Conjugated hepcidin mimetic

Abstract

To provide a hepcidin analog having an improved in vivo half-life, as well as related pharmaceutical compositions and methods of use thereof. [Solution] In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal X1 residue. In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal Asp residue. In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal Glu residue. In certain embodiments, the peptide analog of the present invention includes an amidated C-terminal residue.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of and priority to 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 / 169,538, filed April 1, 2021, and U.S. Provisional Patent Application No. 63 / 325,323, filed March 30, 2022, each of which is incorporated herein by reference in its entirety.

[0002] The present invention relates, in particular, to certain hepcidin peptide analogs comprising both peptide monomers and peptide dimers, as well as their conjugates and derivatives, and compositions comprising such peptide analogs, and also to the use of such peptide analogs in the treatment and / or prevention of various diseases, conditions, or disorders, including polycythemia, e.g., polycythemia vera; iron overload disorders, e.g., hereditary hemochromatosis; iron-overload anemia; and other conditions and disorders described herein. [Background technology]

[0003] 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 transport channel ferroportin, causing its internal translocation 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 physiologically active 25-amino acid form of hepcidin is a simple hairpin with eight cysteine ​​molecules 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 the deletion of five N-terminal amino acid residues results in loss of iron regulatory function. See Nemeth et al. (2006) Blood 107:328-33.

[0004] Abnormal hepcidin activity is associated with iron overload disorders, including hereditary hemochromatosis (HH) and iron-overload anemia. Hereditary hemochromatosis is a hereditary iron overload disorder caused primarily by hepcidin deficiency, or in some cases, hepcidin resistance. This allows for excessive iron absorption from the diet and the development of iron overload. Clinical manifestations of HH may include liver disease (e.g., cirrhosis, NASH, and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is routine venotomy, which places a significant burden on patients. Iron-overload anemia is a hereditary anemia with severe iron overload and ineffective erythropoiesis, such as β-thalassemia. Complications due to iron overload are the main cause of morbidity and death in these patients. Hepcidin deficiency is the main cause of iron overload in patients who have not received blood transfusions and contributes to iron overload in patients who have received transfusions. The current treatment for iron overload in these patients is iron chelation, which is very burdensome, sometimes ineffective, and frequently accompanied by side effects.

[0005] Hepcidin has several limitations that restrict its use as a drug, including a difficult synthesis process that leads to low bioavailability, low injection site reaction, low immunogenicity, and high commercial costs, partly due to protein aggregation and precipitation during folding. There is a need in the art for compounds that have hepcidin activity and also possess other beneficial physical properties such as improved solubility, stability, and / or potency, so that hepcidin-like compounds can be produced inexpensively and used to treat hepcidin-related diseases and disorders, such as those described herein. To address this need, the present invention provides novel peptide analogs, including both peptide monomer analogs and peptide dimer analogs, that possess hepcidin activity and other beneficial properties that make the peptides of the present invention suitable substitutes for hepcidin. [Prior art documents] [Non-patent literature]

[0006] [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 [Overview of the project] [Means for solving the problem]

[0007] The present invention generally relates to peptide analogs comprising both monomers and dimers exhibiting hepcidin activity, and to methods of using the same.

[0008] In one aspect, the present invention relates to formula (I’): R 10 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (I’) (wherein R 1 is hydrogen, C1-C6 alkyl, C6-C 12 aryl, C6-C 12 aryl-C1-C6 alkyl, C1-C 20 alkanoyl, C1-C 20 cycloalkanoyl, C6-C 10 aryl-C1-C6 alkylene-C(O)-, 5- or 6-membered heteroaryl-C(O)-, or 5- or 6-membered heterocycloalkyl-C(O)-, wherein the C1-C 1 alkanoyl, the C6-C 20 aryl-C1-C6 alkylene-C(O)-, the 5- or 6-membered heteroaryl-C(O)-, and the 5- or 6-membered heterocycloalkyl-C(O)- are each optionally substituted with one, two, or three substituents independently selected from halo, OH, CN, C1-C6 alkoxy, C1-C6 alkyl, -COOH, -COO(C1-C6), C1-C6 haloalkyl, and C1-C6 haloalkoxy, R 2 is NH2, substituted amino, OH, substituted hydroxy, C1-C 20 alkylamino, phenyl-C1-C8 alkylene-NH-, wherein the C1-C 20アルキル amino and phenyl-C1-C8 alkylene-NH- are each optionally substituted with substituents independently selected from NH2, -COOH, and phenyl, ​​X1 is either absent, or is 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, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Glu, Aad, Dap, or isoGlu. X2 is Ala, Thr, Gly, N-substituted Gly, Ser, Cys, 4S_Mcp, 4R_Mcp, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, Cys, (D)Cys, aMeCys, hCys, Pen, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or Cys, (D)Cys, aMeCys, hCys, Pen, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, 4S_Mcp, 4R_Mcp, Morph, or Tic. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, NMe-Cys, Ala, Lys, (D)Lys, substituted Lys, or substituted (D)Lys, X7 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, Phe, or DIP. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, 123triazole, Cys, (D)Cys, aMeCys, hCys, Pen, Lys_Me3, X9 is either absent, or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. X12 to X14 are either absent or are each an amino acid independently. The peptide of formula (I') is further optionally conjugated to any of the amino acids, Any of the amino acids in the peptide of formula (I') is optionally substituted with the corresponding (D)-amino acid, or optionally N-substituted. (i) At least one of X1, X3-X5, and X7-X10 is Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp, where the peptide has a mercapto group on the side chain of one of X1, X3-X5, and X7-X10, a mercapto group on the side chain of X6, and L x Combine them, -SLx -S-bonds are formed, or the mercapto group on the side chain of X1, the mercapto group on the side chain of X8, and L x Together, -SL x -S-bonds are formed, or the mercapto group on the side chain of X2, the mercapto group on the side chain of X5, and L x Together, -SL x It is cyclized by forming an -S- bond, in the formula, L x Each of them is independently a bonding or linking group, (ii) The functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. Here, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor It is leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting bhPhe is β-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. The substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu. The substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH, or t-Bu. A substituted Lys, substituted dLys, or substituted NMe-Lys, or substituted NMe-dLys is Lys, dLys, NMe-Lys, or NMe-dLys, where the ε-amino group on the side chain of Lys, dLys, NMe-Lys, or NMe-dLys is covalently bonded to a C1-C6 alkanoyl, a half-life extension portion, or a linking portion covalently bonded to a half-life extension portion, and the linking portion includes one or more linker portions covalently bonded to each other. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123triazole [ka] And, Dab [ka] The present invention provides a hepcidin analog containing a peptide having the amino acid sequence of ( ), or a pharmaceutically acceptable salt or solvate thereof.

[0009] In a particular embodiment, the present invention relates to formula (I): R 1-X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (I) (In the formula, R 1 However, hydrogen, C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, C1-C 20 It is a cycloalkanoyl, R 2 However, it is NH2, substituted amino, OH, or substituted hydroxy, X0 is either absent, or Cys, (D)Cys, aMeCys, hCys, or Pen. X1 is either absent, or is 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, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X2 is Ala, Thr, Gly, N-substituted Gly, or Ser. X3 is Ala, Gly, N-substituted Gly, His, substituted His, or Cys, (D)Cys, aMeCys, hCys, or Pen. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or Cys, (D)Cys, aMeCys, hCys, or Penl. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X6 is Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either absent, or is Ala, Gly, N-substitute Gly, Ile, Val, Leu, NLeu, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X8 is either absent, or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, 123-triazole, or Cys, (D)Cys, aMeCys, hCys, or Pen. X9 is either absent, or Ala, Ile, Gly, N-substitute Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. X12 to X14 are either absent or each is independently one of the amino acids, however, i) The peptide may be further conjugated with any amino acid, ii) Any of the amino acids in the peptide may be the corresponding (D)-amino acid, or may be N-substituted, and iii) Provided that at least one of X0, X1, X3~X5, X7~X10 is Cys, (D)Cys, aMeCys, hCys, or Pen, and Cys, (D)Cys, aMeCys, hCys, or Pen forms a disulfide bond with X6, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, and bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting bhPhe is β-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. The substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu. The substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH, or t-Bu. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123triazole [ka] And, Dab [ka] It contains a hepcidin analog containing the peptide of ( ), or a pharmaceutically acceptable salt or solvate thereof.

[0010] In one embodiment, X0 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X0 and X6 are linked via a disulfide bond.

[0011] In one embodiment, X1 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X1 and X6 are linked via a disulfide bond.

[0012] In one embodiment, X3 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X3 and X6 are linked via a disulfide bond.

[0013] In one embodiment, X4 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X4 and X6 are linked via a disulfide bond.

[0014] In one embodiment, X5 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X5 and X6 are linked via a disulfide bond.

[0015] In one embodiment, X7 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X7 and X6 are linked via a disulfide bond.

[0016] In one embodiment, X8 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X8 and X6 are linked via a disulfide bond.

[0017] In one embodiment, X9 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X9 and X6 are linked via a disulfide bond.

[0018] In one embodiment, X10 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X10 and X6 are linked via a disulfide bond.

[0019] In one embodiment, R 1 This is IVA or isovaleric acid.

[0020] In one embodiment, R 2 is NH2. In one embodiment, R 2 It is OH.

[0021] In any particular embodiment of the hepcidin analogs of the present invention, the substituted Lys or substituted (D)Lys is either directly substituted or substituted via a linker having an acid selected from C12 (lauric acid), C14 (mysteric acid), C16 (palmitic acid), C18 (stearic acid), C20, C12 diacitate, C14 diacitate, C16 diacitate, C18 diacitate, C20 diacitate, biotin, and isovaleric acid, or a residue thereof. In one embodiment, the linker is Ahx, PEG, or PEG-Ahx.

[0022] In any particular embodiment of the hepcidin analogs of the present invention, X8 or X10 is Lys or (D)Lys substituted with L1Z, where L1 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)-CH2-(Peg) n -N(H)] m -or-[C(O)-CH2-CH2-(Peg) n -N(H)] m -, Peg is -OCH2CH2-, m is 1, 2, or 3, n is an integer from 1 to 100K, and Z is the half-life extension portion. In one embodiment, the half-life extension portion is C 10 -C 21 It is Alkanoyl.

[0023] In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal X1 residue. In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal Asp residue. In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal Glu residue.

[0024] In certain embodiments, the peptide analog of the present invention comprises an amidated C-terminal residue.

[0025] In certain embodiments, the peptide analogs of the present invention include or consist of the amino acid sequences or structures shown in Tables 6A to 6B, or variants thereof.

[0026] In related embodiments, the present invention includes polynucleotides encoding a hepcidin analog or dimer (or monomeric subunit of the dimer) peptide of the present invention.

[0027] In further related embodiments, the present invention includes a vector comprising the polynucleotide of the present invention. In certain embodiments, the vector is an expression vector comprising a promoter operably ligated to the polynucleotide in a manner that promotes the expression of the polynucleotide, for example.

[0028] In another embodiment, the present invention includes a pharmaceutical composition comprising a hepcidin analog, dimer, polynucleotide, or vector of the present invention and a pharmaceutically acceptable carrier, excipient, or vehicle.

[0029] In another embodiment, the present invention provides a method for binding to ferroportin or inducing internal migration and degradation of ferroportin, comprising contacting ferroportin with at least one hepcidin analog, dimer, or composition of the present invention.

[0030] In further embodiments, the present invention includes a method for treating an iron metabolic disorder in a subject requiring treatment, comprising providing the subject with an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the pharmaceutical composition is provided to the subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, subarachnoid, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal, or topical administration routes. In certain embodiments, the pharmaceutical composition is provided to the subject by oral or subcutaneous administration routes. In certain embodiments, the iron metabolic disorder is an iron overload disorder. In certain embodiments, the pharmaceutical composition is provided to the subject up to or about twice a day, up to or about once a day, up to or about once every two days, up to or about once a week, or up to or about once a month.

[0031] In certain embodiments, the hepcidin analog is provided to a target in a dosage of approximately 1 mg to approximately 100 mg or approximately 1 mg to approximately 5 mg.

[0032] In another embodiment, the present invention provides a device comprising the pharmaceutical composition of the present invention for selectively delivering a hepcidin analog or dimer of the present invention to a target orally or subcutaneously.

[0033] In yet another embodiment, the present invention includes a kit comprising a pharmaceutical composition of the present invention, packaged together with a reagent, a device, or instructions, or a combination thereof. [Modes for carrying out the invention]

[0034] The present invention relates, in general, to hepcidin analog peptides, as well as methods for producing and using the same. In certain embodiments, the hepcidin analog exhibits one or more hepcidin activities. In certain embodiments, the present invention relates to a hepcidin peptide analog comprising one or more peptide subunits that form a cyclized structure via intramolecular bonding, such as intramolecular disulfide bonds. In certain embodiments, the cyclized structure has increased potency and selectivity compared to non-cyclized hepcidin peptides and their analogs. In certain embodiments, the hepcidin analog peptide of the present invention exhibits an increased half-life compared to hepcidin or previous hepcidin analogs, for example, when delivered orally.

[0035] Definitions and Nomenclature Unless otherwise defined herein, scientific and technical terms used in this application have meanings that are generally understood by those skilled in the art. In general, the nomenclature and techniques used in relation to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry as described herein are well known and commonly used in the art.

[0036] As used herein, the following terms have their respective meanings unless otherwise specified.

[0037] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to mean including the integer (or component) or group of integers (or components) described, but not to exclude any other integer (or component) or group of integers (or components).

[0038] The singular forms "a," "an," and "the" include the plural form unless the context explicitly indicates otherwise.

[0039] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0040] The terms “patient,” “subject,” and “individual” may be used interchangeably and may refer to either a human or a non-human animal. These terms include mammals such as humans, primates, domestic animals (e.g., cattle, 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, and domestic animals.

[0041] As used herein, the term “peptide” broadly refers to a sequence of two or more amino acids linked together by a peptide bond. It should be understood that this term does not imply a specific length of the amino acid polymer, nor is it intended to mean or distinguish whether a polypeptide is produced using recombinant techniques, chemical synthesis, or enzymatic synthesis, or whether it is naturally occurring.

[0042] As used herein, the terms “peptide analog” or “hepcidin analog” broadly refer to peptide monomers and peptide dimers that contain one or more structural features and / or functional activity common to hepcidin or its functional region. In certain embodiments, peptide analogs include peptides that share substantial amino acid sequence identity with hepcidin, e.g., peptides that include one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence of wild-type hepcidin, e.g., human hepcidin. In certain embodiments, peptide analogs include one or more additional modifications, such as conjugation into another compound. Any peptide monomer or peptide dimer of the present invention is encompassed by the term “peptide analog.” In certain cases, “peptide analog” may also be referred to herein as “hepcidin analog,” “hepcidin peptide analog,” or “hepcidin analog peptide,” or alternatively.

[0043] The enumerations used herein, in their definitions of “sequence identity,” “identity percentage,” and “homology percentage,” or for example, “a sequence that is 50% identical,” refer to the degree to which sequences are identical nucleotide-wise or amino acid-wise across a comparison window. Thus, a “sequence identity percentage” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences 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) occurs, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage.

[0044] The calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) may be performed as follows: To determine the percentage of identity of two amino acid sequences or two nucleic acid sequences, these sequences may be aligned for the purpose of optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably 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. Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then these molecules are identical at that position.

[0045] The percentage of identity between two arrays is a function of the number of identical positions shared by those arrays, taking into account the number of gaps that must be introduced for optimal alignment of the two arrays and the length of each gap.

[0046] The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. In some embodiments, the percentage of identity between two amino acid sequences is determined using either a Blossum 62 matrix or a PAM250 matrix, and the Needleman and Wunsch (1970, J.Mol.Biol.48:444-453) algorithm incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix, and the GAP program in the GCG software package, using gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty 12, a gap elongation penalty 4, and a frameshift gap penalty 5. The percentage of identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller (1989, Cabios, 4:11-17) algorithm, which is incorporated into the ALIGN program (version 2.0), and uses the PAM120 weight residue table, gap length penalty 12, and gap penalty 4.

[0047] The peptide sequences described herein can be used as query sequences to search public databases and identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described by Altschul, et al. (1990, J. Mol. Biol, 215:403-10). A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of the present invention. To obtain gapped alignment for comparative purposes, Gapped BLAST can be used as described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST programs and gapped BLAST programs, you can use the default parameters of each program (e.g., XBLAST and NBLAST).

[0048] As used herein, the term “conservative substitution” means that one or more amino acids are replaced by other biologically similar residues. Examples include substitutions of amino acid residues with similar characteristics, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. See, for example, the table below. In some embodiments of the present invention, one or more Met residues are replaced with norleucine (Nle), a biological equivalent of Met, but which, 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, and 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 at residues not typically found in endogenous mammalian peptides and proteins is the conservative substitution of Arg or Lys with, for example, ornithine, canavanine, aminoethylcysteine, or another basic amino acid. In some embodiments, another conserved substitution is the substitution of one or more Pro residues with bhPro, Leu, or D-Npc (isopithecotinic acid). For further information on phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et al., Science 247, 1306-1310, 1990. In the following scheme, conserved amino acid substitutions are grouped by their physicochemical properties: I: neutral, hydrophilic; II: acidic and amide; III: basic; IV: hydrophobic; V: aromatic, bulky amino acids. [Table 1]

[0049] In the following scheme, conserved amino acid substitutions are grouped by their physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. [Table 2]

[0050] As used herein, the terms “amino acid” or “any amino acid” refer to all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and non-natural 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 bind to peptide chains to form the building blocks of various proteins. These are primarily L-stereoisomers, although a small number of D-amino acids are present in bacterial envelopes and some antibiotics. Twenty “standard” natural amino acids are listed in the table above. “Non-standard” natural amino acids are pyrrolicin (found in methanogenic organisms 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). "Unnatural" or "non-natural" amino acids are non-proteinogenic amino acids (i.e., amino acids that are not naturally encoded or found in the genetic code) that are either naturally occurring or chemically synthesized. More than 140 naturally occurring amino acids are known, and thousands of combinations are possible. An example of an "unnatural" amino acid is the β-amino acid (β 3 and β 2 Examples include homo-amino acids, proline and pyruvate derivatives, trisubstituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methylamino acids. Unnatural amino acids also include modified amino acids. Modified amino acids include amino acids that have been chemically modified to include groups, multiple groups, or chemical moieties that do not exist naturally (e.g., natural amino acids).

[0051] As will be apparent to those skilled in the art, the peptide sequences disclosed herein are shown from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end being the C-terminus of the peptide. Some of the sequences disclosed herein incorporate a "Hy" moiety at the amino terminus (N-terminus) and either an "-OH" moiety or an "-NH2" moiety at the carboxyl terminus (C-terminus). In such cases, unless otherwise indicated, the "Hy" moiety at the N-terminus of the sequence in question refers to a hydrogen atom corresponding to the presence of a free primary or secondary amino group at the N-terminus, and the "-OH" moiety or "-NH2" moiety at the C-terminus refers to a hydroxyl group or an amino group corresponding to the presence of an amide (CONH2) group at the C-terminus, respectively. In each of the sequences of the present invention, the C-terminal "-OH" moiety may be substituted with a C-terminal "-NH2" moiety, and vice versa. It is further understood that the portion at the amino or carboxyl terminus may be bonded, for example, covalently, particularly when the amino or carboxyl terminus is bonded to a linker or another chemical portion, such as a PEG portion.

[0052] As used herein, the term "NH2" refers to the free amino group at the amino terminus of a polypeptide. As used herein, the term "OH" refers to the free carboxyl group at the carboxy terminus of a peptide. Furthermore, as used herein, the term "Ac" refers to acetyl protection by acylation of the C-terminus or N-terminus of a polypeptide.

[0053] As used herein, the term "carboxy" refers to -CO2H.

[0054] In most cases, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the nomenclature rules proposed by the IUPAC Committee on Organic Chemical Nomenclature and the IUPAC-IUB Committee on Biochemical Nomenclature, as described 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 used herein and in the appended claims differ from those proposed, they will be made clear to the reader. Some abbreviations useful in describing the present invention are defined in Table 1 below. [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 3-12] [Table 3-13] [Table 3-14] [Table 3-15]

[0055] Throughout this specification, unless naturally occurring amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they shall be represented by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). For less common or naturally occurring amino acids, unless they are referred to by their full names (e.g., sarcosine, ornithine, etc.), they shall be represented by 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-carbohydrate). Commonly used three- or four-letter codes, including rubonate, 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), are used for these residues.

[0056] Furthermore, R 1In all sequences, isovaleric acid or an equivalent may be substituted. In some embodiments in which the peptide of the present invention is conjugated with an acidic compound such as isovaleric acid, isobutyric acid, or valeric acid, the presence of such conjugate is referred to in acid form. Therefore, in some embodiments, instead of indicating the conjugation of isovaleric acid to the peptide by referring to isovaleroyl, in some embodiments, the present application may refer to such conjugate as isovaleric acid.

[0057] For each of the hepcidine analogue formulas provided herein, it is understood that bonds may be indicated or implied by a "-" based on the formula and its components. For example, "B7(L1Z)" is understood to include a bond between B7 and L1 if L1 is present, or 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, or a bond between B5 and Z if L1 is absent. In addition, it is understood that if both L1 and Z are present, a bond exists between them. Thus, the definitions of certain substituents such as B7, L1, and J may include a "-" before and / or after the defined substituent, but in each case, it is understood that the substituent is bonded 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 bonded to Xaa2 and Y1 via a single bond. Thus, the definition of a substituent may or may not include the "-", but it is still understood that it is bonded to an adjacent substituent.

[0058] As used herein, the term “L-amino acid” refers to the “L” isomer form of a peptide, and conversely, the term “D-amino acid” refers to the “D” isomer form of a peptide. In certain embodiments, the amino acid residues described herein are in the “L” isomer form, but the “D” isomer residues may be substituted with any L-amino acid residue, as long as the desired functional group is retained by the peptide.

