Conjugated Hepcidin Mimetics

Abstract

The present invention provides hepcidin analogs with improved in vivo half-lives, as well as related pharmaceutical compositions and methods of use thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 169,545, filed April 1, 2021, and U.S. Provisional Patent Application No. 63 / 325,328, filed March 30, 2022, each of which is incorporated herein by reference in its entirety.

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

[0003] Hepcidin (also called 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 internalization and degradation. Human hepcidin is a 25 amino acid peptide (Hep25). See Krause et al. (2000) FEBS Lett 480:147-150, and Park et al. (2001) J Biol Chem 276:7806-7810. The structure of the bioactive 25 amino acid form of hepcidin is a simple hairpin with eight cysteines forming four disulfide bonds, as described by Jordan et al. J Biol Chem 284:24155-67. The N-terminal region is required for iron regulatory function, and deletion of the five N-terminal amino acid residues results in loss of iron regulatory function. See Nemeth et al. (2006) Blood 107:328-33.

[0004] Abnormal hepcidin activity is associated with iron overload disorders, including hereditary hemochromatosis (HH) and iron-loaded anemia. Hereditary hemochromatosis is a genetic 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 can include liver disease (e.g., cirrhosis NASH and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is regular phlebotomy, which places a great burden on patients. Iron-loaded anemia is a hereditary anemia with ineffective erythropoiesis, such as beta thalassemia, with severe iron overload. Complications due to iron overload are the main cause of morbidity and mortality in these patients. Hepcidin deficiency is the main cause of iron overload in non-transfused patients and contributes to iron overload in transfused patients. The current treatment for iron overload in these patients is iron chelation, which is very burdensome, sometimes ineffective, and associated with frequent side effects.

[0005] Hepcidin has several limitations that limit its use as a drug, including a difficult synthesis process that is due in part to protein aggregation and precipitation during folding, which in turn leads to low bioavailability, low injection site reactions, low immunogenicity, and high cost of goods. There is a need in the art for compounds that have hepcidin activity and also have other beneficial physical properties, such as improved solubility, stability, and / or efficacy, so that hepcidin-like compounds can be produced inexpensively and used to treat hepcidin-related diseases and disorders, such as those described herein. The present invention addresses such needs and provides novel peptide analogs, including both peptide monomer analogs and peptide dimer analogs, that have hepcidin activity and also have other beneficial properties that make the peptides of the invention suitable alternatives to 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 Summary of the Invention [Means for solving the problem]

[0007] The present invention relates generally to peptide analogs, including both monomers and dimers, that exhibit hepcidin activity and methods of use thereof.

[0008] In one embodiment, the present invention provides a method for the preparation of a compound of 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 But hydrogen, C 1 -C 6 Alkyl, C 6 -C 12 Aryl, C 6 -C 12 Aryl-C 1 -C 6 Alkyl, C 1 -C 20 Alkanoyl or C 1 -C 20 is cycloalkanoyl; R 2 But NH 2 , substituted amino, OH, or substituted hydroxy; X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1, or Tet2, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X2 is Ala, t-BuAla, Thr, substituted Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, t-BuAla, Gly, N-substituted Gly, His, or substituted His; X4 is Ala, t-BuAla, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Ala, t-BuAla, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X6 is absent or is any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen; X7 is absent or is Ala, t-BuAla, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X8 is absent or is Ala, t-BuAla, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, or 123 triazole; X9 is absent or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X10 is absent or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys, or (D)Lys; X12 to X14 are each absent or independently any amino acid; 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 or may be N-substituted; and iii) at least two of X1 to X14 are independently Ala or aMeAla, and each side chain methyl C of Ala is C 2 -C 12 Alkanyl or C 2 -C 12 provided that it is cyclized via an alkenyl linker to form a macrocycle; alkanyl is an alkyl chain and alkenyl is an alkyl chain embedded with at least one double bond; Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor. leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and the substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted bhPhe is b-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted Trp is N-methyl-L-tryptophan, a-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; the substituted bhTrp is N-methyl-L b-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid; 123 Triazole [ka] and Dab [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0009] In one embodiment, X1 and X6, X1 and X7, or X1 and X8 are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0010] In one embodiment, X4 and X6 or X4 and X8 are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0011] In one embodiment, X5 and X6 are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0012] In one embodiment, X and X or X and X are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0013] In one embodiment, C 2 -C12 Alkanyl is -CH 2 -(CH 2 ) q -CH 2 -, wherein q is 2 to 10.

