMOC-L-Ile-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of the amino acid FMOC-L-Ile-OH in DMF and 2.5 mL of a 200 and 220 mM solution of the coupling reagent HBTU-DIEA in DMF. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0424] Step 5: Coupling of FMOC-L-Ala-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of 20% piperidine in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Ala-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0425] Step 6: Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of the amino acid FMOC-Pro-OH in DMF and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0426] Step 7: Coupling of FMOC-L-Dpa-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of 20% piperidine in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-DIP-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0427] Step 8: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-His(Trt)-OH and 2.5 mL of a DMF mixture of the coupling reagents HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0428] Step 9: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Thr(tBu)-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0429] Step 10: Coupling of FMOC-L-Glu(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid FMOC-L-Glu(tBu)-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0430] Step 11: Coupling of isovaleric acid: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin with 2.5 mL of a 20% piperidine solution in DMF twice for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of isovaleric acid in DMF and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0431] Step 12: Removal of IvDde and coupling of Fmoc-Ahx-OH: 2-5% The IvDde was removed from the Lys C-terminus of the resin-bound peptide using hydrazine in DMF (4 × 30 min), followed by a DMF wash. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid Fmoc-Ahx-OH and 2.5 mL of a 200 and 220 mM DMF solution of the coupling reagent HBTU-DIEA. The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min) before the next deprotection / coupling cycle was initiated.

[0432] Step 13: Coupling of palmitic acid: Deprotection of the Fmoc group was carried out by treating the swollen Rink Amide resin twice with 2.5 mL of a 20% piperidine solution in DMF for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 x 0.1 min), followed by the addition of 2.5 mL of a 200 mM solution of isovaleric acid in DMF and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 x 0.1 min) before the next deprotection / coupling cycle was initiated.

[0433] Step 14: TFA cleavage and ether precipitation: 10 ml of cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT)] was added to the protected resin-bound peptide and shaken for 2 hours. Cold diethyl ether was added to form a white precipitate, followed by centrifugation. The ether was decanted and discarded, and the precipitate was washed twice more with ether. The resulting white precipitate cake was dissolved in acetonitrile / water (7:3), filtered, and subsequently purified.

[0434] Step 15: RP-HPLC purification: Semi-preparative reverse-phase HPLC was performed on a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of B buffer in A (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at a flow rate of 20 mL / min (preparative).

[0435] Step 16: Final lyophilization and analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions with a purity of greater than 95% were combined. The combined fractions were lyophilized to obtain peptide No. 4 as a white powder with a purity of 97%. Low-resolution LC / MS of the purified peptide No. 4 gave two charge states for the peptide, an M+3 / 3 of 581.5, an M+2 / 2 of 871.70, and a molecular ion of 1741.90 [M+1]. The experimental mass is consistent with the theoretical mass of 1742.09 Da [M+1]. Example 2A: Peptide analogue activity

[0436] The peptide analogs were tested in vitro for induction of internalization of human ferroportin protein. After internalization, the ferroportin protein is degraded. The assay used (FPN activity assay) measures the decrease in receptor fluorescence.

[0437] The cDNA encoding human ferroportin (SLC40A1) was cloned from the cDNA clone from Origene (NM_014585). DNA encoding ferroportin was amplified by PCR using primers that also encoded terminal restriction sites for subcloning but did not contain a stop codon. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker, and the ferroportin reading frame was fused in-frame with the GFP protein. The fidelity of the DNA encoding the protein was confirmed by DNA sequencing. HEK293 cells were transfected with the ferroportin-GFP receptor expression plasmid. Cells were grown in growth medium according to standard protocols and transfected with the plasmid using Lipofectamine (manufacturer's protocol, Invitrogen). Cells stably expressing ferroportin-GFP were selected using G418 in growth medium (only cells that have taken up and integrated the cDNA expression plasmid survive) and sorted several times on a Cytomation MoFlo™ cell sorter to obtain GFP-positive cells (488 nm / 530 nm). Cells were expanded and frozen in aliquots.