[0059] Unless otherwise specified, the L-isomer forms of the native and unnatural amino acids in question that have a chiral center are referred to. Where appropriate, the D-isomer forms of amino acids are represented in the conventional manner by the prefix "D" before the conventional three-letter code (e.g., Dasp, (D)Asp, or D-Asp, Dphe, (D)Phe, or D-Phe).

[0060] As used herein, “Lys lower homologue” refers to an amino acid that has the structure of lysine but has one or more fewer carbon atoms in its side chain compared to lysine.

[0061] As used herein, “higher homologue of Lys” refers to an amino acid that has the structure of lysine but has one or more additional carbon atoms in its side chain compared to lysine.

[0062] As used herein, the term "DRP" refers to disulfide-rich peptides.

[0063] As used herein, the term “dimer” broadly refers to peptides containing two or more monomeric subunits. A particular dimer contains two DRPs. Dimers of the present invention include homodimers and heterodimers. The monomeric subunits of a dimer may be linked at their C-terminus or N-terminus, or via internal amino acid residues. Each monomeric subunit of a dimer may be linked via the same site, or each may be linked via different sites (e.g., C-terminus, N-terminus, or internal site).

[0064] The terms “isoster substitution” or “substitution” are used interchangeably herein to refer to any amino acid or other analogous moiety having chemical and / or structural properties similar to a particular amino acid. In certain embodiments, isoster substitution is a conservative substitution of a native or non-native amino acid.

[0065] As used herein, the term "cyclization" refers to a reaction in which a portion of a polypeptide molecule is linked to another portion of the polypeptide molecule to form a ring closure, for example, by forming a disulfide crosslink or other similar bond.

[0066] As used herein, the term “subunit” refers to one of a pair of polypeptide monomers that are combined to form a dimeric peptide composition.

[0067] As used herein, the term “linker moiety” broadly refers to a chemical structure that can link or bond two peptide monomer subunits together to form a dimer.

[0068] As used herein, the term “linking group” broadly refers to any chemical structure that can covalently link or bond two other molecular fragments together.

[0069] In the context of this invention, the term "solvate" refers to a defined stoichiometric complex formed between a solute (e.g., a hepcidin analog or a pharmaceutically acceptable salt thereof according to the present invention) and a solvent. The solvent in this context may be, for example, water, ethanol, or another pharmaceutically acceptable, typically small-molecule organic species (such as, but not limited to, acetic acid or lactic acid). When the solvent in question is water, such a solvate is usually referred to as a hydrate.

[0070] As used herein, “iron metabolic disorders” include diseases in which abnormal iron metabolism directly causes the disease, or diseases in which dysregulation of blood iron levels causes the disease, or diseases in which iron dysregulation is a consequence of another disease, or diseases in which the disease can be treated by regulating iron levels. More specifically, iron metabolic disorders as disclosed herein include iron overload diseases, iron deficiency disorders, impaired intracellular iron distribution, other iron metabolic disorders, and other disorders potentially related to iron metabolism. Iron metabolism disorders include hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemomoduberin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sideroblastic anemia, porphyria, late-onset cutaneous porphyria, African type iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital erythrodysplasia anemia, hypochromic microcytic anemia, sickle cell anemia, and polycythemia vera (primary and secondary). This includes, for example, secondary polycythemia, chronic obstructive pulmonary disease (COPD), post-renal transplantation, Chuvasi, HIF and PHD mutations, as well as idiopathic myelodysplasia, pyruvate kinase deficiency, obesity iron deficiency, other anemias, benign or malignant tumors that overproduce or induce hepcidin, conditions with hepcidin excess, Friedreich's ataxia, Gracil syndrome, Harrellforden-Spats disease, Wilson's disease, pulmonary hemosiderin deposition, hepatocellular carcinoma, cancer, hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes mellitus, atherosclerosis, neurodegenerative disorders, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0071] In some embodiments, the diseases and disorders are related to iron overload diseases, such as iron hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemomodoverin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0072] In some embodiments, the hepcidin analogs of the present invention are used to treat diseases and disorders not typically identified as iron-related. For example, hepcidin is highly expressed in mouse pancreas, suggesting that diabetes mellitus (type I or II), insulin resistance, glucose intolerance, and other disorders may be ameliorated by treating underlying iron metabolic disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, incorporated herein by reference. Thus, the peptides of the present invention may 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 by using methods known in the art, such as assays that monitor the concentration and expression of hepcidin, hemoduverin, or iron, such as the assay of WO2004 / 092405, incorporated herein by reference, and the assay described in U.S. Patent No. 7,534,764, incorporated herein by reference.

[0073] In certain embodiments of the present invention, iron metabolic disorders are iron overload disorders, including hereditary hemochromatosis, iron-overload anemia, alcoholic liver disease, and chronic hepatitis C.

[0074] As used herein, the term “pharmaceutically acceptable salt” refers to a salt or zwitterionic form of the peptide or compound of the present invention that is suitable for the treatment of diseases without excessive toxicity, irritation, and allergic reactions, corresponds to a reasonable benefit / risk ratio, and is effective for its intended use, and is water-soluble, oil-soluble, or dispersible. This salt may be prepared during the final isolation and purification of the compound, or separately by reacting the amino group with a suitable acid. Typical acid addition salts include acetate, adipine, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, mesilenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Furthermore, the amino groups in the compounds of the present invention can be quaternized with chloride, bromide, and methyl, ethyl, propyl, and butyl iodide; dimethyl, diethyl, dibutyl, and diamyl sulfate; chloride, bromide, and decyl iodide, lauryl, myristyl, and steryl; and benzyl bromide and phenethyl. 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, as well as organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Pharmaceutically acceptable salts are preferably salts selected from, for example, acid addition salts and basic 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 N(R1)(R2)(R3)(R4)+ (where R1, R2, R3, and R4 independently typically represent hydrogen, an optionally substituted C1-6 alkyl, or an 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 more recent editions thereof), Encyclopaedia of Pharmaceutical Technology, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66:2 (1977). For a review of suitable salts, see Handbook of 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. Typical examples include aluminum salts, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, olamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts. Hemi salts of acids and bases, such as hemisulfate salts and hemicalcium salts, can also be formed.

[0075] As used herein, the term "N (alpha) methylation" refers to the methylation of an alpha amine of an amino acid, also commonly known as N-methylation.

[0076] As used herein, the terms “symmethylation” or “Arg-Me-sym” refer to the symmetric methylation of two nitrogen atoms of the guanidine group of arginine. Furthermore, the terms “asymmethylation” or “Arg-Me-asym” refer to the methylation of a single nitrogen atom of the guanidine group of arginine.

[0077] As used herein, the term “acylated organic compound” refers to a variety of compounds having a carboxylic acid functional group used to acylate the N-terminus of an amino acid subunit before forming a C-terminal dimer. Non-limiting examples of acylated 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-fluoromethylbutyrate, and tetrahydro-2H-pyran-4-carboxylic acid.

[0078] "C n-m " or "C n -C m」 The term indicates a range including the endpoints, where n and m are integers representing the number of carbon atoms. For example, C 1-4 , C 1-6 , C 1-20 Examples include C1-C6 and C1-C8.

[0079] The term "alkyl" includes linear or branched acyclic or cyclic saturated aliphatic hydrocarbons having 1 to 24 carbon atoms. Typical linear saturated alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Typical branched saturated alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Typical cyclic saturated alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Typical cyclic unsaturated alkyls include, but are not limited to, cyclopentenyl and cyclohexenyl.

[0080] The term "alkylene" specifically refers to a divalent alkyl group having 1 to 24 carbon atoms. This term is exemplified by groups such as methylene (-CH2-), ethylene (-CH2CH2-), and propylene isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-).

[0081] The term "alkoxy" refers to the "alkyl-O-" group. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.

[0082] The term "halo" refers to atoms that occupy Group VIIA of the periodic table, such as fluoro, chloro, bromo, or iodine.

[0083] The term "haloalkyl" refers to the unbranched or branched alkyl group defined above, in which one or more (e.g., 1 to 6 or 1 to 3) hydrogen atoms are replaced by halogens. For example, if a residue is substituted with two or more halogens, this may be referred to by using a prefix corresponding to the number of halogen moieties bonded. Dihaloalkyl and trihaloalkyl refer to alkyl groups substituted with two ("di") or three ("tri") halo groups, which may, but not necessarily, be the same halogens. Examples of haloalkyls include, for example, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, and 1,2-dibromoethyl.

[0084] The term "haloalkoxy" refers to a radical-OR where R is a haloalkyl group as defined above, such as trifluoromethoxy, 2,2,2-trifluoroethoxy, or difluoromethoxy.

[0085] The term "alkanoyl" refers to an alkanoyl-C(O)-group in which the alkyl group is as defined herein. Typical alkanoyl groups include methanoloyl, ethanoloyl, 3-methylbutanoyl, and CH3(CH2). 14 Examples include C(O)-.

[0086] As used herein, “amide” means a -CONH- bond or an -NHC(O)- bond.

[0087] As used herein, the terms "thio" or "mercapto" mean the -SH group.

[0088] As used herein, the term "N substitution" means that the α-amino group of an amino acid is substituted. In some embodiments, N substitution means that the α-amino group of an amino acid is replaced with one or two C atoms, such as methyl, ethyl, or propyl. 1-6 This means that it will be substituted with an alkyl group.

[0089] As used herein, “aryl” refers to a polyunsaturated aromatic hydrocarbon group having 6 to 14 or 6 to 10 carbon atoms, which may be a single ring or multiple rings (up to three rings) fused together or covalently bonded, either alone or as part of another substituent. Therefore, this term includes, but is not limited to, groups such as phenyl, biphenyl, anthracenyl, and naphthyl. Non-limiting examples of unsubstituted aryl groups include phenyl, 1-naphthyl, 2-naphthyl, and 4-biphenyl.

[0090] As used herein, the term "functional group" on the side chain of an amino acid means -COOH, -NH2, -NH-, -SH, -SCH3, -OH, -C(O)NH2, guanidinyl, imidazoyl, pyrrolidinyl, phenyl, indolyl, and the like. In some embodiments, the functional group includes -COOH, -NH2, OH, SH, guanidinyl, and the like.

[0091] As used herein, “therapeutic effective dose” of the peptide agonist of the present invention is intended to describe an amount of peptide agonist sufficient to treat any of the diseases and disorders described herein (e.g., iron metabolic disorders), but not limited to those listed herein. In certain embodiments, the therapeutic effective dose will achieve a desired benefit / risk ratio applicable to any medical practice.

[0092] Peptide analog of hepcidin The present invention provides hepcidin peptide analogs (collectively referred to as "hepcidin analogs") that may be monomers or dimers.

[0093] In some embodiments, the hepcidin analogs of the present invention bind to ferroportin, for example, human ferroportin. In certain embodiments, the hepcidin analogs of the present invention bind specifically to human ferroportin. As used herein, “specifically bind” means the preferential interaction of a specific binder with a given ligand over other agents in a sample. For example, a specific binder that specifically binds to a given ligand binds to the given ligand in an observable amount or degree more than any nonspecific interaction with any other component in the sample under preferred conditions. Preferred conditions are those that allow for interaction between a given specific binder and a given ligand. These conditions include pH, temperature, concentration, solvent, incubation time, etc., and may vary depending on a given specific binder and ligand pair, but can be readily determined by those skilled in the art. In some embodiments, the hepcidin analogs of the present invention bind to ferroportin with higher specificity than a hepcidin reference compound (for example, any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the present invention exhibit ferroportin specificity that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1000%, or 10,000% higher than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the present invention exhibit ferroportin specificity that is at least about 5 times, or at least about 10, 20, 50, or 100 times higher than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0094] In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity. In some embodiments, this activity is in vitro or in vivo activity, e.g., in vivo or in vitro activity as 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).

[0095] 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 ferroportin binding capacity exhibited by the hepcidin reference compound. In some embodiments, the hepcidin analogs of the present invention exhibit a lower EC50 or IC50 of binding to ferroportin (e.g., human ferroportin) compared to the hepcidin reference compound. 50 (i.e., higher binding affinity). In some embodiments, the hepcidin analogs of the present invention exhibit an EC50 or IC50 that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than that of the hepcidin reference compound in a ferroportin competitive binding assay. 50 It holds.

[0096] In certain embodiments, the hepcidin analogs of the present invention exhibit increased hepcidin activity compared to the hepcidin reference compound. In some embodiments, this activity is in vitro or in vivo activity, for example, the in vivo or in vitro activity described herein. In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity that is 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 120 times, 140 times, 160 times, 180 times, or 200 times higher than the hepcidin reference compound. In certain embodiments, the hepcidin analogs of the present invention exhibit activity at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% higher than that of the hepcidin reference compound.

[0097] In some embodiments, the peptide analogs of the present invention exhibit in vitro activity to induce degradation of human ferroportin protein at a level at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000%, similar to hepcidin reference compounds, and this activity is measured according to the methods described herein.

[0098] In some embodiments, the peptides or peptide dimers of the present invention exhibit in vivo activity that induces a decrease in free plasma iron in an individual at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000%, similar to hepcidin reference compounds, and this activity is measured according to the method described herein.

[0099] In some embodiments, this activity is in vitro or in vivo activity, for example, the in vivo or in vitro activity described herein. In certain embodiments, the hepcidin analogs of the present invention have an activity 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 the hepcidin reference compound, or at least about 10%, 20%, 30%, 40%, 50%, The activity is 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% higher, and this activity is, for example, in vitro activity for inducing the degradation of ferropoctone, as measured according to the examples herein, or this activity is, for example, in vivo activity for reducing free plasma iron, as measured according to the examples herein.

[0100] In some embodiments, the hepcidin analogs of the present invention mimic the hepcidin activity of Hep25, a bioactive human 25-amino acid type, and are referred to herein as “minihepcidins.” As used herein, in certain embodiments, a compound having “hepcidin activity” (e.g., a hepcidin analog) means that the compound has the ability to lower plasma iron concentration in a subject (e.g., mouse or human) when administered in a dose-dependent and time-dependent manner (e.g., parenteral administration). See, for example, Rivera et al. (2005), Blood 106:2196-9. In some embodiments, the peptides of the present invention reduce the plasma iron concentration 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%.

[0101] In some embodiments, the hepcidin analogs of the present invention have in vitro activity, which is assayed by their ability to induce internal translocation and degradation of ferroportin in ferroportin-expressing cells, as taught in Nemeth et al. (2006) Blood 107:328-33. In some embodiments, in vitro activity is measured by dose-dependent loss of fluorescence in cells engineered to present ferroportin fused to green fluorescent protein, as seen in Nemeth et al. (2006) Blood 107:328-33. Aliquots of cells are incubated for 24 hours with reference preparations of Hep25 or minihepcidin at stepwise concentrations. As provided herein, EC 50 The value is provided as the concentration of a given compound (e.g., the hepcidin analog peptide or peptide dimer of the present invention) that induces 50% of the maximum fluorescence loss produced by the reference compound. The EC of the Hep25 preparation in this assay. 50 The EC2 is in the range of 5 to 15 nM, and in certain embodiments, the preferred hepcidin analog of the present invention exhibits an EC2 of approximately 1,000 nM or less in an in vitro activity assay. 50 In certain embodiments, the hepcidin analogs of the present invention have an EC of less than one of approximately 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, for example, in Nemeth et al. (2006) Blood 107:328-33 or in the examples herein). 50 It has. In some embodiments, the hepcidin analog or biotherapeutic composition (e.g., any one of the pharmaceutical compositions described herein) has an EC of about 1 nM or less. 50 Value or IC 50 It has a value.

[0102] Other methods known in the art may be used to calculate the hepcidin activity and in vitro activity of the hepcidin analogs according to the present invention. For example, in certain embodiments, the in vitro activity of the hepcidin analog or reference peptide is measured by their ability to deliver ferroportin into cells, which is determined by immunohistochemistry or flow cytometry using an antibody that recognizes the extracellular epitope of ferroportin. Alternatively, in certain embodiments, the in vitro activity of the hepcidin analog or reference peptide is measured by their dose-dependent ability to inhibit iron efflux from ferroportin-expressing cells preloaded with a radioactive or stable isotope of iron, as seen in Nemeth et al. (2006) Blood 107:328-33.

[0103] In some embodiments, the hepcidin analogs of the present invention exhibit increased stability (e.g., measured by half-life and proteolytic rate) compared to the hepcidin reference compound. In certain embodiments, the stability of the hepcidin analog of the present invention is increased by at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 120 times, 140 times, 160 times, 180 times, or more than 200 times, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or more than 500%. In some embodiments, the stability is the stability described herein. In some embodiments, stability is plasma stability, which is optionally measured, for example, according to the method described herein. In some embodiments, stability is stability upon oral delivery.

[0104] In certain embodiments, the hepcidin analogs of the present invention exhibit a longer half-life than the 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 10 days, at least about 2 The half-life of the hepcidine analog of the present invention is extended due to one or more lipophilic substituents or half-life extenders, for example, its conjugation to any of the lipophilic substituents or half-life extenders disclosed herein. In some embodiments, the half-life of the hepcidin analog of the present invention is extended due to its conjugation to one or more polymer moieties, for example, any of the polymer moieties or half-life-extending moieties disclosed herein. In certain embodiments, the hepcidin analog of the present invention has the above-mentioned half-life under a given set of conditions, in which the temperature is about 25°C, about 4°C, or about 37°C, and the pH is physiological pH or pH about 7.4.

[0105] In certain embodiments, the hepcidin analogs of the invention that include a conjugated half-life extension moiety have an increased serum half-life after oral, intravenous, or subcutaneous administration as compared to analogs that are the same but lack the conjugated half-life extension moiety. In certain embodiments, the serum half-life of the hepcidin analogs of the invention after any one of 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, this is 12 - 168 hours, 24 - 168 hours, 36 - 168 hours, or 48 - 168 hours.

[0106] In certain particular embodiments, the hepcidin analogs of the invention, such as hepcidin analogs that include a conjugated half-life extension moiety, result in a decreased serum iron concentration after oral, intravenous, or subcutaneous administration to a subject. In certain embodiments, the serum iron concentration of the subject is less than 10%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90% of its serum iron concentration when no hepcidin analog is administered to the subject. In certain embodiments, the decrease 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, this is maintained for 12 - 168 hours, 24 - 168 hours, 36 - 168 hours, or 48 - 168 hours. In one embodiment, the serum iron concentration of the subject decreases to less than 20% at about 4 hours or about 10 hours after administration (e.g., intravenous, oral, or subcutaneous) to the subject. In one embodiment, the serum iron concentration of the subject decreases to less than 50% or less than 60% over about 24 - about 30 hours after administration (e.g., intravenous, oral, or subcutaneous).

[0107] In some embodiments, the half-life is measured in vitro using any suitable method known in the art. For example, in some embodiments, the stability of the hepcidin analog of the present invention is determined by incubating the hepcidin analog with pre-warmed human serum (Sigma) at 37°C. Samples are taken at various time points, typically up to 24 hours, and the stability of the sample is analyzed by separating the hepcidin analog from serum proteins and then analyzing the presence of the target hepcidin analog using LC-MS.

[0108] In some embodiments, the stability of the hepcidin analog is measured in vivo using any suitable method known in the art. For example, in some embodiments, the stability of the hepcidin analog is determined in vivo by administering the peptide or peptide dimer to a subject such as a human or any mammal (e.g., mouse), and then the sample is taken from the subject by blood collection at various time points, typically up to 24 hours. Thereafter, the sample is analyzed as described above for the in vitro method of measuring the half-life. In some embodiments, the in vivo stability of the hepcidin analog of the present invention is determined by the method disclosed in the examples herein.

[0109] In some embodiments, the present invention provides hepcidin analogs described herein, which exhibit improved solubility or improved aggregation properties compared to hepcidin reference compounds. Solubility can be determined by any suitable method known in the art. In some embodiments, preferred methods known in the art for determining solubility include incubating the peptide (e.g., the hepcidin analog of the present invention) in various buffers (acetic acid buffer pH 4.0, acetate buffer pH 5.0, phosphate / citrate buffer pH 5.0, phosphate / citrate buffer pH 6.0, phosphate buffer pH 6.0, phosphate buffer pH 7.0, phosphate buffer pH 7.5, strong PBS buffer pH 7.5, Tris buffer pH 7.5, Tris buffer pH 8.0, glycine buffer pH 9.0, water, acetic acid (pH 5.0 and other pH levels known in the art), and testing for aggregation or solubility using standard techniques. These include, but are not limited to, visual precipitation, dynamic light scattering, circular dichroism, and fluorescent dyes for measuring surface hydrophobicity and detecting aggregation or fibrillation. In some embodiments, improved solubility means that the peptide (e.g., the hepcidin analog of the present invention) is more soluble in a given liquid than a hepcidin reference compound.

[0110] In certain embodiments, the present invention provides a hepcidin analog described herein that exhibits solubility in a particular solution or buffer, for example, in water or in buffers known in the art or buffers 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 more than 200 times, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or more than 500% higher than that of a hepcidin reference compound.

[0111] In certain embodiments, the present invention provides a hepcidin analog described herein that exhibits reduced aggregation, with peptide aggregation in solution being 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 than that of a hepcidin reference compound in a particular solution or buffer, for example, water or buffers known in the art or buffers disclosed herein.

[0112] In some embodiments, the present invention provides hepcidin analogs described herein that exhibit lower degradation (i.e., higher degradation stability) than the hepcidin reference compound, for example, degradation of more than 10% or about 10% lower, more than 20% or about 20% lower, more than 30% or about 30% lower, more than 40% or about 40% lower, or more than 50% or about 50% lower. In some embodiments, degradation stability is determined by any preferred method known in the art. In some embodiments, preferred methods known in the art for determining degradation stability include Hawe, which is entirely incorporated herein. The method described in et al. J Pharm Sci, VOL.101, NO.3, 2012, pp. 895-913 is included. In some embodiments, such a method is used to select potent sequences with extended storage life.

[0113] In some embodiments, the hepcidin analogs of the present invention are produced synthetically. In other embodiments, the hepcidin analogs of the present invention are produced recombinantly.