[0014] In one embodiment, C 2 -C 12 Alkenyl is -(CH 2 ) t1 -(CH=CH)-(CH 2 ) t2 In the formula, t1 and t2 each independently represent an integer of 0 to 9.

[0015] In one embodiment, X1 is Glu, X2 is Thr, X4 is Dpa, or X5 is Pro.

[0016] In one embodiment, the peptide is according to formula II: R 1 -Ala'-Thr-His-[Dpa]-Pro-X6-X7-Ala'-X9-X10-X11-X12-X13-X14-R 2 (II) In the formula, R 1 , R 2 X6 to X7, and X9 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0017] In one embodiment, the peptide is according to formula III: R 1 -Glu-Thr-His-Ala'-Pro-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (III) In the formula, R 1 , R 2and X7 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0018] In one embodiment, the peptide is according to formula IV: R 1 -Glu-Thr-His-[Dpa]-Ala'-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (IV) In the formula, R 1 , R 2 and X7 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0019] In one embodiment, the peptide is according to formula V: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala'-Ala'-X8-X9-X10-X11-X12-X13-X14-R 2 (V) In the formula, R 1 , R 2 and X8 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0020] In one embodiment, X8 is Lys or (D)Lys.

[0021] In one embodiment, the peptide is according to formula VI: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala'-X7-Ala'-X9-X10-X11-X12-X13-X14-R 2 (VI) In the formula, R 1 , R 2 and X8 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0022] In one embodiment, R 1 is IVA or isovaleric acid.

[0023] In one embodiment, R 2 NH 2 In one embodiment, R 2 is OH.

[0024] In certain embodiments of any of the hepcidin analogs of the present invention, the substituted Lys or substituted (D)Lys is a Lys or (D)Lys that is directly substituted or substituted via a linker having an acid or residue thereof selected from C12 (lauric acid), C14 (mysteric acid), C16 (palmitic acid), C18 (stearic acid), C20, C12 diacid, C14 diacid, C16 diacid, C18 diacid, C20 diacid, biotin, and isovaleric acid. In one embodiment, the linker is Ahx, PEG, or PEG-Ahx.

[0025] In certain embodiments of any of the hepcidin analogs of the 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)-CH 2 -CH 2 -(Peg) n -N(H)] m - and Peg is -OCH 2 CH 2 -, m is 1, 2, or 3, n is an integer from 1 to 100K, and Z is a half-life extending moiety. In one embodiment, the half-life extending moiety is 10 -C 21 It is an alkanoyl.

[0026] In certain embodiments, a peptide analog or dimer of the invention comprises an isovaleric acid moiety conjugated to the N-terminal X1 residue. In certain embodiments, a peptide analog or dimer of the invention comprises an isovaleric acid moiety conjugated to the N-terminal Asp residue. In certain embodiments, a peptide analog or dimer of the invention comprises an isovaleric acid moiety conjugated to the N-terminal Glu residue.

[0027] In certain embodiments, peptide analogs of the invention comprise an amidated C-terminal residue.

[0028] In a related embodiment, the invention includes polynucleotides that encode the hepcidin analog or dimer (or monomeric subunit of a dimer) peptide of the invention.

[0029] In further related embodiments, the invention includes vectors comprising the polynucleotides of the invention, hi certain embodiments, the vector is an expression vector comprising, for example, a promoter operably linked to the polynucleotide in a manner that facilitates expression of the polynucleotide.

[0030] In another embodiment, the invention includes a pharmaceutical composition comprising a hepcidin analog, dimer, polynucleotide, or vector of the invention and a pharma- ceutically acceptable carrier, excipient, or vehicle.

[0031] In another embodiment, the present invention provides a method of binding to or inducing internalization and degradation of ferroportin comprising contacting ferroportin with at least one hepcidin analog, dimer, or composition of the present invention.

[0032] In further embodiments, the present invention includes a method for treating an iron metabolism disorder in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition of the present invention. In certain embodiments, the pharmaceutical composition is provided to the subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal, or topical administration route. In certain embodiments, the pharmaceutical composition is provided to the subject by oral or subcutaneous administration route. In certain embodiments, the iron metabolism disorder is an iron overload disorder. In certain embodiments, the pharmaceutical composition is provided to the subject at most or about twice a day, at most or about once a day, at most or about once every two days, at most or about once a week, or at most or about once a month.