[0438] To determine the activity of hepcidin analogs (compounds) against human ferroportin, cells were incubated in 96-well plates in standard medium without phenol red. Compounds were added to the desired final concentration for at least 18 hours in the incubator. After incubation, residual GFP fluorescence was determined either by total cell GFP fluorescence (Envision plate reader, 485 / 535 filter pair) or by a Beckman Coulter Quanta™ flow cytometer (expressed as the geometric mean of 485 nm / 525 nm fluorescence intensity). Compounds were added to the desired final concentration for at least 18 hours, but not more than 24 hours, in the incubator.

[0439] In certain experiments, the reference compounds included native hepcidin, minihepcidin, and the minihepcidin analog, R1-minihepcidin. The "RI" in RI-minihepcidin stands for Retro Inverse. Retro-inverse peptides are peptides with the reverse sequence in all D amino acids. For example, Hy-Glu-Thr-His-NH2 becomes Hy-DHis-DThr-DGlu-NH2. The EC values ​​of these reference compounds for ferroportin internalization / degradation were: 50 was determined according to the FPN activity assay described above. These peptides were used as control standards. [Table 11] EC determined for various peptide analogs of the present invention 50 Potency values ​​(nM) are provided in Tables 6A, 6B, and 6C. These values ​​were determined as described herein. Compound ID numbers are indicated by "Compound ID" and reference compounds are indicated by "Reference Compound." The FPN EC determined from these data 50 The values ​​are shown in Tables 6A, 6B and 6C. T47D(MSA)IC 50 Values ​​are shown in Table 6D. If not shown, the data has not yet been determined. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Table 13] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8] [Table 15-1] [Table 15-2]

[0440] Example 2C: Peptide analogue activity The potency of peptides to induce ferroportin internalization was evaluated in a T47D cell-based assay. The T47D cell line (HTB 133, ATCC) is a human breast carcinoma adherent cell line that endogenously expresses ferroportin. In this internalization assay, the potency of test peptides was evaluated in the presence of serum albumin, a major protein component in blood. T47D cells were maintained in RPMI medium (containing the required amount of fetal bovine serum) and passaged periodically. To prepare the assay, cells were seeded into 96-well plates at a density of 80–100 k cells per well in a 100 μL volume and allowed to stand overnight. The following day, test peptides were first prepared in a dilution series (10-point series, starting at approximately 5 μM and typically diluted 3–4 times), all containing 0.5% mouse serum albumin (MSA purified from mouse serum; Sigma, A3139). The test peptide dilution series was incubated at room temperature for 30 minutes. The medium was then aspirated from the 96-cell well plate, and the test peptide series was added. After 1 hour of incubation, the medium containing the test peptide was aspirated, and the AF647-conjugated detection peptide was added at a fixed concentration of 200 nM. The AF647-conjugated detection peptide has previously been demonstrated to bind to ferroportin and cause its internalization. After 2 hours of incubation, the cells were washed again and prepared for flow cytometry analysis. The median fluorescence intensity (MFI) of the AF647-positive population was measured (after removing dead and non-single cells from the analysis). The MFI values ​​were used to generate dose-response curves and obtain IC50 values ​​for the test peptides. IC50 values ​​were calculated using a four-parameter nonlinear fit function in Graphpad Prism (Table 6D). [Table 16-1] [Table 16-2]

[0441] Example 2D LAD2 activity of peptide analogues A key mechanism of anaphylactoid reactions involves the release of anaphylactic mediators, such as histamine and β-hexosaminidase, via direct stimulation of mast cells or basophils. A recent study by McNeil et al. (2015) demonstrated that a specific membrane receptor, MrgprX2, on human mast cells induces anaphylactoid reactions. Laboratory of Allergic Diseases 2 (LAD2) is a human mast cell line derived from a human mast cell sarcoma / leukemia (Kirshenbaum et al., 2003). LAD2 cells are commonly used in the study of anaphylactoid reactions because they share biological properties with primary human mast cells, including overexpression of the MrgprX2 receptor and sensitivity to degranulating peptides (Kulka et al., 2008). For example, the release of anaphylactic mediators such as β-hexosaminidase is assessed fluorimetrically.