[0114] The various hepcidin analog monomers and dimer peptides of the present invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may include, but are not limited to, modified amino acids, unnatural or non-natural amino acids. In certain embodiments, modified amino acids include natural amino acids that have been chemically modified to include groups, multiple groups, or chemical moieties that do not naturally exist 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, the peptide analogs of the present invention include any of those described herein, in which one or more natural amino acid residues of the peptide analog are substituted with unnatural or non-natural amino acids, or D-amino acids.

[0115] In certain embodiments, the hepcidin analogs of the present invention comprise one or more modified amino acids or unnatural amino acids. For example, in certain embodiments, the hepcidin analog comprises one or more of the following: 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 understand that various other substitutions of natural amino acids with other modified or non-natural amino acids may be made to achieve similar desired results, and that such substitutions are within the scope of the teachings and spirit of the present invention.

[0116] The present invention comprises, for example, one of the hepcidin analogs described herein, in free form or salt form.

[0117] The compounds described herein include isotope-labeled compounds that are identical to those enumerated in the various formulas and structures presented herein, but differ in that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that may be incorporated into these compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, for example, 2 H, 3 H, 1 3C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Examples include Cl. Certain isotope-labeled compounds described herein, for example, 3 H and 14 Compounds incorporating radioactive isotopes such as 13C are useful in drug and / or substrate tissue distribution assays. Furthermore, deuterium, i.e., 2 Substitution with isotopes such as hydrogen (H) can lead to certain therapeutic benefits resulting from higher metabolic stability, such as an increased in vivo half-life or a reduced dosage. In certain embodiments, the compound isotope-substituted with deuterium. In more specific embodiments, the most unstable hydrogen atom is substituted with deuterium.

[0118] The hepcidin analog of the present invention comprises either a peptide monomer or dimer as described herein, linked to a linker moiety containing any of the specific linker moieties described herein.

[0119] The hepcidin analogs of the present invention include peptides, such as monomers or dimers, that include a peptide monomer subunit 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 with any of the amino acid sequences shown in Tables 2 and 3 described herein (e.g., any one of the peptides disclosed herein).

[0120] In certain embodiments, the monomeric subunit of the peptide analog of the present invention, or the dimeric peptide analog of the present invention, 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, and optionally one or more additional non-amino acid moieties, such as conjugated chemical moieties, such as half-life extension moieties, PEGs, or linker moieties. In certain embodiments, the monomeric subunit of the 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, the monomeric subunit of the hepcidin analog of the present invention comprises or consists of 10 to 18 amino acid residues and optionally one or more additional non-amino acid moieties, such as a conjugated chemical moiety, such as a PEG or linker moiety. In various embodiments, the monomeric subunit comprises or consists of 7 to 35 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues. In any particular embodiment 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.

[0121] In certain embodiments, the hepcidin analog or dimer of the present invention does not contain any of the compounds described in PCT / US2014 / 030352 or PCT / US2015 / 038370.

[0122] Peptideheptidin analog In certain embodiments, the hepcidin analog of the present invention comprises a single peptide subunit optionally conjugated to an acidic moiety. In certain embodiments, the acidic moiety is conjugated directly or via a linker.

[0123] In one embodiment, the present invention relates to formula (I'): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (I') (In the formula, R 1 However, hydrogen, C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, C1-C 20 Cycloalkanoyl, C6-C 10 The aryl-C1-C6 alkylene-C(O)-, 5-membered or 6-membered heteroaryl-C(O)-, or 5-membered or 6-membered heterocycloalkyl-C(O)-, where R 1 C1-C 20 Alkanoyl, the C6-C 10 Each of the aryl-C1-C6 alkylene-C(O)-, the 5-membered or 6-membered heteroaryl-C(O)-, and the 5-membered or 6-membered heterocycloalkyl-C(O)- is optionally substituted with one, two, or three substituents independently selected from halo, OH, CN, C1-C6 alkoxy, C1-C6 alkyl, -COOH, -COO(C1-C6), C1-C6 haloalkyl, and C1-C6 haloalkoxy. R 2 However, NH2, substituted amino, OH, substituted hydroxy, C1-C 20 Alkylaminophenyl-C1-C8alkylene-NH-, where C1-C 20アルキル The amino and phenyl-C1-C8 alkylene-NH- groups are each optionally substituted with substituents independently selected from NH2, -COOH, and phenyl. X1 is either absent, or is 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, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Glu, Aad, Dap, or isoGlu. X2 is Ala, Thr, Gly, N-substituted Gly, Ser, Cys, 4S_Mcp, 4R_Mcp, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, Cys, (D)Cys, aMeCys, hCys, Pen, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or Cys, (D)Cys, aMeCys, hCys, Pen, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, 4S_Mcp, 4R_Mcp, Morph, or Tic. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, NMe-Cys, Ala, Lys, (D)Lys, substituted Lys, or substituted (D)Lys, X7 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, Phe, or DIP. X8 is absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, 1,2,3-triazole, Cys, (D)Cys, aMeCys, hCys, Pen, Lys_Me3, X9 is absent or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe, X10 is absent or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor-propanoic acid, X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile, X12 to X14 are each absent or are each independently an amino acid, The peptide of formula (I’) is optionally further conjugated to any amino acid, Any one of the amino acids of the peptide of formula (I’) is optionally substituted with the corresponding (D)-amino acid or is optionally N-substituted, (i) At least one of X1, X3 to X5, X7 to X10 is Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp, where the peptide combines the mercapto group on the side chain of one of X1, X3 to X5, and X7 to X10, the mercapto group on the side chain of X6, and L x together to form -S-Lx - form an -S- bond, or the mercapto group on the side chain of X1, the mercapto group on the side chain of X8, and L x together form -S-L x - form an -S- bond, or the mercapto group on the side chain of X2, the mercapto group on the side chain of X5, and L x together form -S-L x - is cyclized by forming an -S- bond, wherein L x is each independently a bond or a linking group, (ii) the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond, where 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-nipetidic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, 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, where the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine, Substituting bhPhe is β-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. The substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu. The substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH, or t-Bu. A substituted Lys, substituted dLys, or substituted NMe-Lys, or substituted NMe-dLys is Lys, dLys, NMe-Lys, or NMe-dLys, where the ε-amino group on the side chain of Lys, dLys, NMe-Lys, or NMe-dLys is covalently bonded to a C1-C6 alkanoyl, a half-life extension portion, or a linking portion covalently bonded to a half-life extension portion, and the linking portion includes one or more linker portions covalently bonded to each other. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123triazole [ka] And, Dab [ka] The present invention provides a hepcidin analog containing a peptide having the amino acid sequence of ( ), or a pharmaceutically acceptable salt or solvate thereof.

[0124] In some embodiments of the hepcidin analog of formula (I') or a pharmaceutically acceptable salt or solvate thereof, the present invention provides a compound of formula (X): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-R 2 (X) (wherein (i) one of X1, X3 to X5, X7 to X10 is Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp, wherein when the peptide has a mercapto group on the side chain of one of X1, X3 to X5, and X7 to X10, a mercapto group on the side chain of X6, and L x are joined together to form -S-L x -S- bond, or the mercapto group on the side chain of X1, the mercapto group on the side chain of X8, and L x are combined to form -S-L x -S- bond, or the mercapto group on the side chain of X2, the mercapto group on the side chain of X5, and L x are combined to form -S-L x -S- bond to cyclize, wherein L x are each independently a bond or a linking group, (ii) the functional groups on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond), and provides a hepcidin analog comprising a peptide having an amino acid sequence.

[0125] In some embodiments of the peptide of formula (I') or formula (X) or a pharmaceutically acceptable salt or solvate thereof, X1 is Asp, isoAsp, Asp(OMe), Glu, bhGlu, Gln, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Glu, Aad, Dap, or isoGlu, X2 is Ala, Thr, Gly, N-substituted Gly, Ser, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Morph, or Tic. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, or NMe-Cys. X7 is absent, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, or Phe. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, 123-triazole, or Lys_Me3. X9 is either absent or replaced by Ala, Ile, Gly, N-substitute Gly, Val, Leu, N-Leu, Phe, bhPhe, Lys, substitute Lys, (D)Lys, substitute (D)Lys NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or Substitution Phe, X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. Mercapto group on the side chain of X6, mercapto group on the side chain of X7, and L x Together, -SL x -S- bond is formed, in the formula, L x However, bonding, or C 1-8 Alkylene, phenylene, [ka] A linking group selected from, where the alkylene and the phenylene are each C 1-6 alkyl, halo, CN, OH, -COOH, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Optionally substituted with one or two substituents independently selected from haloalkoxy or NH2, L a and L b Each of them operates independently, C 1-4It is an alkylene, and the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. In one embodiment, X6 and X7 form a disulfide bond, where X6 is Cys and X7 is Cys. In another embodiment, X6 and X7 form a disulfide bond, where X6 is Cys and X7 is NMe-Cys. In another embodiment, X6 and X7 form a disulfide bond, where X6 is hCys and X7 is hCys. In another embodiment, X6 and X7 form a disulfide bond, where X6 is Cys and X7 is hCys. In another embodiment, X6 and X7 form a disulfide bond, where X6 is Pen and X7 is Cys. In another embodiment, X6 and X7 form a disulfide bond, where X6 is Cys and X7 is Pen. In yet another embodiment, X6 and X7 form a disulfide bond, where X6 is Pen and X7 is Pen.

[0126] In some embodiments of the peptide of formula (I') or formula (X) or its pharmaceutically acceptable salt or solvate, L x is, combined or [ka] And in the formula, L a and L b Each of them operates independently, C 1-6 It is alkyl. In one embodiment, L x This is a bond. In another embodiment, L x teeth, [ka] And in the formula, L a and L b Each of them operates independently, C 1~6 It is alkyl, for example, L a and L b Each of these is CH3. In some embodiments, L x teeth, [ka] And in the formula, L a and L b Each of them operates independently, C 1-6 Alkyl, for example, methyl. In one embodiment, L x This is p-xylene.

[0127] In some embodiments of the peptide of formula (I') or formula (X) or a pharmaceutically acceptable salt or solvate thereof, X1 is Cys, (D)Cys, aMeCys, hCys, Pen, or (D)Pen. X2 is Ala, Thr, Gly, N-substituted Gly, Ser, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Morph, or Tic. X6 is Ala, Lys, (D)Lys, substitute Lys, or substitute (D)Lys. X7 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Phe, or DIP. X8 is Cys, (D)Cys, aMeCys, hCys, or Pen. X9 is either absent or replaced by Ala, Ile, Gly, N-substitute Gly, Val, Leu, N-Leu, Phe, bhPhe, Lys, substitute Lys, (D)Lys, substitute (D)Lys NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or Substitution Phe, X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. The mercapto group on the side chain of X1 and the mercapto group on the side chain of X8 combine to form a disulfide bond. The functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. In one embodiment, X1 and X8 form a disulfide bond, where X1 is hCy and X8 is Cys. In another embodiment, X1 and X8 form a disulfide bond, where X1 is Pen and X8 is Cys. In yet another embodiment, X1 and X8 form a disulfide bond, where X1 is Cys and X8 is dPen. In yet another embodiment, X1 and X8 form a disulfide bond, where X1 is dCys and X8 is dCys. In yet another embodiment, X1 and X8 form a disulfide bond, where X1 is dCys and X8 is Cys. In yet another embodiment, X1 and X8 form a disulfide bond, where X1 is Cys and X8 is hCys. In yet another embodiment, X1 and X8 form a disulfide bond, where X1 is hcy and X8 is Pen. In another embodiment, X1 and X8 form a disulfide bond, where X1 is hCys and X8 is hCys. In yet another embodiment, X1 and X8 form a disulfide bond, where X1 is Cys and X8 is Cys.

[0128] In some embodiments of the peptide of formula (I') or formula (X) or its pharmaceutically acceptable salt or solvate, X1 is 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, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Glu, Aad, Dap, or isoGlu. X2 is Cys, 4S_Mcp, or 4R_Mcp. X3 is Ala, Gly, N-substituted Gly, His, substituted His, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or DIP. X5 is Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, or NMe-Cys. X7 is absent, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, or Phe. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, 123-triazole, or Lys_Me3. X9 is either absent, or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. The mercapto group on the side chain of X2 and the mercapto group on the side chain of X5 combine to form a disulfide bond. The functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. In one embodiment, X2 and X5 form a disulfide bond, where X2 is Cys and X5 is Cys. In another embodiment, X2 and X5 form a disulfide bond, where X2 is Cys and X5 is NMe-Cys. In yet another embodiment, X2 and X5 form a disulfide bond, where X2 is Cys and X5 is hCys. In yet another embodiment, X2 and X5 form a disulfide bond, where X2 is Cys and X5 is 4S_Mcp. In yet another embodiment, X2 and X5 form a disulfide bond, where X2 is Cys and X5 is 4R_Mcp. In yet another embodiment, X2 and X5 form a disulfide bond, where X2 is 4S_Mcp and X5 is 4R_Mcp. In another embodiment, X2 and X5 form a disulfide bond, where X2 is 4S_Mcp and X5 is 4S_Mcp. In yet another embodiment, X2 and X5 form a disulfide bond, where X2 is 4R_Mcp and X5 is 4R_Mcp.

[0129] In some embodiments of the peptide of formula (I') or formula (X) or its pharmaceutically acceptable salts or solvates, the peptide is cyclized by bringing together the functional group on the side chain of X1 and the functional group on the side chain of X10 to form an amide bond, i.e., a -C(O)NH- bond or a -NHC(O)- bond. In certain cases, the peptide is cyclized by bringing together the amino or carboxyl group on the side chain of X1 and the carboxyl or amino group on the same chain of X10 to form an amide bond. In some embodiments, X1 and X10 form an amide bond and X6 and X7 form a disulfide bond, where X1 is Glu and X10 is dK.

[0130] In some embodiments of the peptide of formula (I') or formula (X) or its pharmaceutically acceptable salt or solvate, the peptide has a functional group on the side chain of X1 and R 2 The functional groups are brought together and cyclized by forming an amide bond, i.e., a -C(O)NH- bond or -NHC(O)- bond. In certain cases, petido is cyclized by bringing together the amino or carboxyl group on the side chain of X1 and R 2 The carboxyl group or amino group of the two is cyclized by bringing them together to form an amide bond. In some embodiments, X1 and R 2 X6 and X7 form an amide bond, and X6 and X7 form a disulfide bond. In one embodiment, X1 and R 2 X6 and X7 form an amide bond, and X1 is Glu, R 2 C1-C 20 These are alkylamino or phenyl-C1-C8 alkylene-NH-, each substituted with a substituent independently selected from NH2 and -COOH. In one embodiment, R 2 C is substituted with NH2 or COOH. 1-8 It is an alkylamino. In one embodiment, R 2 It is 2-aminoethylamino.

[0131] In a particular embodiment, the present invention relates to formula (I): R 1 -X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (I) (In the formula, R 1 However, hydrogen, C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, C1-C 20 It is a cycloalkanoyl, R 2 However, it is NH2, substituted amino, OH, or substituted hydroxy, X0 is either absent, or Cys, (D)Cys, aMeCys, hCys, or Pen. X1 is either absent, or is 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, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X2 is Ala, Thr, Gly, N-substituted Gly, or Ser. X3 is Ala, Gly, N-substituted Gly, His, substituted His, or Cys, (D)Cys, aMeCys, hCys, or Pen. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or Cys, (D)Cys, aMeCys, hCys, or Penl. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X6 is Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either absent, or is Ala, Gly, N-substitute Gly, Ile, Val, Leu, NLeu, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X8 is either absent, or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, 123-triazole, or Cys, (D)Cys, aMeCys, hCys, or Pen. X9 is either absent, or Ala, Ile, Gly, N-substitute Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. X12 to X14 are either absent or each is independently one of the amino acids, however, i) The peptide may be further conjugated with any amino acid, ii) Any of the amino acids in the peptide may be the corresponding (D)-amino acid, or may be N-substituted, and iii) Provided that at least one of X0, X1, X3-X5, X7-X10 is Cys, (D)Cys, aMeCys, hCys, or Pen, and Cys, (D)Cys, aMeCys, hCys, or Pen forms a disulfide bond with X6, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor It is leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting bhPhe is β-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. The substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu. The substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH, or t-Bu. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123triazole [ka] And, Dab [ka] It contains a hepcidin analog containing the peptide of ( ), or a pharmaceutically acceptable salt or solvate thereof.

[0132] In one embodiment, X0 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X0 and X6 are linked via a disulfide bond.

[0133] In one embodiment, X1 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X1 and X6 are linked via a disulfide bond.

[0134] In one embodiment, X3 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X3 and X6 are linked via a disulfide bond.

[0135] In one embodiment, X4 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X4 and X6 are linked via a disulfide bond.

[0136] In one embodiment, X5 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X5 and X6 are linked via a disulfide bond.

[0137] In one embodiment, X7 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X7 and X6 are linked via a disulfide bond.

[0138] In one embodiment, X8 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X8 and X6 are linked via a disulfide bond.

[0139] In one embodiment, X9 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X9 and X6 are linked via a disulfide bond.

[0140] In one embodiment, X10 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X10 and X6 are linked via a disulfide bond.

[0141] In one embodiment, X1 is Glu, Dab, Dap, Orn, Lys, or Tet1. X2 is Thr, X3 is His or 1MeHis. X4 is Dpa, X5 is either Ala or Pro. X6 is absent, Ala, Glu, or substituted Lys. X7 is either absent, or Ala, Ile, Lys, substitute Lys, (D)Lys, or substitute (D)Lys. X8 is either absent or is Ala, Ile, Glu, Asp, 123-triazole, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys. X9 is either absent or bhPhe. X10 is either absent, or Ala, Ile, Phe, bhPhe, Lys, substitute Lys, (D)Lys, or substitute (D)Lys. X11 is either absent, or Pro, bhPhe, Lys, substitute Lys, or (D)Lys.

[0142] In one embodiment, X1 is Glu.

[0143] In one embodiment, X2 is Thr.

[0144] In one embodiment, X3 is His.

[0145] In one embodiment, X4 is Phe or Dpa.

[0146] In one embodiment, X5 is Pro.

[0147] In one embodiment, X7 is Ile.

[0148] In one embodiment, X8 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0149] In one embodiment, X9 is Phe or bhF.

[0150] In one embodiment, X10 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0151] In one embodiment, X11 is absent, Arg, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0152] In one embodiment, the peptide is prepared according to formula II, R 1 -Cys-Thr-His-[Dpa]-Pro-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (II) In the formula, R 1 , R 2 X6, X8, and X10-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0153] In one embodiment, the peptide is prepared according to formula III, R 1 -Glu-Thr-Cys-[Dpa]-Pro-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (III) In the formula, R 1 , R 2X6, X8, and X10-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0154] In one embodiment, the peptide is prepared according to formula IV, R 1 -Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (IV) In the formula, R 1 , R 2 X6, X8, and X10-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0155] In one embodiment, the peptide is prepared according to formula V, R 1 -Glu-Thr-His-[Dpa]-Pro-X6-Cys-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (V) In the formula, R 1 , R 2 X6, X8, and X10-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0156] In one embodiment, the peptide is prepared according to formula VI, R 1 -Glu-Thr-His-[Dpa]-Cys-X6-Ile-Cys-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VI) In the formula, R 1 , R 2 X6 and X10-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0157] In one embodiment, the peptide is prepared according to formula VII, R 1-Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-Cys-X11-X12-X13-X14-R 2 (VII) In the formula, R 1 , R 2 X6, X8, X10, and X11-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0158] In one embodiment, the peptide is prepared according to formula VIIIa or formula VIIIb, R 1 -Cys-Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIa) R 1 -[hCys]-Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIb) In the formula, R 1 , R 2 X6, X8, X10, and X11-X14 are as described in claim 1, and Cys and X6 are linked via disulfide bonds.

[0159] In one embodiment, X6 is a pen.

[0160] In one embodiment, X6 is hCys.

[0161] In one embodiment, X6 is aMeCys.

[0162] In one embodiment, X6 is (D)Cys.

[0163] In one embodiment, X6 is Cys.

[0164] In one embodiment, X8 is Lys replaced by Ahx-Palm.

[0165] In one embodiment, X8 is absent, Lys, substitute Lys, (D)Lys, or substitute (D)Lys.

[0166] In one embodiment, X8 does not exist.

[0167] In one embodiment, X8 is (D)Lys.

[0168] In one embodiment, X8 is Lys.

[0169] In one embodiment, X8 is Lys replaced by Ahx-Palm.

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

[0171] In one embodiment, X8 is a conjugated amino acid.

[0172] In one embodiment, X8 is a conjugated Lys or (D)Lys.

[0173] In one embodiment, X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is the linker and Z is the half-life extension portion.

[0174] In one embodiment, X10 is Glu.

[0175] In one embodiment, X10 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0176] In one embodiment, X10 is absent.

[0177] In one embodiment, X10 is (D)Lys.

[0178] In one embodiment, X10 is Lys.

[0179] In one embodiment, X10 is Lys replaced by Ahx-Palm.

[0180] In one embodiment, X10 is Lys(Ahx_Palm).

[0181] In one embodiment, X10 is a conjugated amino acid.

[0182] In one embodiment, X10 is a conjugated Lys or (D)Lys.

[0183] In one embodiment, X10 is Lys(L1Z) or (D)Lys(L1Z), where L1 is the linker and Z is the half-life extension portion.

[0184] In one embodiment, X8 is (D)Lys, and X10 is Lys(L1Z) or (D)Lys(L1Z). In one embodiment, X10 is Lys(L1Z).

[0185] In one embodiment, X8 is Lys(L1Z) or (D)Lys(L1Z), and X10 is absent, Glu, Lys, or (D)Lys. In one embodiment, X8 is Lys(L1Z). In another embodiment, X10 is absent. In yet another embodiment, X10 is (D)Lys. In a particular embodiment, X10 is Glu.

[0186] In one embodiment, L1 is a single bond.

[0187] In one embodiment, L1 is iso-Glu.

[0188] In one embodiment, L1 is Ahx.

[0189] In one embodiment, L1 is iso-Glu-Ahx.

[0190] In one embodiment, L1 is PEG.