[0033] In certain embodiments, the hepcidin analog is provided to a subject in a dosage of about 1 mg to about 100 mg or about 1 mg to about 5 mg.

[0034] In another embodiment, the present invention provides a device comprising a pharmaceutical composition of the present invention for delivery of a hepcidin analog or dimer of the present invention, optionally orally or subcutaneously, to a subject.

[0035] In yet another embodiment, the invention includes a kit containing a pharmaceutical composition of the invention packaged with reagents, devices, or instructions, or a combination thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present invention generally relates to hepcidin analog peptides and methods for making and using the same. In certain embodiments, the hepcidin analogs exhibit one or more hepcidin activities. In certain embodiments, the present invention relates to hepcidin peptide analogs that include one or more peptide subunits that form a cyclized structure via an intramolecular bond, e.g., an intramolecular disulfide bond. In certain embodiments, the cyclized structure has increased potency and selectivity compared to non-cyclized hepcidin peptides and analogs thereof. In certain embodiments, the hepcidin analog peptides of the present invention exhibit increased half-life compared to hepcidin or previous hepcidin analogs, for example, when delivered orally.

[0037] Definitions and Nomenclature Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0038] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0039] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer (or component) or group of integers (or components) but not the exclusion of any other integer (or component) or group of integers (or components).

[0040] The singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0041] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0042] 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, livestock animals (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats). The term "mammal" refers to any mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, etc.

[0043] The term "peptide" as used herein broadly refers to a sequence of two or more amino acids linked together by peptide bonds. It is to be understood that the term does not imply a specific length of a polymer of amino acids, nor is it intended to imply or distinguish whether a polypeptide is produced using recombinant techniques, chemical synthesis, enzymatic synthesis, or is naturally occurring.

[0044] The term "peptide analog" or "hepcidin analog" as used herein broadly refers to peptide monomers and peptide dimers that contain one or more structural features and / or functional activities in common with hepcidin or a functional region thereof. In certain embodiments, peptide analogs include peptides that share substantial amino acid sequence identity with hepcidin, such as peptides that contain one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence of wild-type hepcidin, such as human hepcidin. In certain embodiments, peptide analogs include one or more additional modifications, such as, for example, conjugation to 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 or alternatively referred to herein as "hepcidin analog," "hepcidin peptide analog," or "hepcidin analog peptide."

[0045] The recitation includes the statements "sequence identity", "percent identity", "percent homology" as used herein, or, for example, "a sequence 50% identical to" refers to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid over a comparison window. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window (i.e., window size), and multiplying the result by 100 to obtain the sequence identity percentage.

[0046] Calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. 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, the molecules are identical at that position.

[0047] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0048] Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In some embodiments, percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) algorithm implemented in 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, percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix, and 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 of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller (1989, Cabios, 4:11-17) algorithm as implemented in the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0049] The peptide sequences described herein can be used as query sequences to search against public databases, for example to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215:403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0050] The term "conservative substitution" as used herein means that one or more amino acids are replaced by another biologically similar residue. Examples include substitution 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 invention, one or more Met residues are replaced with norleucine (Nle), which is a biological equivalent of Met but, in contrast to Met, is not easily 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 a residue not normally 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 conservative substitution is the substitution of one or more Pro residues with bhPro or Leu or D-Npc (isonipecotic acid). For more information on phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et.al. Science 247, 1306-1310, 1990. In the following scheme, conservative substitutions of amino acids are grouped by physicochemical properties: I: neutral, hydrophilic, II: acid and amide, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids. [Table 8]

[0051] In the following scheme, conservative substitutions of amino acids are grouped by physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. [Table 9]

[0052] The term "amino acid" or "any amino acid" as used herein refers to any and all amino acids, including naturally occurring amino acids (e.g., a-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 amino acids found in nature, such as the 23 amino acids that are linked into peptide chains to form the building blocks of various proteins. These are primarily L stereoisomers, although a small number of D-amino acids occur in bacterial envelopes and some antibiotics. The 20 "standard" natural amino acids are listed in the table above. The "non-standard" natural amino acids are pyrrolysine (found in methanogens and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes and also in 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. There are over 140 known naturally occurring amino acids, with thousands of possible combinations. Examples of "unnatural" amino acids include the β-amino acids (β 3 and β 2 ), 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-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety that is not naturally found in amino acids.