[0442] The degranulation ability of hepcidin mimetics was evaluated in LAD2 cells. On the day of the assay, serial dilutions of compounds were added to LAD2 cells seeded at 20,000 cells / well in a 96-well plate. After 30 minutes of incubation, the amount of β-hexosaminidase released into the supernatant and in the cell lysate was quantified using the fluorescent substrate 4-methylumbelliferyl-N-acetyl-bD-glucosaminide. A dose-response curve was generated by plotting the percentage of β-hexosaminidase release (y-axis) against the concentration of the test peptide (x-axis). EC 50 Values ​​and standard errors were calculated using XLfit 5.5.0.5 based on the following formula: 4 parameter sigmoidal model: f=(A+((BA) / (1+((C / x)^D)))), where A=Emin, B=Emax, C=EC50 and D=slope. References: McNeil BD et al., Nature, 12, 519 (2015); Kirshenbaum et al. Leukemia Res. 27, 677 (2003); Kulka et al. Immunology 123, 398 (2008).

[0443] Example 3 In vivo validation of peptide analogues Hepcidin analogs of the present invention were tested for in vivo activity to determine their ability to reduce free Fe2+ in serum.

[0444] Hepcidin analogs or vehicle control were administered intravenously or subcutaneously at 1000 nmol / kg to mice (n=3 / group). Serum samples were collected from the hepcidin analog-treated mice at 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 30 hours, 36 hours, and 48 hours post-administration. Plasma / serum iron content was measured using a colorimetric assay on a Cobas c 111 according to the assay manufacturer's instructions (Assay: IRON2:ACN 661).

[0445] In separate experiments, various hepcidin analogs or vehicle control were administered subcutaneously to mice (n=3 / group) at 1000 nmol / kg. Serum samples were collected from vehicle- or hepcidin analog-administered mice 30 and 36 hours after administration. Plasma / serum iron content was measured using a colorimetric assay on a Cobas c 111 according to the assay manufacturer's instructions (Assay: IRON2:ACN 661).

[0446] These studies demonstrated that the hepcidin analogs of the present invention reduced serum iron levels for at least 30 hours, thus demonstrating increased serum stability.

[0447] Example 4 In vitro validation of peptide analogues Based in part on the structure-activity relationships (SAR) determined from the results of the experiments described herein, various hepcidin-like peptides of the invention were synthesized using the methods described in Example 1 and validated for in vitro activity as described in Example 2. Reference compounds included native hepcidin, minihepcidin, R1-minihepcidin, Reference Compound 1, and Reference Compound 2. The EC of the peptides 50 Values ​​are shown in summary Tables 6A-C.

[0448] Example 5 plasma stability Plasma stability experiments were performed to complement the in vivo results and aid in the design of potent and stable ferroportin agonists. In vitro stability studies were first performed on these materials to predict their stability in rat and mouse plasma.

[0449] The peptide of interest (20 μM) was incubated with plasma (Bioreclamation IVT) pre-warmed to 37°C. Aliquots were taken at various time points (e.g., 0, 0.25, 1, 3, 6, and 24 h) up to 24 h and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, containing 1 μM internal standard). The quenched samples were stored at 4°C until the end of the experiment and centrifuged at 17,000 g for 15 min. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The remaining fraction at each time point was calculated based on the peak area ratio (analyte to internal standard) compared to the initial value at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