[0191] In one embodiment, L1 is PEG-Ahx.

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

[0193] In one embodiment, PEG is -[C(O)-CH2-(Peg)nN(H)]m- or -[C(O)-CH2-CH2-(Peg)nN(H)]m-, where Peg is -OCH2CH2-, m is 1, 2, or 3, and n is an integer from 1 to 100, or 10K, 20K, or 30K.

[0194] In one embodiment, m is 1.

[0195] In one embodiment, m is 2.

[0196] In one embodiment, n is 2.

[0197] In one embodiment, n is 4.

[0198] In one embodiment, n is 8.

[0199] In one embodiment, n is 11.

[0200] In one embodiment, n is 12.

[0201] In one embodiment, n is 20K.

[0202] In one embodiment, PEG is 1Peg2, and 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0203] In one embodiment, PEG is 2Peg2, and 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0204] In one embodiment, PEG is 1Peg2-1Peg2, where each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0205] In one embodiment, PEG is 1Peg2-1Peg2, and 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-.

[0206] In one embodiment, PEG is 2Peg4, and 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-.

[0207] In one embodiment, PEG is 1Peg8, and 1Peg8 is -C(O)-CH2-(Peg)8-N(H)- or -[C(O)-CH2-(OCH2CH2)8-NH]-.

[0208] In one embodiment, PEG is 2Peg8, and 2Peg8 is -C(O)-CH2-CH2-(Peg)8-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)8-NH]-.

[0209] In one embodiment, PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-.

[0210] In one embodiment, PEG is 2Peg11, and 2Peg11 is -C(O)-CH2-CH2-(Peg)11-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)11-NH]-.

[0211] 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]-.

[0212] In one embodiment, when PEG is bound to Lys, the -C(O)- of PEG is bound to the Ne of Lys.

[0213] In one embodiment, when PEG is bound to isoGlu, the -N(H)- of PEG is bound to the -C(O)- of isoGlu.

[0214] In one embodiment, when PEG is bound to Ahx, the -N(H)- of PEG is bound to the -C(O)- of Ahx.

[0215] In one embodiment, when PEG is bound to Palm, the -N(H)- of PEG is bound to the -C(O)- of Palm.

[0216] In one embodiment, Z is a Palm.

[0217] In one embodiment, -L1Z is PEG11_OMe, PEG12_C18 acid, 1PEG2_1PEG2_Ahx_Palm, 1PEG2_Ahx_Palm, Ado_Palm, Ahx_Palm, Ahx_PEG20K, PEG12 Ahx IsoGlu Behenic Acid 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 Iso Glue_Palm, -1PEG2_1PEG2_Dap_C18_Dioxide, -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_Diacid, -1PEG8_IsoGlu_C18_Diacid, -IsoGlu_C18_diacid, -PEG12_Ahx_C18_Diacid, -PEG12_C16_Diacid, -PEG12_C18_diacid, -1PEG2_1PEG2_1PEG2_C18_Diacid, -1PEG2_1PEG2_1PEG2_IsoGlu_C18_Diacid, -PEG12_IsoGlu_C18_Diacid, -PEG4_IsoGlu_C18_Diacid, or -PEG4_PEG4_IsoGlu_C18_diacid, Here, PEG11_OMe-[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)-CH2-CH2-(OCH2CH2)8-NH-, 1PEG8 is -[C(O)-CH2-(OCH2CH2)8-NH-, PEG12 is -[C(O)-CH2-CH2-(OCH2CH2) 12 It is -NH- and Ado is -[C(O)-(CH2)] 11 -NH]- Cn acid is -C(O)(CH2) n-2 -CH3, and C18 acid is -C(O)-(CH2) 16 -Me, Palm is -C(O)-(CH2) 14 -Me, isoGlu is isoglutamic acid. isoGlu_Palm [ka] And, Ahx is -[C(O)-(CH2)5-NH]-, Cn diacid is -C(O)-(CH2) n-2 -COOH, where n is 10, 12, 14, 16, 18, or 22.

[0218] In one embodiment, X6 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n It is a diacid, and Lys(1PEG2_1PEG2_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0219] In one embodiment, X6 or X10 is (D)Lys(1PEG2_1PEG2_IsoGlu_C n (D)Lys(1PEG2_1PEG2_IsoGlu_C) n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0220] In one embodiment, X6 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.

[0221] In one embodiment, X6 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.

[0222] In one embodiment, X6 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.

[0223] In one embodiment, X6 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.

[0224] In one embodiment, X6 or X10 is (D)Lys(IsoGlu_C n (D)Lys(IsoGlu_C) n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0225] In one embodiment, X6 or X10 is Lys(PEG12_IsoGlu_C n It is a diacid, and Lys(PEG12_IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0226] In one embodiment, X6 or X10 is (D)Lys(PEG12_IsoGlu_C n (D)Lys(PEG12_IsoGlu_C) n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0227] In one embodiment, X6 or X10 is Lys(PEG4_IsoGlu_C n It is a diacid, and Lys(PEG4_IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0228] In one embodiment, X6 or X10 is (D)Lys(PEG4_IsoGlu_C n (D)Lys(PEG4_IsoGlu_C) n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0229] In one embodiment, X6 or X10 is Lys(PEG4_PEG4_IsoGlu_C n It is a diacid, and Lys(PEG4_PEG4_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0230] In one embodiment, X6 or X10 is (D)Lys(PEG4_PEG4_IsoGlu_C n (D)Lys(PEG4_PEG4_IsoGlu_C) n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0231] In one embodiment, X6 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.

[0232] In one embodiment, X6 or X10 is (D)Lys(IsoGlu_C n(D)Lys(IsoGlu_C) n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0233] In one embodiment, X6 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.

[0234] In one embodiment, X6 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.

[0235] In one embodiment, X6 or X10 is (D)Lys(PEG12_Ahx_C n (Diacid) and (D)Lys(PEG12_Ahx_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0236] In one embodiment, X6 or X10 is Lys(PEG12_C n _Diacid) and Lys(PEG12_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0237] In one embodiment, X6 or X10 is (D)Lys(PEG12_C n (Diacid) and (D)Lys(PEG12_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0238] In one embodiment, R 2 It is NH2.

[0239] In one embodiment, R 2 It is a substituted amino acid.

[0240] In one embodiment, R 2 It is an N-alkylamino compound.

[0241] In one embodiment, R 2 This is an N-alkylamino, where the alkyl group is either further substituted or unsubstituted.

[0242] In one embodiment, R 2 This is an N-alkylamino, where alkyl is further substituted with an aryl or heteroaryl.

[0243] In one embodiment, R 2 is an alkylamino, where the alkyl is either unsubstituted or substituted with an aryl, and the alkyl is ethyl, propyl, butyl, or pentyl.

[0244] In one embodiment, R 2is an alkylamino, where the alkyl is either unsubstituted or substituted with phenyl, and the alkyl is ethyl, propyl, butyl, or pentyl.

[0245] In one embodiment, R 2 It is OH.

[0246] In one embodiment, R 1 C1-C 20 It is Alkanoyl.

[0247] In one embodiment, R 1 This is IVA or isovaleric acid.

[0248] In one embodiment, the peptide is a linear peptide.

[0249] In one embodiment, the peptide is a lactam.

[0250] In one embodiment, the peptide is a lactam, where one of the free -NH2 groups is cyclized with one of the free -C(O)2H groups.

[0251] This disclosure comprises a peptide, or a hepcidin analog comprising a peptide, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide is one of the peptides listed in Tables 6A to 6B.

[0252] This disclosure includes a peptide, or a hepcidin analog comprising a peptide, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide is ID number 3 [ka] Isovaleric acid-ETH-[Cys]-P-[Cys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, ID number 4 [ka] Iso-glycine-ETH-[Dpa]-[Cys]-[Cys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2、 ID number 5

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[0253] In some embodiments, the present invention provides peptides listed in Table 7, or pharmaceutically acceptable salts and solvates thereof.

[0254] In some embodiments, the present invention provides peptides listed in Table 6C, or pharmaceutically acceptable salts and solvates thereof, where Cys and Cys, dCys and dCys, Cys and dCys, Cys and dPen, Cys and hCys, hcys and hCys, or hCys and Pen are cyclized to form a disulfide bond. In certain embodiments, mercapto groups on the side chains of amino acid pairs in the peptides listed in Table 6C, such as Cys and Cys, dCys and dCys, Cys and dCys, Cys and dPen, Cys and hCys, hcys and hCys, or hCys and Pen, come together to form a disulfide bond.

[0255] In some embodiments, the present invention provides peptides listed in Table 6D, or pharmaceutically acceptable salts and solvates thereof, where Cys and Cys, Cys and NMe-Cys, Cys and hCys, Cys and dCys, Cys and 4S_Mcp, Cys and 4R_Mcp, or 4S_Mcp and 4R_Mcp are cyclized to form a disulfide bond. In certain embodiments, the mercapto groups on the side chains of amino acid pairs in the peptides listed in Table 6D, for example, Cys and Cys, Cys and NMe-Cys, Cys and hCys, Cys and dCys, Cys and 4S_Mcp, Cys and 4R_Mcp, or 4S_Mcp and 4R_Mcp, combine to form a disulfide bond.

[0256] In some embodiments, the present invention provides peptides listed in Table 6E, or pharmaceutically acceptable salts and solvates thereof, where Cys and Cys, Cys and Pen, Cys and hCys, hcys and hCys, Cys and NMe_Cys, or Pen and Pen in the peptide are cyclized to form a disulfide bond. In certain embodiments, amino acid pairs in the peptides listed in Table 6D, for example, Cys and Cys, Cys and Pen, Cys and hC ys, hcys and hCys, Cys and NMe_Cys, or Pen and the mercapto group on the side chain of Pen combine to form a disulfide bond. In some embodiments, the present invention provides the peptides listed in Table 6E, where (i) Cys and Cys in the peptide are cyclized to form a disulfide bond, and (ii) the carboxyl group on the side chain of Glu and the amino group on the side chain of dLys in the peptide combine to form an amide bond, i.e., a -C(O)NH- bond. In certain embodiments, the present invention provides the peptides listed in Table 6E, where (i) Cys and Cys in the peptide are cyclized to form a disulfide bond, and (ii) the carboxyl group on the side chain of Glu and the amino group of N_ethylamine combine to form an amide bond, i.e., a -C(O)NH- bond. In other embodiments, the present invention provides the peptides listed in Table 6E, where Cys and Cys in the peptide are cyclized via a p-xylene linker, [ka] To form a bond.

[0257] In one embodiment, R 2 In another embodiment, R 2 is a substituted amino acid. In another embodiment, R 2 is an alkylamino or (substituted alkyl)amino. In another embodiment, R 2 These are methylamino, ethylamino, propylamino, benzylamino, or phenethylamino.

[0258] In one embodiment, R 2 It is OH.

[0259] In one embodiment, R 1 C1-C 20 It is Alkanoyl.

[0260] In one embodiment, R 1 This is IVA or isovaleric acid.

[0261] In a particular embodiment of any of the peptide analogs having any of the various formulas described herein, R 1 The amides are selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and conjugated amides of lauric acid, hexadecanoic acid, and γ-Glu-hexadecanoic acid.

[0262] In certain embodiments, the substitution Lys is 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 Lys is substituted with an alkanoyl or any other as described herein. In one embodiment, Lys is N ε It will be replaced with.

[0263] In a particular embodiment, the substitution (D)Lys is 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 (D)Lys is substituted with an alkanoyl or any other as described herein. In one embodiment, (D)Lys is N ε It will be replaced with.

[0264] In a particular embodiment, C10 -C 20 Alkanoyl is Palm.

[0265] In certain embodiments, the present invention includes a polypeptide comprising an amino acid sequence described in Tables 6A to 6B, or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity with any of those amino acid sequences.

[0266] In certain embodiments, the present invention includes a hepcidin analog having the structure described below or comprising the amino acid sequence described below.

[0267] In certain embodiments, the present invention includes a hepcidin analog having the structure described below or comprising the amino acid sequence described below.

[0268] In a particular embodiment, the peptide is one of the peptides having an FPN activity of less than 100 nM. In another embodiment, the peptide is one of the peptides having an FPN activity of less than 50 nM. In yet another embodiment, the peptide is one of the peptides having an FPN activity of less than 20 nM. In yet another embodiment, the peptide is one of the peptides having an FPN activity of less than 10 nM. In a more specific embodiment, the peptide is one of the peptides having an FPN activity of less than 5 nM.

[0269] Peptide analog conjugates In certain embodiments, the hepcidin analogs of the present invention, including both monomeric and dimeric forms, contain one or more conjugated chemical substituents, such as lipophilic substituents and polymeric substituents, collectively referred herein as half-life extension moieties. While we do not wish to be bound by any particular theory, it is thought that the lipophilic substituents bind to albumin in the bloodstream, thereby protecting the hepcidin analog from enzymatic degradation and thus extending its half-life. In addition, it is thought that the polymeric substituents extend the half-life, reduce clearance in the bloodstream, and, in some cases, enhance permeability through the epithelium and retention in the lamina propria. Furthermore, it is also conceivable that these substituents may, in some cases, enhance permeability through the epithelium and retention in the lamina propria. Those skilled in the art will be well aware of suitable techniques for preparing the compounds used in connection with the present invention. For non-limiting examples of suitable chemistry, see, for example, WO98 / 08871, WO00 / 55184, WO00 / 55119, Madsen et al (J.Med.Chem.2007,50,6126-32), and Knudsen et al.2000 (J.Med Chem.43,1664-1669).

[0270] 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 another half-life extender. 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 moieties, if present, may provide a gap between the hepcidin analog and the lipophilic substituent.

[0271] In certain embodiments, the lipophilic substituent or half-life extender comprises a hydrocarbon chain having 4 to 30 carbon atoms, for example, at least 8 or 12 carbon atoms, preferably 24 or fewer carbon atoms, or 20 or fewer carbon atoms. The hydrocarbon chain may be linear or branched, and may be saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted with a portion that forms part of the bond to the amino acid side chain or spacer, for example, 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, and therefore the hydrocarbon chain may form part of an alkanoyl group, for example, palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0272] The lipophilic substituent may be conjugated to any amino acid side chain in the hepcidine analog of the present invention. In certain embodiments, the amino acid side chain includes a carboxyl group, hydroxyl group, thiol group, amide group, or amine group for forming an ester, sulfonyl ester, thioester, amide, or sulfonamide with a spacer or lipophilic substituent. For example, the lipophilic substituent may be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys, or Dbu, Dpr, or Orn. ​​In certain embodiments, the lipophilic substituent is conjugated to Lys. The amino acid represented as Lys in any of the formulas provided herein may be substituted, for example, with Dbu, Dpr, or Orn to which the lipophilic substituent is added.

[0273] In further embodiments of the present invention, or / or in addition, the side chains of one or more amino acid residues in the hepcidin analog of the present invention may be conjugated to a polymer moiety or other half-life extension moiety, for example, to increase solubility and / or half-life and / or bioavailability in vivo (e.g., plasma). Such modifications are also known to reduce the clearance (e.g., renal clearance) of therapeutic proteins and therapeutic peptides.

[0274] As used herein, "polyethylene glycol" or "PEG" refers to the general formula H-(O-CH2-CH2) nPEG is a polyether compound of -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), and as used herein, PEO, PEE, or POG refer to oligomers or polymers of ethylene oxide, depending on their molecular weight. While these three names are chemically synonymous, PEG tends to refer to oligomers and polymers with molecular weights less than 20,000 g / mol, PEO to polymers with molecular weights greater than 20,000 g / mol, and POE to polymers of any molecular weight. PEG and PEO are liquid or low-melting-point solids, depending on their molecular weight. Throughout this disclosure, these three names are used without distinction. 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. PEG and PEO with different molecular weights are used in different applications and have different physical properties (e.g., viscosity) due to chain length effects, but their chemical properties are nearly identical. The polymer portion is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymer portions include polyethylene glycol (PEG), homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Int. J. Hematology 68:1 (1998), Bioconjugate Chem. 6:150 (1995), and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992). PEGs prepared for the purpose of extending half-life, such as mono-methoxy-terminated polyethylene glycol (mPEG), and monoactivated alkoxy-terminated polyalkylene oxides (POAs), as well as bisactivated polyethylene oxides (glycols) or other PEG derivatives, are also intended. The preferred polymers vary substantially by weight, with a range of about 200 to about 40,000 being typically selected for the purposes of the present invention. In certain embodiments, PEG having a molecular weight of 200 to 2,000 daltons or 200 to 500 daltons is used.Depending on the initiator used in the polymerization process, different forms of PEG may be used. For example, common initiators include monofunctional methyl ether PEG or methoxypoly(ethylene glycol), abbreviated as mPEG. Other suitable initiators are known in the art and are suitable for use in the present invention.

[0275] Low molecular weight PEGs are also available as pure oligomers, referred to as monodisperse, homogeneous, or individual. These are used in certain embodiments of the present invention.

[0276] PEG is available in different forms: branched PEG has 3 to 10 PEG chains arising from a central core group, star-shaped PEG has 10 to 100 PEG chains arising from a central core group, and comb-shaped PEG has multiple PEG chains that are usually grafted onto a polymer backbone. PEG can also be linear. The number often included in the name of PEG indicates its average molecular weight (for example, PEG with n=9 has an average molecular weight of approximately 400 daltons and would be labeled PEG400).

[0277] As used herein, “PEGylation” is the act of coupling a PEG structure to a hepcidin analog of the present invention (e.g., covalently), and in certain embodiments, is referred to as the “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 extension portion is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, this 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.

[0278] In some embodiments, the present invention comprises hepcidin analog peptides (or dimers thereof) conjugated to PEG, which are covalently bonded, for example, via amides, thiols, by click chemistry, or by any other suitable means known in the art. In certain embodiments, the PEG is bonded via amide bonds, and therefore certain PEG derivatives used are appropriately functionalized. For example, in certain embodiments, PEG11, which is O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol, has both an amine and a carboxylic acid to bond to the peptide of the present invention. In certain embodiments, PEG25 has a diacid moiety and 25 glycol moieties.

[0279] Other suitable polymer moieties include poly-amino acids, such as poly-lysine, poly-aspartic acid, and poly-glutamic acid (see, for example, 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 moieties may be linear or branched. In some embodiments, they have molecular weights of 500 to 40,000 Da, for example, 500 to 10,000 Da, 1,000 to 5,000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da).

[0280] In some embodiments, the hepcidine analog of the present invention may comprise two or more such polymer moieties, in which case the total molecular weight of all such moieties is generally within the above range.

[0281] In some embodiments, the polymer moiety may be covalently coupled to an amino, carboxyl, or thiol group of an amino acid side chain. Certain examples include the thiol group of a Cys residue and the epsilon-amino group of a Lys residue, and the carboxyl groups of Asp and Glu residues may also be involved.

[0282] Those skilled in the art will be well aware of suitable techniques that can be used to carry out coupling reactions. For example, the PEG moiety having a methoxy group can be coupled to a Cys thiol group via a maleimide bond using reagents commercially available from Nektar Therapeutics AL. For details of suitable chemistry, see also WO2008 / 101017 and the references above. Maleimide-functionalized PEG may also be conjugated to the sulfhydryl group of the Cys residue's side chain.

[0283] As used herein, disulfide bond oxidation may occur in a single step or as a two-step process. In the case of a single oxidation step, as used herein, the trityl protecting group is often used during construction, allowing for deprotection during cleavage, followed by solution oxidation. If a second disulfide bond is required, there is a choice between spontaneous oxidation or selective oxidation. In the case of selective oxidation requiring an orthogonal protecting group, Acm and trityl are used as protecting groups for cysteine. Cleavage results in the removal of one protecting pair of cysteine, allowing oxidation of this pair. A second oxidative deprotection step of the cysteine ​​protecting Acm group then takes place. In the case of spontaneous oxidation, the trityl protecting group is used for all cysteine, allowing for spontaneous folding of the peptide.

[0284] Those skilled in the art will be well aware of suitable techniques that can be used to carry out the oxidation step.

[0285] In certain embodiments, the hepcidin analog of the present invention may include, but is not limited to, Ahx-Palm, PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-lauric acid, isoGlu-mysteric acid, and isoGlu-isovaleric acid, and comprises a half-life extension moiety.

[0286] In certain embodiments, the hepcidin analog comprises a half-life extension portion having the structure shown below, where n is 0 to 24 or n is 14 to 24. [ka] .

[0287] In certain embodiments, the hepcidin analog of the present invention comprises a conjugated half-life extension portion shown in Table 2. [Table 5-1] [Table 5-2]

[0288] In certain embodiments, the half-life extension portion is directly conjugated to the hepcidine analog, while in other embodiments, the half-life extension portion is conjugated to the hepcidine analog peptide via a linker portion, for example, one of those shown in Table 3. [Table 6-1] [Table 6-2]

[0289] Referring to the linker structure shown in Table 3, references to n=1 to 24 or n=1 to 25 (for example, in L4 or L5) indicate that n can be any integer within the enumerated range. Additional linker parts may be used, as shown in the "Abbreviations" table.

[0290] In certain embodiments, the hepcidin analog of the present invention comprises one of the linker moieties shown in Table 3 and one of the half-life extension moieties shown in Table 2, each comprising one of the following combinations shown in Table 4. [Table 7-1] [Table 7-2] [Table 7-3]

[0291] In certain embodiments, the hepcidin analog comprises two or more linkers. In certain embodiments, the two or more linkers are concatemerized, i.e., linked to one another.

[0292] In related embodiments, the present invention includes a polynucleotide encoding a polypeptide having a peptide sequence present in any of the hepcidin analogs described herein.

[0293] In addition, the present invention includes vectors containing the polynucleotide of the present invention, such as expression vectors.

[0294] Treatment method In some embodiments, the present invention provides a method for treating a subject suffering from a disease or disorder associated with hepcidin signaling dysregulation, the method comprising administering the subject a hepcidin analog of the present invention. 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, the method comprising administering to the individual a hepcidin analog or composition of the present invention in an amount sufficient to bind to and stimulate ferroportin in the subject or to mimic hepcidin. In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by iron metabolism dysregulation, the method comprising administering to the subject a hepcidin analog or composition of the present invention.