[0053] As will be apparent to one of skill in the art, the peptide sequences disclosed herein are presented from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C-terminus of the peptide. Some of the sequences disclosed herein contain a "Hy" moiety at the amino terminus (N-terminus) of the sequence and an "-OH" or "-NH 2 In such cases, unless otherwise indicated, a "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 a "-OH" moiety or a "-NH 2 " moieties are the amides (CONH) at the C-terminus, 2 In each of the sequences of the invention, a C-terminal "-OH" moiety refers to a C-terminal "-NH 2 " moiety, and vice versa. It is further understood that the moiety at the amino or carboxy terminus can be a bond, e.g., a covalent bond, especially in situations where the amino or carboxy terminus is attached to a linker or another chemical moiety, e.g., a PEG moiety.

[0054] As used herein, "NH 2 The term "" refers to the free amino group present at the amino terminus of a polypeptide. The term "OH" as used herein refers to the free carboxy group present at the carboxy terminus of a peptide. Additionally, the term "Ac" as used herein refers to acetyl protection by acylation of the C-terminus or N-terminus of a polypeptide.

[0055] As used herein, the term "carboxy" refers to -CO 2 Point to H.

[0056] In most cases, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions proposed by the IUPAC Commission on Organic Chemical Nomenclature and the IUPAC-IUB Commission on Biochemical Nomenclature 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 in this specification and the appended claims differ from those suggestions, they will be made clear to the reader. Some abbreviations useful in describing the present invention are defined in Table 1 below. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12]

[0057] Throughout this specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are 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 non-naturally occurring amino acids, unless they are referred to by their full name (e.g., sarcosine, ornithine, etc.), the abbreviations are 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-carboxylate), β-Glu (β-aminobutyric acid ... Frequently used three-letter or four-letter codes are used for these residues, including 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).

[0058] Furthermore, R 1 may be substituted with isovaleric acid or equivalents in all sequences. In some embodiments where the peptides of the invention are conjugated to an acidic compound, such as, for example, isovaleric acid, isobutyric acid, valeric acid, etc., the presence of such conjugates is referred to in acid form. Thus, by way of example and not by way of limitation, instead of indicating the conjugation of isovaleric acid to a peptide by referring to isovaleroyl, in some embodiments the application may refer to such conjugates as isovaleric acid.

[0059] For each of the hepcidin analog formulas provided herein, it is understood that bonds may be indicated or implied with "-" based on the formula and 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 a bond exists between L1 and Z when both are present. Thus, for example, the definition 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 attached to Xaa2 and Y1 via a single bond. Thus, the definition of a substituent may or may not include a "-", but it is still understood to be attached to an adjacent substituent.

[0060] As used herein, the term "L-amino acid" refers to the "L" isomeric form of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomeric form of a peptide. In certain embodiments, the amino acid residues described herein are in the "L" isomeric form, although the "D" isomeric form of the residue can be substituted for any L-amino acid residue, so long as the desired functional group is retained by the peptide.

[0061] Unless otherwise indicated, the L-isomer forms of the natural and unnatural amino acids in question having 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).

[0062] As used herein, a "lower homologue of Lys" refers to an amino acid that has the structure of lysine but has one or more fewer carbons in the side chain compared to lysine.

[0063] As used herein, a "higher homologue of Lys" refers to an amino acid that has the structure of lysine but has one or more additional carbon atoms in the side chain compared to lysine.

[0064] As used herein, the term "DRP" refers to a disulfide-rich peptide.

[0065] The term "dimer" as used herein broadly refers to a peptide that includes two or more monomeric subunits. A particular dimer includes 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 may be linked via internal amino acid residues. The monomeric subunits of a dimer may each be linked via the same site, or each may be linked via different sites (e.g., C-terminus, N-terminus, or internal site).

[0066] The terms "isosteric replacement" or "isosteric substitution" are used interchangeably herein to refer to any amino acid or other analog moiety that has similar chemical and / or structural properties to a particular amino acid. In certain embodiments, an isosteric substitution is a conservative substitution with a natural or unnatural amino acid.

[0067] The term "cyclization" as used herein refers to a reaction in which a portion of a polypeptide molecule is linked to another portion of a polypeptide molecule to form a closed ring, e.g., by forming a disulfide bridge or other similar bond.

[0068] The term "subunit" as used herein refers to one of a pair of polypeptide monomers that are combined to form a dimeric peptide composition.