[0450] Example 6 Serum iron depletion in mice A hepcidin mimetic compound designed for oral stability was tested for systemic absorption via PO administration in a wild-type mouse model, C57BL / 6. Animals were acclimated to normal rodent chow for 4-5 days and fasted overnight before the start of the study. Groups of four animals each received either vehicle or compound. Compounds were formulated in saline at a concentration of 5 mg / mL. Mice were administered the solution by oral gavage in a volume of 200 μl per animal weighing 20 g. Each group received one dose of compound at 50 mg / kg / dose. The group labeled vehicle received the formulation alone. Blood was collected 4 hours after administration, and serum was prepared for PK and PD measurements. Compound concentrations were measured by mass spectrometry, and iron concentrations in the samples were measured using a colorimetric method on a Roche Cobas C system.

[0451] Example 7 Serum iron depletion in mice In a separate experiment, a new set of compounds was tested for systemic absorption via PO administration in a wild-type mouse model, C57BL / 6. Animals were acclimated to normal rodent chow for 4–5 days prior to the start of the study. The night before the first dose, mice were switched to a low-iron diet (2 ppm iron), and this diet was maintained for the remainder of the study. Groups of five animals each received either vehicle or compound. Compounds were formulated at 30 mg / mL in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withdrawn approximately 2 h before each dose to ensure the stomach was clear of food residue before PO administration. Mice were administered the solution by oral gavage in a volume of 200 μl per 20 g animal. Each group received two doses of compound at 300 mg / kg / dose on consecutive days. The group labeled vehicle received the formulation alone. Blood was drawn 4.5 hours after the last dose, and serum was prepared for measurement of PD. Serum iron concentrations were measured using a colorimetric method on a Roche cobas c system.

[0452] Example 8 Pharmacodynamic effects of representative compounds on their ability to reduce serum iron in mice In a second in vivo study, representative compounds were tested for pharmacodynamic effects, comparing a single dose of 300 mg / kg / dose with two doses of 300 mg / kg QD (once daily) for two days. C57BL / 6 mice were acclimated to normal rodent chow for 4–5 days prior to the start of the study. The night before the first dose, mice were switched to a low-iron diet (2 ppm iron), and this diet was maintained for the remainder of the study. Groups of five animals each received either vehicle or compound. Compounds were formulated in 0.7% NaCl + 10 mM sodium acetate buffer at a concentration of 30 mg / mL. Food was withdrawn approximately 2 hours before each dose to ensure the stomach was clear of food residue before PO administration. Mice were administered the solution by oral gavage in a volume of 200 μl per 20 g animal.

[0453] Example 9 PK / PD effects of oral administration of representative compounds of the present invention in mice In another in vivo study using a healthy wild-type mouse model, C57 / BL6, representative compounds were administered multiple times over a three-day period to test their PK and PD effects. During acclimation, mice were maintained on normal rodent chow and switched to an iron-deficient diet (containing approximately 2 ppm iron) overnight before the first dose. Groups of five mice each received a total of six doses of either representative compounds of the present invention at various dose strengths or vehicle BID over a three-day period. Mice were orally gavaged with representative compounds formulated in 0.7% saline and 10 mM sodium acetate. Each group received either vehicle, 150 mg / kg / dose BID, 75 mg / kg / dose BID, 37.5 mg / kg / dose BID, or 18.75 mg / kg / dose BID. An additional group received 100 mg / kg / day BID in addition to the compound in drinking water (DW) for a total dose of 100 mg / kg / day, resulting in a total dose of 300 mg / kg / day. Three hours after the final dose, the vehicle group receiving iron and all compound-treated groups were administered an iron solution by oral gavage at 4 mg / kg iron per animal. Blood was collected 90 minutes after iron administration to prepare serum for PK and PD measurements. Compound concentrations were measured by mass spectrometry, and iron concentrations in the samples were measured using a colorimetric assay on a Roche Cobas C system.