[0295] In some embodiments, the methods of the present invention include providing a hepcidin analog or a composition of the present invention to a subject requiring it. In certain embodiments, the subject requiring it is diagnosed with, or determined to be at risk of developing, a disease or disorder characterized by iron level dysregulation (e.g., iron metabolic disorders or disorders, diseases or disorders related to iron overload, and diseases or disorders related to abnormal hepcidin activity or expression). In certain embodiments, the subject is a mammal (e.g., human).

[0296] In certain embodiments, the disease or disorder is an iron metabolic disorder, such as an iron overload disorder, an iron deficiency disorder, an iron distribution disorder, or another iron metabolic disorder, and other disorders potentially related to iron metabolism. In certain embodiments, iron metabolic disorders include hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemomoduberin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, beta-thalassemia, sideroblastic anemia, porphyria, late-onset cutaneous porphyria, African-type iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital erythrodysplasia anemia, hypochromic microcytic anemia, sickle cell anemia, polycythemia vera (primary and secondary), and secondary erythrocyte Cyltonia, for example, chronic obstructive pulmonary disease (COPD), post-kidney transplantation, Chuvasi, HIF and PHD mutations, as well as idiopathic myelodysplasia, pyruvate kinase deficiency, hypochromic microcytic anemia, transfusion-dependent anemia, hemolytic anemia, obesity iron deficiency, other anemias, benign or malignant tumors that overproduce or induce hepcidin, conditions with hepcidin excess, Friedreich's ataxia, Gracil syndrome, Harrellforden-Spats disease, Wilson's disease, pulmonary hemosiderin deposition, hepatocellular carcinoma, cancer (e.g., liver cancer), hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes mellitus, atherosclerosis, neurodegenerative disorders, dementia, multiple sclerosis, Parkinson's disease, Huntington's disease, or Alzheimer's disease.

[0297] In certain embodiments, the disease or disorder is related to iron overload diseases, such as iron hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemomodoverin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0298] In certain embodiments, the disease or disorder is one that is not typically identified as iron-related. For example, hepcidin is highly expressed in mouse pancreas, suggesting that diabetes mellitus (type I or II), insulin resistance, glucose intolerance, and other disorders may be ameliorated by treating underlying iron metabolic disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, incorporated herein by reference. Thus, the peptides of the present invention may 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 by using methods known in the art, such as assays that monitor the concentration and expression of hepcidin, hemoduverin, or iron, such as the assay of WO2004 / 092405, incorporated herein by reference, and the assay described in U.S. Patent No. 7,534,764, incorporated herein by reference.

[0299] In a particular embodiment, the disease or disorder is postmenopausal osteoporosis.

[0300] In certain embodiments of the present invention, iron metabolic disorders are iron overload disorders, including hereditary hemochromatosis, iron-overload anemia, alcoholic liver disease, heart disease and / or heart failure, cardiomyopathy, and chronic hepatitis C.

[0301] In certain embodiments, any of these diseases, disorders, or indications are caused by or related to hepcidin deficiency or iron overload.

[0302] In some embodiments, the method of the present invention includes providing a hepcidin analog (i.e., a first therapeutic agent) to a subject in need of it, in combination with a second therapeutic agent. In certain embodiments, the second therapeutic agent is provided to the subject before and / or simultaneously with and / or after the administration of the pharmaceutical composition to the subject. In certain embodiments, the second therapeutic agent is an iron chelating agent. In certain embodiments, the second therapeutic agent is selected from deferoxamine and deferasirox (Exjade®), which are iron chelating agents. In other embodiments, the method includes administering a third therapeutic agent to the subject.

[0303] The present invention provides a composition (e.g., a pharmaceutical composition) comprising one or more hepcidin analogs of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent. A pharmaceutically acceptable carrier, diluent, or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride.

[0304] The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers. Therapeutic pharmaceutically acceptable carriers are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, slightly acidic or physiological pH sterile salines and phosphate-buffered salines may be used. Suitable pH buffers may be, for example, phosphate buffer, citrate buffer, acetate buffer, tris(hydroxymethyl)aminomethane (TRIS) buffer, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) buffer, ammonium bicarbonate buffer, diethanolamine buffer, histidine buffer, arginine buffer, lysine buffer, or acetate buffer (e.g., as sodium acetate), or mixtures thereof. The term further encompasses any carriers listed in the United States Pharmacopeia for use in animals, including humans.

[0305] In certain embodiments, the composition comprises two or more hepcidin analogs disclosed herein. In certain embodiments, the combination is selected from (i) any two or more hepcidin analog peptide monomers shown herein, (ii) any two or more hepcidin analog peptide dimers disclosed herein, (iii) any one or more hepcidin analog peptide monomers disclosed herein, and any one or more hepcidin analog peptide dimers disclosed herein.

[0306] It should be understood that including a hepcidin analog of the present invention (i.e., one or more hepcidin analog peptide monomers or one or more hepcidin analog peptide dimers of the present invention) in a pharmaceutical composition also includes including pharmaceutically acceptable salts or solvates 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.

[0307] 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, for example, the treatment methods 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 disclosed herein or elsewhere (see, for example, the treatment methods 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.

[0308] The hepcidin analogs of the present invention are suitable for administration with or without storage and can be formulated as pharmaceutical compositions typically comprising a therapeutically effective amount of at least one hepcidin analog of the present invention together with a pharmaceutically acceptable carrier, excipient, or vehicle.

[0309] In some embodiments, the hepcidin analog pharmaceutical composition of the present invention is a unit dosage form. In such a dosage form, the composition is divided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form may be presented as a packaged preparation, which package contains, for example, a separate amount of the preparation, such as packaged tablets, capsules, or powders in a vial or ampoule. The unit dosage form may be, for example, a capsule, cachet, or tablet itself, or any appropriate number of these packaged forms. The unit dosage form may also be provided in the form of a single-dose injection form, for example, in the form of a pen-type device containing a liquid-phase (typically aqueous) composition. The composition may be formulated for any preferred route and means of administration, for example, any one of the routes and means of administration disclosed herein.

[0310] In certain embodiments, the hepcidin analog, or the pharmaceutical composition containing the hepcidin analog, is suspended in a sustained-release matrix. As used herein, a sustained-release matrix is ​​a matrix made of materials, typically polymers, that are degradable by enzymatic hydrolysis, acid-base hydrolysis, or dissolution. Once inserted into the body, this matrix acts upon enzymes and body fluids. The sustained-release matrix is ​​selected from biocompatible materials, such as liposomes, polylactides (polylactic acid), polyglycolides (glycolic acid polymers), polylactide coglycolides (lactic acid / glycolic acid copolymers), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids, such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. One embodiment of the biodegradable matrix is ​​a matrix of any one of the following: polylactide, polyglycolide, or polylactidecoglycolide (lactic acid / glycolic acid copolymer).

[0311] In certain embodiments, the composition is administered parenterally, subcutaneously, or orally. In certain embodiments, the composition is administered orally, intracapsularly, vaginally, intraperitoneally, rectally, topically (including by powder, ointment, drops, suppositories, or transdermal patch, including intravitreal, intranasal, and inhalation delivery), or buccally. As used herein, the term “parenteral” refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intra-articular injections and infusions. Accordingly, in certain embodiments, the composition is formulated for delivery by any of these routes of administration.

[0312] In certain embodiments, a pharmaceutical composition for parenteral injection comprises a pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, or sterile powder for reconstitution into a sterile injectable solution or dispersion immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), carboxymethylcellulose and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Sustained absorption of the injectable pharmaceutical form may be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0313] Injectable depot formulations include those prepared by forming a microcapsule matrix of hepcidine analogs in one or more biodegradable polymers such as polylactide-polyglycolide, poly(orthoester), poly(anhydride), and (poly)glycol, for example, PEG. The release rate of the hepcidine analog can be controlled depending on the ratio of the peptide to the polymer and the properties of the specific polymer used. Injectable depot formulations can also be prepared by encapsulating the hepcidine analogs in liposomes or microemulsions compatible with body tissues.

[0314] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media immediately before use.

[0315] The hepcidin analog of the present invention may also be administered via liposomes or other lipid-based carriers. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by monolayers or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, metabolizable lipid capable of forming liposomes can be used. The composition in liposomal form may contain stabilizers, preservatives, excipients, etc., in addition to the hepcidin analog of the present invention. In certain embodiments, the lipids include phospholipids containing both natural and synthetic phosphatidylcholine (lecithin) and serine. Methods for forming liposomes are known in the art.

[0316] Pharmaceutical compositions used in the present invention, which are suitable for parenteral administration, may generally include sterile aqueous solutions and / or suspensions of peptide inhibitors that have been made isotonic with the recipient's blood using sodium chloride, glycerin, glucose, mannitol, sorbitol, etc.

[0317] In some embodiments, the present invention provides pharmaceutical compositions for oral delivery. The compositions and hepcidin analogs of the present invention may 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 understand that the hepcidin analogs of the present invention may be modified or integrated into systems or delivery vehicles that are not disclosed herein but are well known in the art and suitable for use in the oral delivery of peptides.

[0318] In certain embodiments, the oral formulation may include an adjuvant to artificially increase intestinal permeability (e.g., resorcinol and / or nonionic surfactants, e.g., polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether), and / or an enzyme inhibitor to inhibit enzymatic degradation (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF), or tracylol). In certain embodiments, the hepcidin analog in solid dosage form for oral administration may be mixed with at least one additive, e.g., sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, or glyceride. These dosage forms may also contain other types of additives, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens and sorbic acid, antioxidants such as ascorbic acid, alpha-tocopherol and cysteine, disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavorings, or perfusion agents.

[0319] In certain embodiments, an oral dosage form or unit dose suitable for use with the hepcidin analog of the present invention may include a mixture of the hepcidin analog with non-pharmacological components or excipients, as well as other non-reusable materials that may be considered either components or packaging. The oral composition may comprise at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, an oral dosage form is provided that comprises an effective amount of the hepcidin analog, and this dosage form comprises at least one of pills, tablets, capsules, gels, pastes, beverages, syrups, ointments, and suppositories. In some cases, an oral dosage form is provided that is designed and configured to achieve delayed release of the hepcidin analog in the small intestine and / or colon of the subject.

[0320] In one embodiment, an oral pharmaceutical composition comprising the hepcidin analog of the present invention comprises an enteric coating designed to delay the release of the hepcidin analog in the small intestine. In at least some embodiments, a pharmaceutical composition is provided comprising the hepcidin analog of the present invention and a protease inhibitor such as aprotinin in a delayed-release pharmaceutical formulation. In some cases, the pharmaceutical composition of the present invention comprises an enteric coating that is soluble in gastric juice at a pH of about 5.0 or higher. In at least one embodiment, a pharmaceutical composition is provided comprising an enteric coating comprising a polymer having a dissociable carboxylic acid group, such as a cellulose derivative, e.g., hydroxypropyl methylcellulose phthalate, cellulose phthalate acetate, and cellulose trimellitate acetate, and similar cellulose derivatives, and other carbohydrate polymers.

[0321] In one embodiment, the pharmaceutical composition comprising the hepcidin analog of the present invention is provided with an enteric coating, which is designed to protect and release the pharmaceutical composition in a controlled manner within the lower digestive system of the target and to avoid systemic side effects. In addition to the enteric coating, the hepcidin analog of the present invention may be encapsulated, coated, bound, or otherwise associated within any suitable oral drug delivery system or component. For example, in some embodiments, the hepcidin analog of the present invention is provided with a lipid carrier system comprising at least one of polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0322] 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, thereby protecting the hepcidin analog from protein degradation in the small intestine and / or colon. The hepcidin analog of the present invention may be further formulated for use in compatible carrier systems designed to increase solubility and enhance intestinal absorption of peptides. These methods include the use of liposomes, micelles, and nanoparticles to increase the gastrointestinal permeability of peptides.

[0323] Various biological response systems may be combined with one or more hepcidin analogs of the present invention to provide orally delivered pharmaceuticals. In some embodiments, the hepcidin analogs of the present invention are used in combination with biological response systems such as hydrogen-bonding hydrogels and mucosal adhesive polymers (e.g., PEG, poly(methacrylic acid) [PMAA], cellulose, Eudragit®, chitosan, and alginates) to provide orally administered therapeutic agents. Other embodiments include methods for optimizing or extending the drug residence time of the hepcidin analogs disclosed herein, wherein the surface of the hepcidin analog is modified to include mucosal adhesive properties by hydrogen bonding, polymers having linked mucins, and / or hydrophobic interactions. These modified peptide molecules may exhibit increased drug residence time in a target according to the desired features of the present invention. Furthermore, the targeted mucosal adhesive system may specifically bind to receptors on the surface of intestinal cells and M cells, thereby further increasing the uptake of particles containing the hepcidin analog.

[0324] Other embodiments include methods for oral delivery of the hepcidin analog of the present invention, wherein the hepcidin analog is provided to subjects in combination with a permeabiliser that facilitates the transport of peptides across the intestinal mucosa by increasing paracellular or transcellular permeability. For example, in one embodiment, the permeabiliser is combined with the hepcidin analog, wherein the permeabiliser comprises at least one of long-chain fatty acids, bile salts, amphiphilic surfactants, and chelating agents. In one embodiment, a permeabiliser comprising sodium N-[hydroxybenzoyl)amino]caprylate is used to form a weak non-covalent bond with the hepcidin analog of the present invention, wherein the permeabiliser prefers membrane transport and further dissociation once it reaches the bloodstream. 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. Furthermore, in at least one embodiment, a non-covalent bond is provided between the peptide inhibitor of the present invention and a permeabilis enhancer selected from the group consisting of cyclodextrin (CD) and dendrimers, wherein the permeabilis enhancer reduces peptide aggregation and increases the stability and solubility of the hepcidine analog molecule.

[0325] Other embodiments of the present invention provide methods for treating a subject using a hepcidin analog of the present invention having an extended half-life. In one embodiment, the present invention provides a hepcidin analog having a half-life of at least several hours to one day in vitro or in vivo sufficient for a therapeutically effective dose once daily (qd) or twice daily (bid) administration (for example, when administered to a human subject). In another embodiment, the hepcidin analog has a half-life of three days or more sufficient for a therapeutically effective dose once weekly (qw) administration. In yet another embodiment, the hepcidin analog has a half-life of eight days or more sufficient for a therapeutically effective dose every other week (biw) or once monthly administration. In yet another embodiment, the hepcidin analog is derivatized or modified to have a longer half-life compared to an underivativeed or unmodified hepcidin analog. In yet another embodiment, the hepcidin analog includes one or more chemical modifications to increase the serum half-life.

[0326] When used in at least one of the therapeutic or delivery systems described herein, the hepcidin analogs of the present invention may be used in pure form or, if such form exists, in a pharmaceutically acceptable salt form.

[0327] Dosage The total daily dose of the hepcidin analogs and compositions of the present invention may be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutically effective dose level for any particular subject depends on a variety of factors, including a) the disorder being treated and its severity, b) the activity of the specific compound used, c) the specific composition used, the patient's age, weight, overall health, sex, and diet, d) the time of administration, the route of administration, and the excretion rate of the specific hepcidin analog used, e) the duration of treatment, f) any drugs used in combination with or concurrently with the specific hepcidin analog used, and similar factors well known in the medical technology.

[0328] In certain embodiments, the total daily dose of the hepcidin analog of the present invention administered to a human or other mammalian host in a single dose or divided dose may be, for example, 0.0001 to 300 mg / kg body weight or 1 to 300 mg / kg body weight. In certain embodiments, the dosage of the hepcidin analog of the present invention is administered in one or more doses, such as 1 to 3 doses, ranging from about 0.0001 to about 100 mg / kg body weight per day, for example, about 0.0005 to about 50 mg / kg body weight per day, for example, about 0.001 to about 10 mg / kg body weight per day, for example, about 0.01 to about 1 mg / kg body weight per day. In certain embodiments, the total dosage may be, 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 in a human patient, about once or twice a week. In certain embodiments, the total dosage per human patient is, for example, in the range of approximately 1 mg to 5 mg, or approximately 1 mg to 3 mg, or approximately 2 mg to 3 mg, once a week.

[0329] In various embodiments, the hepcidin analogs of the present invention may be administered continuously (e.g., by intravenous administration or another continuous drug administration method) or at intervals, typically at regular time intervals, depending on the dosage and pharmaceutical composition selected by those skilled in the art as desirable for a particular subject. Regular dosing intervals may include, for example, once daily, twice daily, once every two days, once every three days, once every four days, once every five days, or once every six days, once or twice a week, once or twice a month, and so on.

[0330] Such a periodic hepcidin analog administration regimen of the present invention may be advantageously interrupted for a period of time, for example, under certain circumstances such as during long-term chronic administration, so that the subject receiving the medication reduces the level of the drug or stops taking the drug (often referred to as taking a “drug-free period”). Drug-free periods are useful, for example, to maintain or restore sensitivity to the drug, or to mitigate undesirable side effects of long-term chronic treatment of a subject using the drug. The timing of the drug-free period depends on the timing of the periodic administration regimen and the purpose of taking the drug-free period (e.g., to restore drug sensitivity and / or to mitigate undesirable side effects of continuous long-term administration). In some embodiments, the drug-free period may be a reduction in the dosage of the drug (e.g., until it falls below a therapeutically effective dose over a certain time interval). In other embodiments, the administration of the drug is suspended over a certain time interval before administration is resumed (e.g., at a lower or higher dose and / or at a higher frequency) using the same or a different administration regimen. Thus, drug-free periods of the present invention can be selected from a wide range of periods and administration regimens. Exemplary drug-free periods include periods of two days or more, one week or more, or one month or more, up to a maximum of approximately 24 months. Therefore, for example, a regular once-daily dosing regimen using the peptide, peptide analog, or dimer of the present invention may be interrupted by a drug-free period of, for example, one week, two weeks, or four weeks, after which the aforementioned regular dosing regimen (e.g., a once-daily or once-weekly dosing regimen) is resumed. Various other drug-free periods are expected to be useful for the administration of the hepcidin analog of the present invention.

[0331] Therefore, this hepcidin analog can be delivered by a dosing regimen that includes two or more dosing phases separated by their respective drug-free periods.

[0332] During each administration phase, the hepcidin analog is administered to the recipient in a therapeutically effective dose according to a predetermined administration pattern. The administration pattern may include continuous administration of the drug to the recipient over the duration of the administration phase. Alternatively, the administration pattern may include administration of multiple doses of the hepcidin analog to the recipient, with intervals equal to the administration interval between doses.

[0333] The administration pattern may include at least 2 doses per administration phase, at least 5 doses per administration phase, at least 10 doses per administration phase, at least 20 doses per administration phase, at least 30 doses per administration phase, or more.

[0334] Depending on the specific drug formulation, bioavailability, and pharmacokinetic profile of the hepcidin analog of the present invention, this dosing interval may be once daily, twice daily, once every two days, once every three days, once every four days, once every five days, or once every six days, once or twice a week, once or twice a month, or a regular interval with even less frequent dosing.

[0335] The administration phase may have a duration of 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.

[0336] If the dosing pattern includes multiple doses, the duration of the following drug-free interval phase shall be longer than the dosing interval used in that dosing pattern. If the dosing interval is irregular, the duration of the drug-free interval phase may be longer than the average interval between doses throughout the dosing phase. Alternatively, the duration of the drug-free interval may be longer than the longest interval between consecutive doses during the dosing phase.

[0337] The duration of the drug-free period may be at least twice the relevant dosing interval (or its average), at least three times, at least four times, at least five times, at least ten times, or at least twenty times the relevant dosing interval or its average.

[0338] Within these constraints, the drug-free period phase may have a duration of 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.

[0339] The administration regimen includes at least two administration phases. The consecutive administration phases are separated by their respective rest periods. Therefore, the administration regimen may include 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 or more administration phases, each separated by its respective rest period phase.

[0340] The continuous dosing phase may utilize the same dosing pattern, although this may not always be desirable or necessary. However, when other drugs or active agents are administered in combination with the hepcidin analog of the present invention, typically the same drug or active agent combination is administered in the continuous dosing phase. In certain embodiments, the recipient is human.

[0341] In some embodiments, the present invention provides compositions and agents comprising at least one hepcidin analog disclosed herein. In some embodiments, the present invention provides a method for producing an agent comprising at least one hepcidin analog disclosed herein for the treatment of iron metabolic disorders such as iron overload disease. In some embodiments, the present invention provides a method for producing an agent comprising at least one hepcidin analog disclosed herein for the treatment of diabetes mellitus (type I or type II), insulin resistance, or glucose intolerance. Also provided are methods for treating iron metabolic disorders in subjects, e.g., mammalian subjects, preferably human subjects, 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 dose. Also provided are methods for treating diabetes mellitus (type I or type II), insulin resistance, or glucose intolerance in subjects, e.g., mammalian subjects, preferably human subjects, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or the composition is administered in a therapeutically effective dose.

[0342] In some embodiments, the present invention provides a process for producing hepcidin analogs or hepcidin analog compositions (e.g., pharmaceutical compositions) as disclosed herein.

[0343] In some embodiments, the present invention provides a device comprising at least one hepcidin analog or a pharmaceutically acceptable salt or solvate thereof for target delivery of the hepcidin analog.

[0344] In some embodiments, the present invention provides a method for conjugating ferroportin or inducing internal migration and degradation of ferroportin, comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0345] In some embodiments, the present invention provides a method for binding to ferroportin and blocking pore and efflux transporter functions without causing internal migration of ferroportin. Such a method involves contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0346] In some embodiments, the present invention provides a kit comprising at least one hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) as disclosed herein, packaged together with reagents, devices, instructions, or a combination thereof.

[0347] In some embodiments, the present invention provides a method for administering the hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) of the present invention to a subject via an implant or osmotic pump, by a cartridge or micropump, or by other means known to those skilled in the art. In some embodiments, the present invention provides a complex comprising ferroportin, preferably at least one hepcidin analog disclosed herein conjugated to human ferroportin, or an antibody, for example, an antibody that specifically conjugates to a hepcidin analog disclosed herein, Hep25, or a combination thereof.