[0069] The term "linker moiety" as used herein refers broadly to a chemical structure capable of linking or bonding two peptide monomer subunits together to form a dimer.

[0070] The term "solvate" in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (e.g., a hepcidin analog according to the present invention or a pharma- ceutically acceptable salt thereof) and a solvent. The solvent in this context may be, for example, water, ethanol, or another pharma- ceutically 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.

[0071] As used herein, "iron metabolism disorders" include disorders in which abnormal iron metabolism directly causes the disorder, or disorders in which blood iron levels are dysregulated and cause the disorder, or disorders in which iron dysregulation is the result of another disorder, or disorders in which the disorder may be treated by regulating iron levels, etc. More specifically, iron metabolism disorders according to the present disclosure include iron overload disorders, iron deficiency disorders, iron biodistribution disorders, other iron metabolism disorders, and other disorders potentially related to iron metabolism, etc. Disorders of iron metabolism include hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemojuvelin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, sideroblastic anemia, porphyria, porphyria cutanea tarda, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital dyserythroplastic anemia, hypochromic microcytic anemia, sickle cell disease, polycythemia vera (primary and secondary) Hepcidin is a neurodegenerative disorder characterized by a wide range of conditions, including: chronic obstructive pulmonary disease (COPD), post-renal transplant, Chuvash, HIF and PHD mutations, as well as idiopathic myelodysplasia, pyruvate kinase deficiency, obesity iron deficiency, other anemias, benign or malignant tumors that overproduce hepcidin or induce its overproduction, conditions with hepcidin excess, Friedreich's ataxia, Greisl's syndrome, Hallervorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer, hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes, atherosclerosis, neurodegenerative disorders, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0072] In some embodiments, the disease and disorder is associated with iron overload diseases, such as ferrohemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0073] In some embodiments, the hepcidin analogs of the present invention are used to treat diseases and disorders that are not typically identified as iron-related. For example, hepcidin is highly expressed in mouse pancreas, suggesting that diabetes (type I or type II), insulin resistance, glucose intolerance, and other disorders may be improved by treating the underlying iron metabolism disorder. 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 using methods known in the art, such as assays known in the art that monitor hepcidin, hemojuvelin, or iron concentration and expression, such as those in WO2004 / 092405, incorporated herein by reference, and those described in U.S. Patent No. 7,534,764, incorporated herein by reference.

[0074] In certain embodiments of the invention, the iron metabolism disorder is an iron overload disorder, including hereditary hemochromatosis, iron-loading anemia, alcoholic liver disease, and chronic hepatitis C.

[0075] The term "pharmaceutically acceptable salts" as used herein refers to water- or oil-soluble or dispersible salts or zwitterionic forms of the peptides or compounds of the invention that are suitable for the treatment of diseases without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio, and effective for their intended use. The salts can be prepared during the final isolation and purification of the compounds, or can be prepared separately by reacting the amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, 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. Also, the amino group in the compound of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chloride, bromide, and iodide; dimethyl, diethyl, dibutyl, and diamyl sulfate; decyl chloride, bromide, and iodide, lauryl, myristyl, and steryl; and benzyl and phenethyl bromide. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. The pharmaceutically acceptable salt can be suitably, for example, a salt selected from acid addition salts and base salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts.Examples of basic salts include salts in which the cation is selected from alkali metal cations such as sodium or potassium ions, alkaline earth metal cations such as calcium or magnesium ions, and substituted ammonium ions such as ions of the type N(R1)(R2)(R3)(R4)+, where R1, R2, R3, and R4 independently typically represent hydrogen, optionally substituted C1-6 alkyl, or optionally substituted C2-6 alkenyl. Examples of relevant C1-6 alkyl groups include methyl, ethyl, 1-propyl, and 2-propyl groups. Examples of potentially relevant C2-6 alkenyl groups include ethenyl, 1-propenyl, and 2-propenyl. Other examples of pharma- ceutically acceptable salts are found 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). Also see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002) for a review of suitable salts. Other suitable base salts are formed from bases which form non-toxic salts. Representative examples include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases can also be formed, such as, for example, hemisulfate and hemicalcium salts.

[0076] The term "N (alpha) methylation" as used herein refers to the methylation of the alpha amine of an amino acid, also commonly referred to as N-methylation.

[0077] As used herein, the term "sym methylation" or "Arg-Me-sym" refers to symmetric methylation of the two nitrogens of the guanidine group of arginine. Additionally, the term "asym methylation" or "Arg-Me-asym" refers to methylation of a single nitrogen of the guanidine group of arginine.