[0454] Example 10 Serum iron depletion in mice In a separate screen, a new set of compounds was tested for pharmacodynamic effects when administered orally to a wild-type mouse model, C57BL / 6. Animals were acclimated to normal rodent chow for 4–5 days prior to the start of the study. A group of five animals designated to receive two doses of a representative compound was fed an iron-deficient diet (containing 2 ppm iron) the night before the first dose, while all other groups designated to receive a single dose of another compound were treated with the iron-deficient diet for two nights prior to compound administration. The compound concentration in the dosing solution was 30 mg / mL and formulated in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withdrawn approximately 2 h before all doses to ensure the stomach was clear of food residue before PO administration. Mice were administered the solution by oral gavage in a volume of 200 μl per 20 g animal. The group designated as vehicle received the formulation alone. Blood was drawn 4.5 hours after the last dose, and serum was prepared for measurement of PD. Serum iron concentrations were measured using a colorimetric method on a Roche cobas c system.

[0455] Example 11 Stability in simulated gastric fluid Blank SGF was prepared by adding 2 g of sodium chloride, 7 mL of hydrochloric acid (37%) to a final volume of 1 L of water and adjusting the pH to 1.2.

[0456] SGF was prepared by dissolving 320 mg of pepsin (Sigma® P6887, derived from porcine gastric mucosa) in 100 mL of blank SGF and stirring for 30 minutes at room temperature. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at −20°C.

[0457] Experimental compounds of interest (at a concentration of 20 μM) were incubated with SGF pre-warmed to 37°C. Aliquots were taken at various time points (e.g., 0, 0.25, 1, 3, 6, and 24 h) up to 24 h and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, containing 1 μM internal standard). The quenched samples were stored at 4°C until the end of the experiment and centrifuged at 4,000 rpm for 10 min. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The remaining fraction at each time point was calculated based on the peak area ratio (analyte to internal standard) compared to the initial value at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

[0458] Example 12 Stability in simulated intestinal fluid Blank FaSSIF was prepared by dissolving 0.348 g NaOH, 3.954 g sodium phosphate monobasic monohydrate, and 6.186 g NaCl in a final volume of 1 liter of water (pH adjusted to 6.5).

[0459] FaSSIF was prepared by dissolving 1.2 g of porcine pancreatin (Chem-supply, PL378) in 100 mL of blank FaSSIF and stirring for 30 min at room temperature. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at −20°C.

[0460] Experimental compounds of interest (20 μM) were incubated with FaSSIF (1% pancreatin in the final incubation mixture) pre-warmed to 37°C. Aliquots were taken at various time points (e.g., 0, 0.25, 1, 3, 6, and 24 h) up to 24 h and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, containing 1 μM internal standard). The quenched samples were stored at 4°C until the end of the experiment and centrifuged at 4,000 rpm for 10 min. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The remaining fraction at each time point was calculated based on the peak area ratio (analyte to internal standard) compared to the initial value at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

[0461] Example 13 Modification experiments for peptides prone to "non-specific binding" The target compound (at a concentration of 20 μM) was mixed with pre-warmed FaSSIF (1% pancreatin in the final working solution). The solution mixture was aliquoted and incubated at 37°C. The number of aliquots required was equal to the number of time points (e.g., 0, 0.25, 1, 3, 6, and 24 hours). One aliquot was taken at each time point and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, containing 1 μM internal standard). The remaining steps were the same as in the comprehensive experiment.

[0462] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety.

[0463] At least some of the chemical names and sequences of the present invention presented and described herein may have been generated automatically using commercially available chemical naming software programs and have not been independently verified. In the event of a difference between a given chemical name or sequence and a depicted structure, the depicted structure shall prevail. In chemical structures in which a chiral center is present but no specific stereochemistry is depicted for the chiral center, both enantiomers associated with the chiral structure are encompassed by the structure. Similarly, for peptides in which E / Z isomers exist but are not specifically mentioned, both isomers are specifically disclosed and intended to be encompassed.

[0464] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims

[Claim 1] The invention described in this specification.

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