[0348] In some embodiments, the hepcidin analog of the present invention is measured at less than 500 nM (e.g., EC) in an FPN internal transfer assay. 50) has. As those skilled in the art will understand, the function of the hepcidin analog depends on the tertiary structure of the hepcidin analog and the presented binding surface. Therefore, it is possible to maintain the function by making slight modifications to the sequence encoding a hepcidin analog that does not affect the fold or a hepcidin analog that is not present on the binding surface. In other embodiments, the present invention provides a hepcidin analog having 85% or more (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 of the hepcidin analogs described herein, which exhibit activity (e.g., hepcidin activity) or alleviate the symptoms of a disease or indication in which hepcidin is involved.

[0349] In other embodiments, the present invention provides a hepcidin analog having 85% or more (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 of the hepcidin analogs presented herein, or to a peptide conforming to any one of the formulas or hepcidin analogs described herein.

[0350] In some embodiments, the hepcidin analogs of the present invention may comprise a functional fragment or variant 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.

[0351] In addition to the methods described herein in the examples, hepcidin analogs of the present invention may be produced using 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, JKSetlow, incorporated herein by reference. See 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. The hepcidine analogs of the present invention can be purified using protein purification techniques known in the art, such as reversed-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, incorporated herein by reference. Alternatively, the hepcidin analogs of the present invention can be prepared by recombinant DNA techniques known in the art. Therefore, polynucleotides encoding the polypeptides of the present invention are contemplated herein. In certain preferred embodiments, the polynucleotides are isolated. As used herein, “isolated polynucleotide” means a polynucleotide in an environment different from the environment in which it naturally occurs. [Examples]

[0352] The following examples demonstrate certain specific embodiments of the present invention. Unless otherwise described in detail, the following examples were performed using routine standard techniques well known to those skilled in the art. It should be understood that these examples are for illustrative purposes only and are not intended to be definitive in any way regarding the conditions or scope of the present invention. Therefore, they should not be construed as limiting the scope of the present invention. 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-Hydoxysuccinimide 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)

[0353] In peptide sequences provided herein, where K() indicates a compound or chemical group presented in parentheses immediately following a lysine residue, it should be understood that the compound or chemical group in parentheses is a side chain conjugated to the lysine residue. Therefore, for example, and not limited to, K-[(PEG8)]- indicates that the PEG8 portion is conjugated to this lysine side chain.

[0354] Palm: Shows the conjugation of palmitic acid (palmitoyl).

[0355] Synthesis Protocol-1 Synthesis of peptide monomers The peptide monomers of the present invention were synthesized using the Merrifield solid-phase synthesis technique on a Protein Technology Symphony multi-channel synthesizer. The peptides were constructed using HBTU (O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate) and diisopropylethylamine (DIEA) coupling conditions. For some amino acid couplings, PyAOP (7-azabenzotriazole-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 a C-terminal amide, and Wang resin preloaded with an N-α-Fmoc protected amino acid was used for peptides with a C-terminal acid. Coupling reagents (premixture of HBTU and DIEA) 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 were identified and screened based on medical chemistry optimization and / or phage presentation to identify those with excellent binding and / or inhibitory properties.

[0356] construction The peptides were constructed using the standard Symphony protocol. The peptide sequences were constructed as follows: The resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4 mL of DMF, followed by treatment with 2.5 mL of 20% 4-methylpiperidine (Fmoc deprotected) for 10 minutes. The resin was then filtered, washed twice with 4 mL of DMF, and treated again with piperidine for a further 30 minutes. The resin was washed three times again with 4 mL of DMF, followed by the addition of 2.5 mL of amino acids and 2.5 mL of HBTU-DIEA mixture. After frequent stirring for 45 minutes, the resin was filtered and washed three times with 4 mL of DMF each. For typical peptides of the present invention, double coupling was performed. After the coupling reaction was complete, the resin was washed three times with 4 mL of DMF each, and then the next amino acid coupling was performed.

[0357] Cutting After the peptide construction was complete, 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). The cleavage reagent successfully cleaved the peptide from the resin and all remaining side-chain protecting groups.

[0358] The cleaved peptides were precipitated in cold diethyl ether and subsequently washed twice with ethyl ether. The filtrate was poured out, a second aliquot of cold ether was added, and this procedure was repeated. The crude peptides were dissolved in acetonitrile / water (7:3 with 1% TFA) solution and filtered. The linear peptides were then purified after their quality was verified using electrospray ionization mass spectrometry (ESI-MS) (Micromass / Waters ZQ).

[0359] purification Analytical reverse-phase high-performance liquid chromatography (HPLC) was performed using a Gemini C18 column (4.6 mm × 250 mm) (Phenomenex). Half-sample reverse-phase HPLC was performed using a Gemini 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was achieved using a linear gradient of buffer B in buffer 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 (analysis) and 20 mL / min (preparation). Separation was achieved using a linear gradient of buffer B in buffer 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 (analysis) and 15 mL / min (preparation).

[0360] Synthesis Protocol-2 Synthesis of peptide monomers The peptide monomers of the present invention were synthesized using a standard Fmoc solid-phase synthesis technique on a CEM Liberty Blue® microwave peptide synthesizer. The peptides were constructed using Oxyma / DIC (cyanohydroxyiminoethyl acetate / diisopropylcarbodiimide) with microwave heating. Rink Amide-MBHA resin (100-200 mesh, 0.66 mmol / g) was used for peptides with a C-terminal amide, and Wang resin preloaded with N-α-Fmoc-protected amino acids was used for peptides with a C-terminal acid. Oxyma was prepared as a 1M DMF solution containing 0.1M DIEA. DIC was prepared as a 0.5M DMF solution. Amino acids were prepared at 200 mM. Peptide inhibitors of the present invention were identified and screened based on medicinal chemistry optimization and / or phage presentation to identify those with excellent binding and / or inhibitory properties.

[0361] construction Peptides were prepared using the standard CEM Liberty Blue™ protocol. The peptide sequences were constructed 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. The peptides were constructed by repeating Fmoc deprotection and Oxyma / DIC coupling cycles. For deprotection, a 20% 4-methylpiperidine DMF solution 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. Then, for most amino acids, 5 equivalents of amino acids, Oxyma, and DIC were added to the reaction vessel, and the mixed reaction was rapidly heated to 90°C for 4 minutes under microwave irradiation. For arginine and histidine residues, milder conditions using temperatures of 75°C and 50°C for 10 minutes, respectively, were used to prevent racemization. Rare and expensive amino acids were manually coupled overnight at room temperature, often using only 1.5–2 equivalents of reagent. Difficult couplings were often double-coupled by repeating 4-minute cycles at 90°C. After coupling, the resin was washed with DMF, and the entire cycle was repeated until the desired peptide construction was complete.

[0362] Cutting After peptide construction was complete, the peptide was cleaved from the resin by treatment with a standard 91:5:2:2 TFA / H2O / TIPS / DODT cleavage cocktail for 2 hours. If two or more Arg(Pbf) residues were present, cleavage was continued for an additional hour.

[0363] The cleaved peptides were precipitated in cold diethyl ether. The filtrate was decanted and removed, and a second aliquot of cold ether was added, and this procedure was repeated. Subsequently, the quality of the linear peptides was verified using electrospray ionization mass spectrometry (ESI-MS) (Waters®, Micromass®, ZQ®), and then purified.

[0364] purification Analytical reverse-phase high-performance liquid chromatography (HPLC) was performed using a Gemini® C18 column (4.6 mm × 250 mm) (Phenomenex). Half-sample reverse-phase HPLC was performed using a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex) or a Jupiter® 10 μm, 300 Å C18 column (21.2 mm × 250 mm) (Phenomenex). Separation was achieved using a linear gradient of buffer B in buffer 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 (analysis) and 20 mL / min (preparation).

[0365] Example 1A Synthesis of peptide analogs Unless otherwise specified, the reagents and solvents used below were standard laboratory reagents or commercially available analytical grade reagents, and were used without further purification.

[0366] Solid-phase synthesis procedure for peptides Method A The peptide analogs of the present invention were chemically synthesized using an optimized 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol. For the C-terminal amide, link amide resins were used, but Wang resins and trityl resins were also used to produce the C-terminal acid. The side-chain protecting groups were as follows: Glu, Thr, and Tyr: Ot-butyl; Trp and Lys: t-Boc (t-butyloxycarbonyl); Arg: N-gamma-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; His, Gln, Asn, Cys: trityl. For selective disulfide crosslinking, Acm (acetamidomethyl) was also used as a Cys protecting group. For coupling, a 4-10-fold excess DMF solution containing Fmoc amino acids, 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.

[0367] Method B Alternatively, peptides were synthesized using a CEM Liberty Blue microwave-assisted peptide synthesizer. Using Liberty Blue, FMOC deprotection was performed by adding a 20% 4-methylpiperzine DMF solution with a 0.1 M Oxyma DMF solution, followed by heating to 90°C for 4 minutes using microwave irradiation. After DMF washing, FMOC-amino acids were synthesized with 0.2 M amino acids (4-6 equivalents) and 0.5 M Coupling was performed by adding DIC (4-6 equivalents) and 4-6 equivalents of 1M Oxyma (containing 0.1M DIEA) (all in DMF solution). The coupling solution was heated to 90°C for 4 minutes using microwave radiation. When coupling with Arg or other sterically hindered amino acids, the second coupling method was used. When coupling with histidine, the reaction mixture was heated to 50°C for 10 minutes. This cycle was repeated until the full-length peptide was obtained.

[0368] Peptide cleavage procedure from resin Side-chain deprotection and cleavage of the peptide analog of the present invention (e.g., Compound 2) were achieved by stirring the dry resin for 2-4 hours in a solution containing trifluoroacetic acid, water, ethanedithiol, and tri-isopropylsilane (90:5:2.5:2.5). After removing the TFA, the peptide was precipitated using ice-cold diethyl ether. The solution was centrifuged to decant the ether, followed by a second diethyl ether wash. The peptide was dissolved in an acetonitrile / water (1:1) solution containing 0.1% TFA (trifluoroacetic acid), and the resulting solution was filtered. The quality of the linear peptide was evaluated using electrospray ionization mass spectrometry (ESI-MS).

[0369] Peptide purification procedure The peptide of the present invention (e.g., compound 2) was purified 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 the linear peptide was achieved using preparative RP-HPLC with a C18 column (5 μm, 250 × 21.2 mm) at a flow rate of 20 mL / min. Separation was achieved using a linear gradient of buffer B in buffer A (buffer A: 0.05% TFA aqueous solution, buffer B: 0.043% TFA, 90% acetonitrile aqueous solution).

[0370] Those skilled in the art will understand that the compounds of the present invention can be produced using standard peptide synthesis methods.

[0371] Conjugation of the half-life extension portion Peptide conjugation was performed on resin. Lys(ivDde) ​​was used as the major amino acid. After constructing the peptide on the resin, selective deprotection of the ivDde group was performed for 5 minutes using a 2% hydrazine DMF solution for 5 minutes three times. The conjugated peptide was obtained by linker activation and acylation using 1-2 equivalents of HBTU and DIEA for 3 hours, followed by Fmoc removal and then a second acylation with a lipid acid.

[0372] Example 1B Synthesis of peptide ID number 31 Isovaleric acid-ETH-[Dpa]-P-[Cys]-[Cys]-[Lys(1PEG2_1PEG2_IsoGlu_C18_diacid)]-[bhPhe]-[(D)Lys]-NH2 The TFA salt of peptide number 31 was synthesized on Rink amide resin. Upon completion, 50 mg of 95% ultrapure peptide number 31 was isolated as a white powder. Peptide number 31 was constructed on Rink Amide MBHA (100-200 mesh, 0.66 mmol / g) resin using standard Fmoc protection synthesis conditions. The constructed 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. The pure fraction was lyophilized to obtain the final product peptide number 31.

[0373] Peptide construction Swelling resin: 200 mg of Rink Amide MBHA solid-phase resin (0.66 mmol / g load) was transferred to a 250 mL reaction vessel. The resin was swelled with 60 mL of DMF (2 hours).

[0374] Step 1: Coupling of FMOC-(D)Lys(Boc)-OH: Deprotection of the Fmoc group was achieved by treating a swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-(D)Lys(Boc)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0375] Step 2: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-βhomo-L-Phe-OH amino acid DMF solution (200 mM) and 2.5 mL of HBTU-DIEA coupling reagent DMF mixed solution (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0376] Step 3: Coupling of FMOC-L-Lys(IvDde)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of DMF solution of the amino acid FMOC-L-Lys(IvDde)-OH (200 mM) and 2.5 mL of DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0377] Step 4: Coupling of FMOC-L-Cys(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-L-Cys(Trt)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0378] Step 5: Coupling of FMOC-L-Cys(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of DMF solution of the amino acid FMOC-L-Cys(Trt)-OH (200 mM) and 2.5 mL of DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0379] Step 6: Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-Pro-OH amino acid DMF solution (200 mM) and 2.5 mL of HBTU-DIEA coupling reagent DMF mixed solution (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0380] Step 7: Coupling of FMOC-L-DIP-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-L-DIP-OH amino acid DMF solution (200 mM) and 2.5 mL of HBTU-DIEA coupling reagent DMF mixed solution (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0381] Step 8: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-L-His(Trt)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0382] Step 9: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of DMF solution of the amino acid FMOC-L-Thr(tBu)-OH (200 mM) and 2.5 mL of DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0383] Step 9': Coupling of FMOC-L-Glu(tBu)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of amino acid (2S)-2-[[(9H-FMOC-L-Glu(tBu)-OH) in DMF (200 mM) solution and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0384] Step 10: Isovaleric acid coupling: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of isovaleric acid DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0385] Step 11: IvDde removal and coupling of Fmoc-1PEG2-OH: IvDde was removed from the Lys C-terminus of the resin-bound peptide using a 2-5% hydrazine DMF solution (four 30-minute washes), followed by DMF washing. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1-minute washes), followed by the addition of 2.5 mL of amino acid Fmoc-1PEG2-OH DMF solution (200 mM) and 2.0 mL of coupling reagent HBTU-DIEA DMF mixed solution (200 mM 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 (three 0.1-minute washes), and then the next deprotection / coupling cycle was started.

[0386] Step 12: Coupling of Fmoc-1PEG2-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of Fmoc-1PEG2-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0387] Step 13: Coupling of Fmoc-L-Glu-OtBu: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of Fmoc-Glu-OtBu DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0388] Step 14: tBuO2C(CH2) 16 CO2H coupling: Deprotection of the Fmoc group was achieved by treating a swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by 2.5 mL of tBuO2C(CH2) 16A CO2H DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM and 220 mM) were added. 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0389] Step 15: TFA cleavage and ether precipitation: 10 ml of the cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT)] was added to the protective resin-bound peptide and shaken for 2 hours. Cold diethyl ether was added, a white precipitate was formed, and then the mixture was centrifuged. 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, oxidized, and purified.

[0390] Step 16: Dilute the peptide solution in acetonitrile / water (7:3) to 250 mL. Add the I2 MeOH solution dropwise until the brown color persists. Stir the mixture for 2-5 minutes, then add ascorbic acid in small amounts until the solution becomes clear. Filter the solution and prepare for purification by RP-HPLC.

[0391] Step 17: RP-HPLC purification: Half-portion reverse-phase HPLC was performed using a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was achieved using a linear gradient of buffer B in buffer 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 (prepared).

[0392] Step 18: Final Lyophilization and Analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions with a purity of over 95% were combined. The combined fractions were lyophilized to obtain peptide number 31 as a white powder with a purity of over 95%. Low-resolution LC / MS of the purified peptide number 31 revealed one of the major charge states of the peptide, [M+2H] 2+ A mass of 1064.3 was obtained. The experimental mass is consistent with the theoretical molecular weight of 2126.6 Da.

[0393] Example 1C Synthesis of peptide number 42 Isovaleric acid-[Glu_OMe]-TH-[Dpa]-P-[Cys]-[Cys]-[Lys(1PEG2_1PEG2_Dap_C18_diacid)]-[bhPhe]-[(D)Lys]-NH2 The TFA salt of peptide number 42 was synthesized on Rink amide resin. Upon completion, 52 mg of 95% ultrapure peptide number 42 was isolated as a white powder.

[0394] Peptide number 42 was constructed on Rink Amide MBHA (100-200 mesh, 0.66 mmol / g) resin using standard Fmoc protection synthesis conditions. The constructed 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. The pure fraction was lyophilized to obtain the final product peptide number 42.

[0395] Peptide construction Swelling resin: 200 mg of Rink Amide MBHA solid-phase resin (0.66 mmol / g load) was transferred to a 250 mL reaction vessel. The resin was swelled with 60 mL of DMF (2 hours).

[0396] Step 1: Coupling of FMOC-(D)Lys(Boc)-OH: Deprotection of the Fmoc group was achieved by treating a swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-(D)Lys(Boc)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0397] Step 2: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-βhomo-L-Phe-OH amino acid DMF solution (200 mM) and 2.5 mL of HBTU-DIEA coupling reagent DMF mixed solution (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0398] Step 3: Coupling of FMOC-L-Lys(IvDde)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of DMF solution of the amino acid FMOC-L-Lys(IvDde)-OH (200 mM) and 2.5 mL of DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0399] Step 4: Coupling of FMOC-L-Cys(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-L-Cys(Trt)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0400] Step 5: Coupling of FMOC-L-Cys(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of DMF solution of the amino acid FMOC-L-Cys(Trt)-OH (200 mM) and 2.5 mL of DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0401] Step 6: Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-Pro-OH amino acid DMF solution (200 mM) and 2.5 mL of HBTU-DIEA coupling reagent DMF mixed solution (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0402] Step 7: Coupling of FMOC-L-DIP-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-L-DIP-OH amino acid DMF solution (200 mM) and 2.5 mL of HBTU-DIEA coupling reagent DMF mixed solution (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0403] Step 8: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of FMOC-L-His(Trt)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0404] Step 9: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of DMF solution of the amino acid FMOC-L-Thr(tBu)-OH (200 mM) and 2.5 mL of DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0405] Step 9': Coupling of FMOC-L-Glu(OMe)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of amino acid (2S)-2-[[(9H-FMOC-L-Glu(OMe)-OH) in DMF (200 mM) solution and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0406] Step 10: Isovaleric acid coupling: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of isovaleric acid DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0407] Step 11: IvDde removal and coupling of Fmoc-1PEG2-OH: IvDde was removed from the Lys C-terminus of the resin-bound peptide using a 2-5% hydrazine DMF solution (four 30-minute washes), followed by DMF washing. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1-minute washes), followed by the addition of 2.5 mL of amino acid Fmoc-1PEG2-OH DMF solution (200 mM) and 2.0 mL of coupling reagent HBTU-DIEA DMF mixed solution (200 mM 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 (three 0.1-minute washes), and then the next deprotection / coupling cycle was started.

[0408] Step 12: Coupling of Fmoc-1PEG2-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of Fmoc-1PEG2-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0409] Step 13: Coupling of Fmoc-L-Dap(Boc)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively, twice. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by the addition of 2.5 mL of Fmoc-Dap(Boc)-OH DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0410] Step 14: tBuO2C(CH2) 16 CO2H coupling: Deprotection of the Fmoc group was achieved by treating a swollen Rink Amide resin with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three washes of 0.1 minutes each), followed by 2.5 mL of tBuO2C(CH2) 16A CO2H DMF solution (200 mM) and 2.5 mL of a DMF mixed solution of the coupling reagent HBTU-DIEA (200 mM and 220 mM) were added. 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 (three washes of 0.1 minutes each), and then the next deprotection / coupling cycle was started.

[0411] Step 15: TFA cleavage and ether precipitation: 10 ml of the cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT)] was added to the protective resin-bound peptide and shaken for 2 hours. Cold diethyl ether was added, a white precipitate was formed, and then the mixture was centrifuged. 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, oxidized, and purified.

[0412] Step 16: Dilute the peptide solution in acetonitrile / water (7:3) to 250 mL. Add the I2 MeOH solution dropwise until the brown color persists. Stir the mixture for 2-5 minutes, then add ascorbic acid in small amounts until the solution becomes clear. Filter the solution and prepare for purification by RP-HPLC.

[0413] Step 17: RP-HPLC purification: Half-portion reverse-phase HPLC was performed using a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was achieved using a linear gradient of buffer B in buffer 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 (prepared).

[0414] Step 18: Final freeze-drying and analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions with a purity of over 95% were combined. The combined fractions were freeze-dried to obtain peptide number 42 as a white powder with a purity of over 95%. Low-resolution LC / MS of the purified peptide number 42 revealed the two charge states of the peptide: [M+3H] 3+ 700.0 and molecular ion [M+2H] 2+ A mass of 1049.4 was obtained. The experimental mass is consistent with the theoretical molecular weight of 2097.6 Da.

[0415] Example 2 Activity of peptide analogs Peptide analogs were tested in vitro for induction of internal translocation of human ferroporin protein. After internal translocation, the ferroporin protein is degraded. The assay used (FPN activity assay) measures the decrease in receptor fluorescence.

[0416] The cDNA encoding human ferroportin (SLC40A1) was cloned from Origene's cDNA clone (NM_014585). The ferroportin-encoding DNA was amplified by PCR using primers that also encoded the terminal restriction sites for subcloning but did not contain stop codons. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker so that the ferroportin reading frame was fused in-frame with the GFP protein. The fidelity of the DNA encoding this protein was confirmed by DNA sequencing. HEK293 cells were used for transfection with the ferroportin-GFP receptor expression plasmid. These cells were grown in growth medium according to a standard protocol and transfected with the plasmid using lipofectamine (manufacturer's protocol, Invitrogen). Cells stably expressing ferroportin-GFP were selected in growth medium using G418 (in which case only cells that incorporated the cDNA expression plasmid survived), and sorted several times on a Cytomation MoFlo® cell sorter to obtain GFP-positive cells (488nm / 530nm). The cells were grown and frozen in aliquots.

[0417] To determine the activity of hepcidin analogs (compounds) against human ferroportin, these cells were incubated in 96-well plates in standard medium without phenol red. The compound was added to the incubator for at least 18 hours until the desired final concentration was reached. After incubation, residual GFP fluorescence was determined by either whole-cell GFP fluorescence (Envision plate reader, 485 / 535 filter pair) or Beckman Coulter Quanta® flow cytometer (expressed as the geometric mean of fluorescence intensity at 485 nm / 525 nm). The compound was added to the incubator for at least 18 hours and no more than 24 hours until the desired final concentration was reached.

[0418] In certain experiments, the reference compounds included natural hepcidin, minihepcidin, and R1-minihepcidin, an analog of minihepcidin. "RI" in RI-minihepcidin stands for retroinverse. A retroinverse peptide is a peptide that has the reverse sequence of all D amino acids. One example is Hy-Glu-Thr-His-NH2 becoming Hy-DHis-DThr-DGlu-NH2. The EC of these reference compounds for ferroportin internal translocation / degradation. 50 These were determined according to the FPN activity assay described above. These peptides served as control standards. [Table 8] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 10]

[0419] In Tables 6A-6E, for the FPN and T47D internal transfer assays, IC 50 The symbols representing values ​​have the following meanings: ****=IC 50 ***=IC 50 If the value is greater than 10 nM and less than or equal to 100 nM, then **=IC 50 * = greater than 500 nM. If not specified, the data was unavailable. [Table 11-1] Table 11-2 Table 12-1 Table 12-2 Table 12-3 Table 13-1 Table 13-2 Table 13-3 Table 13-4 Table 13-5 Table 13-6 Table 13-7 Table 13-8 Table 13-9 Table 13-10 Table 13-11 Table 13-12 Table 13-13 [Table 13-14] [Table 13-15] [Table 13-16] [Table 13-17] [Table 13-18] [Table 13-19] [Table 13-20] [Table 13-21]

[0420] Example 2C Activity of peptide analogs The efficacy of a peptide in inducing ferroportin internalization was evaluated using a T47D cell-based assay. The T47D cell line (HTB 133, ATCC) is a human breast cancer adherent cell line that endogenously expresses ferroportin. In this internalization assay, the efficacy of the test peptide 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 periodically subcultured. In preparation for this assay, cells were seeded in 96-well plates at a density of 80-100k cells / well per 100µl volume and left to stand overnight. The following day, the test peptide was first prepared in a dilution series (10-point series, starting concentration approximately 5μM, typically 3-4 times dilution steps), and all were diluted with 0.5% mouse serum albumin (MSA, Sigma, A3139 purified from mouse serum). The test peptide was prepared using [method / method]. The test peptide dilution series was incubated at room temperature for 30 minutes. Then, the medium was aspirated from the 96-well cell plate and the test peptide dilution series was added. After incubation for 1 hour, the medium containing the test peptide was aspirated and removed, and the AF647 conjugate detection peptide was added at a fixed concentration of 200 nM. It had been previously verified that the AF647 conjugate detection peptide binds to ferroportin and induces its internal translocation. After incubating the cells for 2 hours in preparation for flow cytometry analysis, they were washed again. The median fluorescence intensity (MFI) of the AF647-positive population was measured (after removing dead cells and non-singlets from the analysis). Dose-response curves were generated using the MFI values ​​to obtain the IC50 potency of the test peptide. The IC50 potency was calculated using a 4-parameter nonlinear fit function in Graphpad Prism (Tables 6A-B).

[0421] Example 2D LAD2 activity of peptide analogs In anaphylactoid reactions, the primary mechanism requires direct stimulation of mast cells or basophils, leading to the release of anaphylaxis mediators such as histamine and β-hexosaminidase. Recent research by McNeil et al. (McNeil BD et al., 2015) reported that MrgprX2, a specific membrane receptor on human mast cells, induces anaphylactoid reactions. The LAD2 (Allergy Disease Research Laboratory 2) human mast cell line (Kirshenbaum et al., 2003), derived from human mast cell sarcoma / leukemia, is commonly used in anaphylactoid reaction studies because its biological characteristics, including overexpression of the MrgprX2 receptor and sensitivity to degranulation peptides, are identical to those of primary human mast cells (Kulka et al., 2008). The release of anaphylaxis mediators such as β-hexosaminidase is evaluated by fluorescence quantification.

[0422] The degranulation ability of hepcidin mimetic compounds was evaluated in LAD2 cells. On the day of the assay, serial dilutions of the compound were added to LAD2 cells seeded at 20,000 cells / well in 96-well plates. After incubation for 30 minutes, the amount of β-hexosaminidase released into the supernatant and cell lysates was quantified using the fluorescent substrate 4-methylumbelliferyl-N-acetyl-bD-glucosaminide. A dose-response curve was generated by plotting the % of β-hexosaminidase release (y-axis) against the concentration of the peptide tested (x-axis). EC 50 The values ​​and standard errors were calculated using XLfit 5.5.0.5 based on the following equation: 4-parameter sigmoid 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).

[0423] Example 3 In vivo validation of peptide analogs The hepcidin analogs of the present invention were tested for in vivo activity, and their ability to reduce free Fe2+ in serum was determined.

[0424] Hepcidin analogs or vehicle controls were administered intravenously or subcutaneously to mice (n=3 / group) at a dose of 1000 nmol / kg. Serum samples were collected from the mouse group administered with hepcidin analogs at 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 30 hours, 36 hours, and 48 hours post-administration. Iron content in plasma / serum was measured using a colorimetric assay in Cobas c 111 according to the manufacturer's instructions for the assay (Assay: IRON2: ACN 661).

[0425] In another experiment, various hepcidin analogs or vehicle controls were subcutaneously administered to mice (n=3 / group) at a dose of 1000 nmol / kg. Serum samples were collected from the groups of mice that received the vehicle or hepcidin analog at 30 and 36 hours post-administration. Iron content in plasma / serum was measured using a colorimetric assay on a Cobas c 111 according to the manufacturer's instructions for the assay (Assay: IRON2: ACN 661).

[0426] These studies demonstrate that the hepcidin analog of the present invention lowers serum iron levels for at least 30 hours, thus demonstrating increased serum stability.

[0427] Example 4 In vitro validation of peptide analogs Based in part on the structure-activity relationships (SARs) determined from the experimental results described herein, various hepcidin-like peptides of the present invention were synthesized using the method described in Example 1, and their in vitro activity was tested as described in Example 2. Reference compounds included natural hepcidin, minihepcidin, R1-minihepcidin, reference compound 1, and reference compound 2. IC of the peptides... 50 Value or EC 50 The values ​​are summarized in Tables 6A to 6E.

[0428] Example 5 plasma stability To complement in vivo results and aid in the design of potent and stable ferroportin agonists, plasma stability experiments were conducted. Ex vivo stability studies were performed first in rat and mouse plasma to predict stability in these matrices.

[0429] The target peptide (20 μM) was incubated with pre-warmed plasma (Bioreclamation IVT) at 37°C. Aliquots were taken at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid with a 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 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The residual percentage at each time point was calculated based on the peak area ratio (analyte relative to internal standard) to the initial level at zero. The half-life was calculated by fitting a linear exponential decay equation using GraphPad.

[0430] Example 6 Decreased serum iron 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. These animals were acclimatized to a normal rodent diet for 4-5 days prior to the start of the study and fasted overnight before the start of the study. Each group of four animals was administered either the vehicle or the compound. The compound was formulated in physiological saline at a concentration of 5 mg / mL. Mice were administered the solution via oral gastric tube feeding at a volume of 200 μl per 20 g animal. Each group received a single dose of the compound at a dose of 50 mg / kg / dose. The group marked "vehicle" received only the formulation. 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 colorimetric analysis on a Roche-cobas c system.

[0431] Example 7 Decreased serum iron in mice In another experiment, a new set of compounds was tested for systemic absorption via PO administration in a wild-type mouse model C57BL / 6. These animals were acclimatized to a normal rodent diet for 4-5 days prior to the start of the study. The night before the first dose, the mice were switched to a low-iron diet (containing 2 ppm iron) and this diet was maintained for the remainder of the study. Each group of five animals received either the vehicle or the compound. The compound was formulated at a concentration of 30 mg / mL in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withheld approximately 2 hours before each dose to ensure that there were no food particles in the stomach before PO administration. Mice were administered the solution via oral gastric tube feeding at a volume of 200 μl per 20 g animal. Each group received two consecutive daily doses of the compound at a dose of 300 mg / kg / dose. The group marked "vehicle" received only the formulation. Blood samples were collected 4.5 hours after the final dose, and serum was prepared for PD measurement. Serum iron concentration was measured using a colorimetric method on a Roche-Cobas c system.

[0432] Example 8 Pharmacodynamic effects of representative compounds on serum iron reduction ability in mice In the second in vivo study, representative compounds were tested for pharmacodynamic effects using a single dose of 300 mg / kg / dose versus two doses of 300 mg / kg over a 2-day QD (once daily) period. C57BL / 6 mice were acclimatized to a normal rodent diet for 4-5 days prior to the start of the study. The night before the first dose, the mice were switched to a low-iron diet (containing 2 ppm iron), and this diet was maintained for the remainder of the study. Each group of five animals received either the vehicle or the compound. The compound was formulated at a concentration of 30 mg / mL in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withheld approximately 2 hours before each dose to ensure that there were no food particles in the stomach before PO administration. Mice were administered the solution via oral gastric tube feeding at a volume of 200 μl per 20 g animal.

[0433] 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 tested for their PK and PD effects after multiple administrations over a 3-day period. Mice were maintained on a normal rodent diet during acclimatization and switched to an iron-deficient diet (containing approximately 2 ppm iron) the night before the first administration. Each group of five mice received a total of six doses of either the vehicle or a representative compound of the present invention in different dose intensities over a 3-day period. Mice were administered by oral gastric tube feeding with a representative compound formulated in 0.7% saline and 10 mM sodium acetate. Different groups received either the vehicle, 150 mg / kg / dose BID, 75 mg / kg / dose BID, 37.5 mg / kg / dose BID, or 18.75 mg / kg / dose BID. In addition to the compound / drinking water (DW) solution totaling 100 mg / kg / day, the additional group received 100 mg / kg / dose BID, resulting in a total dose of 300 g / kg / day. Three hours after the final dose, the vehicle group marked as iron-loaded and all compound-administered groups received an iron solution at 4 mg / kg per animal via oral gastric tube feeding. Blood was collected 90 minutes after iron loading, 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 colorimetric analysis on a Roche-cobas c system.

[0434] Example 10 Decreased serum iron in mice In a separate triage, a new set of compounds was tested for its pharmacodynamic effects when orally administered to wild-type mouse model C57BL / 6. These animals were acclimatized to a normal rodent diet 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 were given an iron-deficient diet (containing 2 ppm iron) the night before the first dose, while all other groups designated to receive one dose of a different compound were treated with an iron-deficient diet for two nights prior to compound administration. The concentration of the compounds in the administration solution was 30 mg / mL and was formulated in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withheld approximately 2 hours before each administration to ensure that there were no food particles in the stomach before PO administration. Mice were administered the solution via oral gastric tube feeding at a volume of 200 μl per 20 g animal. Groups marked as "vehicle" received only the formulation. Blood samples were collected 4.5 hours after the final dose, and serum was prepared for PD measurement. Serum iron concentration was measured using a colorimetric method on a Roche-Cobas c system.

[0435] Example 11 Stability in simulated gastric juice A blank SGF was prepared by adding 2 g of sodium chloride and 7 mL of hydrochloric acid (37%) to a final water volume of 1 L, and the pH was adjusted to 1.2.

[0436] SGF was prepared by dissolving 320 mg of pepsin (Sigma®, P6887, derived from porcine gastric mucosa) in 100 mL of blank SGF and stirring at room temperature for 30 minutes. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at -20°C.

[0437] The target experimental compound (at a concentration of 20 μM) was incubated with pre-warmed SGF at 37°C. Aliquots were taken at various time points up to a maximum of 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid containing a 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 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The residual percentage at each time point was calculated based on the peak area ratio (analyte relative to the internal standard) to the initial level at zero. The half-life was calculated by fitting a linear exponential decay equation using GraphPad.

[0438] Example 12 Stability in simulated intestinal fluid A blank FaSSIF was prepared by dissolving 0.348 g of NaOH, 3.954 g of sodium phosphate monobasic monohydrate, and 6.186 g of NaCl in 1 liter of final water (the pH was adjusted to 6.5).

[0439] FaSSIF was prepared by dissolving 1.2 g of porcine pancreas (Chem-supply, PL378) in 100 mL of blank FaSSIF and stirring at room temperature for 30 minutes. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at -20°C.

[0440] The target experimental compound (20 μM) was incubated at 37°C with pre-warmed FaSSIF (1% pancreatin in the final incubation mixture). Aliquots were taken at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid with a 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 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The residual percentage at each time point was calculated based on the peak area ratio (analyte relative to internal standard) to the initial level at zero. The half-life was calculated by fitting a linear exponential decay equation using GraphPad.

[0441] Example 13 Modification experiments on peptides that tend to bind nonspecifically. 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 equivalent to the number of time points (e.g., 0, 0.25, 1, 3, 6, and 24 hours). At each time point, one aliquot was taken and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid with a 1 μM internal standard). The remaining steps were the same as in a general experiment.

[0442] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this application datasheet are incorporated herein by reference in their entirety.

[0443] At least some of the chemical names or sequences of the compounds of the present invention provided and described in this application may have been automatically generated using commercially available chemical naming software programs and have not been independently verified. In the event of any discrepancy between a given chemical name or sequence and a given structure, the given structure shall prevail. In chemical structures where a chiral center is present but no specific stereochemistry is shown for the chiral center, both enantiomers related to the chiral structure are encompassed by that structure. Similarly, for peptides where E / Z isomers exist but are not specifically mentioned, both isomers are specifically disclosed and covered.

[0444] As described above, specific embodiments of the present invention have been illustrated herein for illustrative purposes, but it will be understood that various modifications may be made without departing from the spirit and scope of the invention. The present invention provides, for example, the following items: (Item 1) Equation (I'): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (I') (In the formula, R 1 However, hydrogen, C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, C1-C 20 Cycloalkanoyl, C6-C 10 The aryl-C1-C6 alkylene-C(O)-, 5-membered or 6-membered heteroaryl-C(O)-, or 5-membered or 6-membered heterocycloalkyl-C(O)-, where R 1 The aforementioned C1-C 20 Alkanoyl, the C6-C 10The aryl-C1-C6 alkylene-C(O)-, the 5-membered or 6-membered heteroaryl-C(O)-, and the 5-membered or 6-membered heterocycloalkyl-C(O)- are each optionally substituted with one, two, or three substituents independently selected from halo, OH, CN, C1-C6 alkoxy, C1-C6 alkyl, -COOH, -COO(C1-C6), C1-C6 haloalkyl, and C1-C6 haloalkoxy. R 2 However, NH2, substituted amino, OH, substituted hydroxy, C1-C 20 Alkylaminophenyl-C1-C8alkylene-NH-, where C1-C 20アルキル The amino and phenyl-C1-C8 alkylene-NH- groups are each optionally substituted with substituents independently selected from NH2, -COOH, and phenyl. X1 is either absent, or is 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, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Glu, Aad, Dap, or isoGlu. X2 is Ala, Thr, Gly, N-substituted Gly, Ser, Cys, 4S_Mcp, 4R_Mcp, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, Cys, (D)Cys, aMeCys, hCys, Pen, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or Cys, (D)Cys, aMeCys, hCys, Pen, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, 4S_Mcp, 4R_Mcp, Morph, or Tic. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, NMe-Cys, Ala, Lys, (D)Lys, substituted Lys, or substituted (D)Lys, X7 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, Phe, or DIP. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, 123triazole, Cys, (D)Cys, aMeCys, hCys, Pen, Lys_Me3, X9 is either absent, or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Cys, (D)Cys, aMeCys, hCys, Pen, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. X12 to X14 are either absent or are each an amino acid independently. The peptide of formula (I') is further optionally conjugated to any of the amino acids, Any of the amino acids in the peptide of formula (I') is optionally substituted with the corresponding (D)-amino acid, or optionally N-substituted, (i) At least one of X1, X3-X5, and X7-X10 is Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp, where the peptide has a mercapto group on the side chain of one of X1, X3-X5, and X7-X10, a mercapto group on the side chain of X6, and L x Combine them, -SL x -S-bonds are formed, or the mercapto group on the side chain of X1, the mercapto group on the side chain of X8, and L x Together, -SL x -S-bonds are formed, or the mercapto group on the side chain of X2, the mercapto group on the side chain of X5, and L x Together, -SL x It is cyclized by forming an -S- bond, in the formula, L x Each of them is independently a bonding or linking group, (ii) The functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. Here, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor It is leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting bhPhe is β-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. The substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu. The substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH, or t-Bu. A substituted Lys, substituted dLys, or substituted NMe-Lys, or substituted NMe-dLys is Lys, dLys, NMe-Lys, or NMe-dLys, where the ε-amino group on the side chain of Lys, dLys, NMe-Lys, or NMe-dLys is covalently bonded to a C1-C6 alkanoyl, a half-life extension portion, or a linking portion covalently bonded to a half-life extension portion, and the linking portions include one or more linker portions covalently bonded to each other. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123triazole [ka] And, Dab [ka] A hepcidin analog containing a peptide having the amino acid sequence of ( ), or a pharmaceutically acceptable salt or solvate thereof. (Item 2) Formula (X): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-R 2 (X) (In the formula, (i) One of X1, X3-X5, and X7-X10 is Cys, (D)Cys, aMeCys, hCys, Pen, (D)Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp, where the peptide has a mercapto group on the side chain of one of X1, X3-X5, and X7-X10, a mercapto group on the side chain of X6, and L x Combine them, -SL x -S-bonds are formed, or the mercapto group on the side chain of X1, the mercapto group on the side chain of X8, and L x Together, -SL x-S-bonds are formed, or the mercapto group on the side chain of X2, the mercapto group on the side chain of X5, and L x Together, -SL x It is cyclized by forming an -S- bond, in the formula, L x Each of them is independently a bonding or linking group, (ii) A hepcidin analog described in item 1, or a pharmaceutically acceptable salt or solvate thereof, comprising a peptide containing an amino acid sequence in which the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. (Item 3) Formula I: R 1 -X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (I) (In the formula, R 1 However, hydrogen, C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, C1-C 20 It is a cycloalkanoyl, R 2 However, it is NH2, substituted amino, OH, or substituted hydroxy, X0 is either absent, or Cys, (D)Cys, aMeCys, hCys, or Pen. X1 is either absent, or is 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, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X2 is Ala, Thr, Gly, N-substituted Gly, or Ser. X3 is Ala, Gly, N-substituted Gly, His, substituted His, or Cys, (D)Cys, aMeCys, hCys, or Pen. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or Cys, (D)Cys, aMeCys, hCys, or Penl. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X6 is Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either absent, or is Ala, Gly, N-substitute Gly, Ile, Val, Leu, NLeu, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X8 is either absent, or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, 123-triazole, or Cys, (D)Cys, aMeCys, hCys, or Pen. X9 is either absent, or Ala, Ile, Gly, N-substitute Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or Cys, (D)Cys, aMeCys, hCys, or Pen. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. X12 to X14 are either absent or each is independently one of the amino acids. however, 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 of the amino acid, or may be N-substituted, and iii) Provided that at least one of X0, X1, X3-X5, X7-X10 is Cys, (D)Cys, aMeCys, hCys, or Pen, and Cys, (D)Cys, aMeCys, hCys, or Pen forms a disulfide bond with X6, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor It is leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting bhPhe is β-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. The substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu. The substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH, or t-Bu. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123triazole [ka] And, Dab [ka] A hepcidin analog described in item 1, or a pharmaceutically acceptable salt or solvate thereof, comprising a peptide that conforms to ( ). (Item 4) A hepcidin analog as described in item 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X0 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X0 and X6 are linked via a disulfide bond. (Item 5) A hepcidin analog as described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X1 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X1 and X6 are linked via a disulfide bond. (Item 6) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X3 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X3 and X6 are linked via a disulfide bond. (Item 7) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X4 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X4 and X6 are linked via a disulfide bond. (Item 8) A hepcidin analog as described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X5 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X5 and X6 are linked via a disulfide bond. (Item 9) A hepcidin analog as described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X7 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X7 and X6 are linked via a disulfide bond. (Item 10) A hepcidine analog as described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X8 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X8 and X6 are linked via a disulfide bond. (Item 11) A hepcidine analog as described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X9 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X9 and X6 are linked via a disulfide bond. (Item 12) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is Cys, (D)Cys, aMeCys, hCys, or Pen, and X10 and X6 are linked via a disulfide bond. (Item 13) A hepcidin analog as described in any one of items 1-4 and 6-12, where X1 is Glu, or a pharmaceutically acceptable salt or solvate thereof. (Item 14) A hepcidin analog described in any one of items 1 to 13, wherein X2 is Thr, or a pharmaceutically acceptable salt or solvate thereof. (Item 15) A hepcidin analog described in any one of items 1 to 14, or a pharmaceutically acceptable salt or solvate thereof, wherein X3 is His. (Item 16) A hepcidin analog as described in any one of items 1 to 15, or a pharmaceutically acceptable salt or solvate thereof, wherein X4 is Phe or Dpa. (Item 17) X5 is Pro, and is a hepcidin analog described in any one of items 1 through 16, or a pharmaceutically acceptable salt or solvate thereof. (Item 18) A hepcidin analog described in any one of items 1 to 17, or a pharmaceutically acceptable salt or solvate thereof, wherein X7 is Ile. (Item 19) A hepcidin analog as described in any one of items 1 to 18, or a pharmaceutically acceptable salt or solvate thereof, wherein X8 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 20) A hepcidin analog as described in any one of items 1 to 19, or a pharmaceutically acceptable salt or solvate thereof, wherein X9 is Phe or bhF. (Item 21) A hepcidin analog according to any one of items 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 22) A hepcidin analog according to any one of items 1 to 21, or a pharmaceutically acceptable salt or solvate thereof, wherein X11 is absent, Arg, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 23) The aforementioned peptide, according to formula II, R 1 -Cys-Thr-His-[Dpa]-Pro-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (II) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 24) The aforementioned peptide, according to formula III, R 1 -Glu-Thr-Cys-[Dpa]-Pro-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (III) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 25) The peptide, according to formula IV, R 1 -Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (IV) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 26) The peptide, according to formula V, R 1 -Glu-Thr-His-[Dpa]-Pro-X6-Cys-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (V) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 27) The peptide, according to formula VI, R 1 -Glu-Thr-His-[Dpa]-Cys-X6-Ile-Cys-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VI) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 28) The peptide, according to formula VII, R 1 -Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-Cys-X11-X12-X13-X14-R 2 (VII) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 29) The peptide is determined according to formula VIIIa or formula VIIIb, R 1 -Cys-Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIa) R 1 -[hCys]-Glu-Thr-His-[Dpa]-Cys-X6-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIb) A hepcidin analog described in any one of items 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein Cys and X6 are linked via a disulfide bond. (Item 30) X1 is 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, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Glu, Aad, Dap, or isoGlu, X2 is Ala, Thr, Gly, N-substituted Gly, Ser, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Morph, or Tic. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, or NMe-Cys. X7 is absent, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, or Phe. X8 is either absent, or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, 123-triazole, or Lys_Me3. X9 is either absent, or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. Mercapto group on the side chain of X6, mercapto group on the side chain of X7, and L x Together, -SL x -S- bond is formed, in the formula, L x However, bonding, or C 1-8 Alkylene, phenylene, [ka] A linking group selected from, where the alkylene and the phenylene are each C 1-6 alkyl, halo, CN, OH, -COOH, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Optionally substituted with one or two substituents independently selected from haloalkoxy or NH2, L a and L b Each of them operates independently, C 1-4 It is alkylene, A hepcidin analog according to item 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. (Item 31) L x A hepcidin analog according to any one of items 1, 2, and 30, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound is bonded to or p-xylene. (Item 32) X1 is Cys, (D)Cys, aMeCys, hCys, Pen, or (D)Pen. X2 is Ala, Thr, Gly, N-substituted Gly, Ser, NMe-Thr, aMePro, Hyp_3R, HyP_3S, hSer, or Thr_Me. X3 is Ala, Gly, N-substituted Gly, His, substituted His, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or DIP. X5 is Ala, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Morph, or Tic. X6 is Ala, Lys, (D)Lys, substitute Lys, or substitute (D)Lys. X7 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Phe, or DIP. X8 is Cys, (D)Cys, aMeCys, hCys, or Pen. X9 is either absent, or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. The mercapto group on the side chain of X1 and the mercapto group on the side chain of X8 combine to form a disulfide bond. A hepcidin analog according to item 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. (Item 33) X1 is 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, Tet2, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Glu, Aad, Dap, or isoGlu, X2 is Cys, 4S_Mcp, or 4R_Mcp, X3 is Ala, Gly, N-substituted Gly, His, substituted His, 3Pal, 4Pal, BIP, Ala_3Quin, Trp_5OH, or His_1Me. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, 2Pal, or DIP. X5 is Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, 4S_Mcp, or 4R_Mcp. X6 is Cys, (D)Cys, aMeCys, hCys, Pen, dPen, or NMe-Cys. X7 is absent, Cys, (D)Cys, aMeCys, hCys, Pen, NMe-Cys, or Phe. X8 is either absent, or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, 123-triazole, or Lys_Me3. X9 is either absent, or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, NMe-Phe, aMePhe, Dip, dDIP, BIP, aMePhe, or substituted Phe. X10 is either absent, or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, Tle, substituted (D)Lys, NMe-Lys, NMe-dLys, substituted NMe-Lys, substituted NMe-dLys, or Mor_propanoic acid. X11 is either absent, or Ala, Pro, bhPhe, Lys, substitute Lys, (D)Lys, NMe-Phe, NMe-dSer, NMe-dGln, NMe-dLeu, NMe-dTyr, 1Nal, or NMe-Ile. The mercapto group on the side chain of X2 and the mercapto group on the side chain of X8 combine to form a disulfide bond. A hepcidin analog according to item 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. (Item 34) X6 is Cys, X7 is Cys, or X6 is Cys and X7 is NMe-Cys, or X6 is hCys, X7 is hCys, or X6 is Cys, and X7 is hCys. X6 is Pen, X7 is Cys, or X6 is Cys, X7 is Pen, or A hepcidin analog as described in any one of items 1, 2, and 30-31, where X6 is Pen and X7 is Pen, or a pharmaceutically acceptable salt or solvate thereof. (Item 35) X1 is hCy and X8 is Cys, or X1 is Pen, X8 is Cys, or X1 is Cys, X8 is dPen, or X1 is dCys, X8 is dCys, or X1 is dCys and X8 is Cys, or X1 is Cys, X8 is hCys, or X1 is hcy, X8 is Pen, or X1 is hCys, X8 is hCys, or A hepcidin analog as described in any one of items 1, 2, and 32, or a pharmaceutically acceptable salt or solvate thereof, wherein X1 is Cys and X8 is Cys. (Item 36) X2 is Cys, X5 is Cys, or Cys and X5 are NMe-Cys, or X2 is Cys and X5 is hCys, or X2 is Cys, X5 is 4S_Mcp, or X2 is Cys, X5 is 4R_Mcp, or X2 is 4S_Mcp and X5 is 4R_Mcp, or X2 is 4S_Mcp, X5 is 4S_Mcp, or A hepcidin analog described in any one of items 1, 2, and 33, or a pharmaceutically acceptable salt or solvate thereof, wherein X2 is 4R_Mcp and X5 is 4R_Mcp. (Item 37) A hepcidin analog according to any one of items 1, 2, and 30-36, wherein the functional group on the side chain of X1 and the functional group on the side chain of X10 optionally combine to form an amide bond, or a pharmaceutically acceptable salt or solvate thereof. (Item 38) A hepcidin analog according to any one of items 1, 2, and 30-37, wherein X1 and X10 form an amide bond, or a pharmaceutically acceptable salt or solvate thereof. (Item 39) A hepcidin analog according to any one of items 1, 2, and 30-38, wherein X1 is Glu and X10 is (D)Lys, or a pharmaceutically acceptable salt or solvate thereof. (Item 40) A hepcidin analog according to any one of items 1, 2, and 30-36, or a pharmaceutically acceptable salt or solvate thereof, wherein the functional group on the side chain of X1 and the functional group of R2 optionally combine to form an amide bond. (Item 41) X1 and R 2 A hepcidin analog according to any one of items 1, 2, 30-36, and 40, wherein the hepcidin analog forms an amide bond, or a pharmaceutically acceptable salt or solvate thereof. (Item 42) X1 is Glu, R 2 C is substituted with NH2 or -COOH 1-8 an alkylamino hepcidin analog as described in any one of items 1, 2, 30-36, and 40-41, or a pharmaceutically acceptable salt or solvate thereof. (Item 43) R 2 A hepcidin analog described in any one of items 1, 2, 30-36, and 40-42, wherein is 2-aminoethylamino, or a pharmaceutically acceptable salt or solvate thereof. (Item 44) A hepcidin analog described in any one of items 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is Pen. (Item 45) A hepcidin analog described in any one of items 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is hCys. (Item 46) A hepcidin analog described in any one of items 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is aMeCys. (Item 47) A hepcidin analog described in any one of items 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is (D)Cys. (Item 48) A hepcidin analog described in any one of items 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is Cys. (Item 49) A hepcidin analog described in any one of items 1-31 or 33-48, in which X8 is replaced with Ahx-Palm, or a pharmaceutically acceptable salt or solvate thereof. (Item 50) A hepcidine analog as described in any one of items 1-31, 33-48, or a pharmaceutically acceptable salt or solvate thereof, wherein X8 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 51) A hepcidin analog, or a pharmaceutically acceptable salt or solvate thereof, as described in any one of items 1-31 or 33-48, in which X8 is absent. (Item 52) A hepcidin analog described in any one of items 1-31 or 33-48, wherein X8 is (D)Lys, or a pharmaceutically acceptable salt or solvate thereof. (Item 53) A hepcidin analog described in any one of items 1-31 or 33-48, where X8 is Lys, or a pharmaceutically acceptable salt or solvate thereof. (Item 54) A hepcidin analog described in any one of items 1-31 or 33-48, in which X8 is replaced with Ahx-Palm, or a pharmaceutically acceptable salt or solvate thereof. (Item 55) A hepcidin analog described in any one of items 1-31 or 33-48, where X8 is Lys(Ahx_Palm), or a pharmaceutically acceptable salt or solvate thereof. (Item 56) A hepcidin analog as described in any one of items 1-31 or 33-48, in which X8 is a conjugated amino acid, or a pharmaceutically acceptable salt or solvate thereof. (Item 57) A hepcidin analog as described in any one of items 1-31 or 33-48, or a pharmaceutically acceptable salt or solvate thereof, wherein X8 is conjugated Lys or (D)Lys. (Item 58) A hepcidin analog described in any one of items 1-31 or 33-48, where X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extension portion, or a pharmaceutically acceptable salt or solvate thereof. (Item 59) A hepcidin analog described in any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is Glu. (Item 60) A hepcidin analog according to any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (Item 61) A hepcidin analog described in any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, in which X10 is absent. (Item 62) A hepcidin analog described in any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is (D)Lys. (Item 63) A hepcidin analog described in any one of items 1 to 58, where X10 is Lys, or a pharmaceutically acceptable salt or solvate thereof. (Item 64) A hepcidin analog described in any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is replaced with Ahx-Palm. (Item 65) A hepcidin analog described in any one of items 1 to 58, where X10 is Lys(Ahx_Palm), or a pharmaceutically acceptable salt or solvate thereof. (Item 66) A hepcidin analog described in any one of items 1 to 58, in which X10 is a conjugated amino acid, or a pharmaceutically acceptable salt or solvate thereof. (Item 67) A hepcidin analog as described in any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is conjugated Lys or (D)Lys. (Item 68) A hepcidin analog described in any one of items 1 to 58, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extension portion. (Item 69) A hepcidin analog described in item 58 or 66, wherein L1 is a single bond. (Item 70) A hepcidin analog as described in item 58 or 66, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is iso-Glu. (Item 71) A hepcidin analog as described in item 58 or 66, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is Ahx. (Item 72) A hepcidin analog as described in item 58 or 66, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is iso-Glu-Ahx. (Item 73) A hepcidin analog as described in item 58 or 66, wherein L1 is PEG, or a pharmaceutically acceptable salt or solvate thereof. (Item 74) A hepcidin analog as described in item 58 or 66, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is PEG-Ahx. (Item 75) A hepcidin analog as described in item 58 or 66, where L1 is iso-Glu-PEG-Ahx, or a pharmaceutically acceptable salt or solvate thereof. (Item 76) A hepcidine analog as described in any one of items 70 to 75, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is -[C(O)-CH2-(Peg)nN(H)]m- or -[C(O)-CH2-CH2-(Peg)nN(H)]m-, Peg is -OCH2CH2-, m is 1, 2, or 3, and n is an integer from 1 to 100, or 10K, 20K, or 30K. (Item 77) A hepcidin analog as described in item 76, where m is 1, or a pharmaceutically acceptable salt or solvate thereof. (Item 78) A hepcidin analog as described in item 76, where m is 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 79) A hepcidin analog as described in item 76, where n is 2, or a pharmaceutically acceptable salt or solvate thereof. (Item 80) A hepcidin analog as described in item 76, where n is 4, or a pharmaceutically acceptable salt or solvate thereof. (Item 81) A hepcidin analog as described in item 76, where n is 8, or a pharmaceutically acceptable salt or solvate thereof. (Item 82) A hepcidin analog as described in item 76, where n is 11, or a pharmaceutically acceptable salt or solvate thereof. (Item 83) A hepcidin analog as described in item 76, where n is 12, or a pharmaceutically acceptable salt or solvate thereof. (Item 84) A hepcidin analog as described in item 76, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 20K. (Item 85) A hepcidin analog described in any one of items 73 to 84, wherein PEG is 1Peg2 and 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-, or a pharmaceutically acceptable salt or solvate thereof. (Item 86) A hepcidine analog described in any one of items 73 to 84, wherein PEG is 2Peg2 and 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-, or a pharmaceutically acceptable salt or solvate thereof. (Item 87) A hepcidine analog as described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1Peg2-1Peg2, and each PEG is -C(O)-CH2-CH2-(Peg)2-N(H)-. (Item 88) A hepcidine analog described in any one of items 73 to 84, wherein PEG is 1Peg2-1Peg2 and 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-, or a pharmaceutically acceptable salt or solvate thereof. (Item 89) A hepcidine analog described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2Peg4 and 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-. (Item 90) A hepcidine analog described in any one of items 73 to 84, wherein PEG is 1Peg8 and 1Peg8 is -C(O)-CH2-(Peg)8-N(H)- or -[C(O)-CH2-(OCH2CH2)8-NH]-, or a pharmaceutically acceptable salt or solvate thereof. (Item 91) A hepcidine analog described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2Peg8 and 2Peg8 is -C(O)-CH2-CH2-(Peg)8-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)8-NH]-. (Item 92) A hepcidine analog described in any one of items 73 to 84, wherein PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-, or a pharmaceutically acceptable salt or solvate thereof. (Item 93) A hepcidine analog described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2Peg11 and 2Peg11 is -C(O)-CH2-CH2-(Peg)11-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)11-NH]-. (Item 94) A hepcidine analog as described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof, 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 95) If PEG is bound to Lys, the -C(O)- of PEG is bound to the Ne of Lys, a hepcidin analog as described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof. (Item 96) If PEG is bound to isoGlu, the -N(H)- of PEG is bound to the -C(O)- of isoGlu, a hepcidin analog as described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof. (Item 97) If PEG is bound to Ahx, the -N(H)- of PEG is bound to the -C(O)- of Ahx, a hepcidin analog as described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof. (Item 98) If PEG is bound to Palm, the -N(H)- of PEG is bound to the -C(O)- of Palm, a hepcidin analog as described in any one of items 73 to 84, or a pharmaceutically acceptable salt or solvate thereof. (Item 99) A hepcidin analog described in any one of items 73-84, where Z is Palm, or a pharmaceutically acceptable salt or solvate thereof. (Item 100) -L1Z is, PEG11_OMe, PEG12_C18 acid, 1PEG2_1PEG2_Ahx_Palm, 1PEG2_Ahx_Palm, Ado_Palm, Ahx_Palm, Ahx_PEG20K, PEG12 Ahx IsoGlu Behenic Acid 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 Iso Glue_Palm, -1PEG2_1PEG2_Dap_C18_Dioxide, -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_Diacid, -1PEG8_IsoGlu_C18_Diacid, -IsoGlu_C18_diacid, -PEG12_Ahx_C18_Diacid, -PEG12_C16_Diacid, -PEG12_C18_diacid, -1PEG2_1PEG2_1PEG2_C18_Diacid, -1PEG2_1PEG2_1PEG2_IsoGlu_C18_Diacid, -PEG12_IsoGlu_C18_Diacid, -PEG4_IsoGlu_C18_Diacid, or -PEG4_PEG4_IsoGlu_C18_diacid, Here, PEG11_OMe-[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)-CH2-CH2-(OCH2CH2)8-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 acid is -C(O)-(CH2) 16 -Me, Palm is -C(O)-(CH2) 14 -Me, isoGlu is isoglutamic acid. isoGlu_Palm [ka] And, Ahx is -[C(O)-(CH2)5-NH]-, Cn diacid is -C(O)-(CH2) n-2 A hepcidin analog as described in item 58 or 68, which is -COOH, where n is 10, 12, 14, 16, 18, or 22, or a pharmaceutically acceptable salt or solvate thereof. (Item 101) X6 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n It is a diacid, and Lys(1PEG2_1PEG2_IsoGlu_C n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 102) X6 or X10 is (D)Lys(1PEG2_1PEG2_IsoGlu_C n (D)Lys(1PEG2_1PEG2_IsoGlu_C) n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 103) X6 or X10 is Lys(1PEG8_IsoGlu_C n _Diacid) and Lys(1PEG8_IsoGlu_C n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 104) X6 or X10 is (D)Lys(1PEG8_IsoGlu_C n (Diacid) and (D)Lys(1PEG8_IsoGlu_C n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 105) X6 or X10 is Lys(1PEG2_1PEG2_Dap_C n _Diacid) and Lys(1PEG2_1PEG2_Dap_C n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 106) X6 or X10 is Lys(IsoGlu_C n _Diacid) and Lys(IsoGlu_C n(Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 107) X6 or X10 is (D)Lys(IsoGlu_C n (D)Lys(IsoGlu_C) n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 108) X6 or X10 is Lys(PEG12_IsoGlu_C n It is a diacid, and Lys(PEG12_IsoGlu_C n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 109) X6 or X10 is (D)Lys(PEG12_IsoGlu_C n (D)Lys(PEG12_IsoGlu_C) n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 110) X6 or X10 is Lys(PEG4_IsoGlu_C n It is a diacid, and Lys(PEG4_IsoGlu_C n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 111) X6 or X10 is (D)Lys(PEG4_IsoGlu_C n (D)Lys(PEG4_IsoGlu_C) n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 112) X6 or X10 is Lys(PEG4_PEG4_IsoGlu_C n It is a diacid, and Lys(PEG4_PEG4_IsoGlu_C n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 113) X6 or X10 is (D)Lys(PEG4_PEG4_IsoGlu_C n(D)Lys(PEG4_PEG4_IsoGlu_C) n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 114) X6 or X10 is Lys(IsoGlu_C n _Diacid) and Lys(IsoGlu_C n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 115) X6 or X10 is (D)Lys(IsoGlu_C n (D)Lys(IsoGlu_C) n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 116) X6 or X10 is Lys(PEG12_Ahx_C n _Diacid) and Lys(PEG12_Ahx_C n (Diacid) [ka] A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 117) X6 or X10 is Lys(PEG12_Ahx_C n _Diacid) and Lys(PEG12_Ahx_C n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 118) X6 or X10 is (D)Lys(PEG12_Ahx_C n (Diacid) and (D)Lys(PEG12_Ahx_C n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 119) X6 or X10 is Lys(PEG12_C n _Diacid) and Lys(PEG12_C n (Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 120) X6 or X10 is (D)Lys(PEG12_C n (Diacid) and (D)Lys(PEG12_C n(Diacid) [ka] ; A hepcidin analog as described in item 58 or 68, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 10, 12, 14, 16, or 18. (Item 121) R 2 A hepcidin analog described in any one of items 1 to 120, wherein the hepcidin is NH2, or a pharmaceutically acceptable salt or solvate thereof. (Item 122) R 2 A hepcidin analog described in any one of items 1 to 120, wherein is a substituted amino acid. or its pharmaceutically acceptable salts or solvates. (Item 123) R 2 A hepcidin analog described in any one of items 1 to 120, wherein is an N-alkylamino compound, or a pharmaceutically acceptable salt or solvate thereof. (Item 124) R 2 A hepcidin analog as described in any one of items 1 to 120, or a pharmaceutically acceptable salt or solvate thereof, wherein is an N-alkylamino, where the alkyl is further substituted or unsubstituted. (Item 125) R 2 A hepcidine analog according to any one of items 1 to 120, or a pharmaceutically acceptable salt or solvate thereof, wherein is an N-alkylamino and the alkyl is further substituted with an aryl or heteroaryl. (Item 126) R 2 A hepcidin analog as described in any one of items 1 to 120, or a pharmaceutically acceptable salt or solvate thereof, wherein is an alkylamino, where the alkyl is unsubstituted or substituted with an aryl, and the alkyl is ethyl, propyl, butyl, or pentyl. (Item 127) R 2A hepcidin analog as described in any one of items 1 to 120, or a pharmaceutically acceptable salt or solvate thereof, wherein is an alkylamino, where the alkyl is unsubstituted or substituted with phenyl, and the alkyl is ethyl, propyl, butyl, or pentyl. (Item 128) R 2 A hepcidin analog described in any one of items 1 to 120, or a pharmaceutically acceptable salt or solvate thereof, wherein the OH group is OH. (Item 129) R 1 C1-C 20 A hepcidin analog described in any one of items 1 to 128, which is an alkanoyl, or a pharmaceutically acceptable salt or solvate thereof. (Item 130) R 1 A hepcidin analog as described in any one of items 1 to 128, or a pharmaceutically acceptable salt or solvate thereof, wherein is IVA or isovaleric acid. (Item 131) A hepcidin analog according to any one of items 1 to 128, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide is a linear peptide. (Item 132) A hepcidin analog according to any one of items 1 to 128, wherein the peptide is a lactam, or a pharmaceutically acceptable salt or solvate thereof. (Item 133) The peptide is a lactam, wherein any free-NH2 is cyclized with any free-C(O)2H, as described in any one of items 1 to 128, a hepcidine analog, or a pharmaceutically acceptable salt or solvate thereof. (Item 134) A hepcidin analog described in any one of items 1 to 3, comprising or consisting of a peptide, wherein the peptide is one of the peptides listed in Tables 6A to 6B, or a pharmaceutically acceptable salt or solvate thereof. (Item 135) The hepcidin analog described in any one of items 1 to 3, comprising or consisting of the peptides listed in Tables 6C to 6E and Table 7, or a pharmaceutically acceptable salt or solvate thereof. (Item 136) A peptide is contained in or consists of a peptide, and the peptide is ID number 3 [ka] Isovaleric acid-ETH-[Cys]-P-[Cys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, ID number 4 [ka] Isovaleric acid-ETH-[Dpa]-[Cys]-[Cys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, ID number 5 [ka] Isovaleric acid-ETH-[Dpa]-[Cys]-[Cys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, ID number 6 [ka] Isovaleric acid-ETH-[Dpa]-P-[Cys]-[Cys]-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, ID number 7 [ka] Iso-glycine-ETH-[Dpa]-P-[Cys]-I-[Cys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2、 ID number 21

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Claims

[Claim 1] The invention described herein.

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