[0078] The term "acylating organic compound" as used herein refers to various compounds having a carboxylic acid functional group that are used to acylate the N-terminus of an amino acid subunit prior to forming a C-terminal dimer. Non-limiting examples of acylating organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3,3,3-trifluoropropionic acid, 3-fluoromethylbutyric acid, and tetrahydro-2H-pyran-4-carboxylic acid.

[0079] The term "alkyl" includes straight-chain or branched, non-cyclic or cyclic, saturated aliphatic hydrocarbons having 1 to 24 carbon atoms. n-m " denotes a range inclusive of the endpoints, where n and m are integers and indicate the number of carbons. Examples include 1-4 , C 1-6 , C 1-8 , C 1-20 "C n-m The term "alkyl" refers to an alkyl group having n to m carbon atoms. For example, "C 1-6"Alkyl" refers to a hydrocarbon radical, straight or branched, having 1 to 6 carbon atoms obtained by removal of one hydrogen atom from a single carbon atom of a parent alkane. Representative straight chain saturated alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, and representative branched saturated alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative cyclic saturated alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, and representative cyclic unsaturated alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

[0080] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and having at least one double bond. For example, (C 2 -C 6 ) Alkenyl is meant to include ethenyl, propenyl, and the like. In some embodiments, alkenyl has 2-24 carbon atoms.

[0081] The term "alkylene" specifically refers to a divalent alkyl group having 1 to 24 carbon atoms. This term includes methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), -(CH 2 ) 4 -, -(CH 2 ) 6 -, propylene isomers (e.g., -CH 2 CH 2 CH 2 - and -CH(CH 3 )CH 2 -).

[0082] The term "alkenylene" refers to a divalent alkenyl group having at least one carbon-carbon double bond and, in particular, having 2 to 24 carbon atoms. This term includes -CH=CH-, -CH 2 CH=CH-, -C(CH 3 )=CH-, -CH 2 C(=CH 2 )C(=CH 2 )CH 2 -, -CH 2 CH=CHCH 2 -, -(CH 2 )C(=CH 2 )C(=CH 2 )(CH 2 ) 2 -.

[0083] The term "administering" or "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0084] As used herein, a "therapeutically effective amount" of a peptide agonist of the invention is intended to describe a sufficient amount of the peptide agonist to treat a hepcidin-related disease, including, but not limited to, any of the diseases and disorders described herein (e.g., iron metabolism diseases). In certain embodiments, a therapeutically effective amount will achieve a desired benefit / risk ratio applicable to any medical treatment.

[0085] Peptide analogs of hepcidin The present invention provides peptide analogs of hepcidin (collectively, "hepcidin analogs"), which may be monomeric or dimeric.

[0086] In some embodiments, the hepcidin analogs of the present invention bind to ferroportin, e.g., human ferroportin. In certain embodiments, the hepcidin analogs of the present invention specifically bind to human ferroportin. As used herein, "specifically binds" refers to the preferential interaction of a specific binding agent with a given ligand over other agents in a sample. For example, a specific binding agent that specifically binds to a given ligand binds to the given ligand in an amount or degree that is more observable than the amount or degree of any non-specific interactions with other components in the sample under suitable conditions. Suitable conditions are conditions that allow for the interaction between a given specific binding agent and a given ligand. These conditions include pH, temperature, concentration, solvent, incubation time, etc., and may vary for a given specific binding agent and ligand pair, but can be readily determined by one of skill in the art. In some embodiments, the hepcidin analogs of the present invention bind to ferroportin with greater specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the invention exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1000%, or 10,000% greater ferroportin specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the invention exhibit at least about 5-fold, or at least about 10-, 20-, 50-, or 100-fold greater ferroportin specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0087] In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity. In some embodiments, the activity is an in vitro or in vivo activity, such as an in vivo or in vitro activity described herein. In some embodiments, the hepcidin analogs of the present invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99% of the activity exhibited by a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0088] In some embodiments, the hepcidin analogs of the invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99% of the ferroportin binding capacity exhibited by the hepcidin reference compound. In some embodiments, the hepcidin analogs of the invention exhibit a lower EC50 or IC for binding to ferroportin (e.g., human ferroportin) compared to the hepcidin reference compound. 50 In some embodiments, the hepcidin analogs of the invention have an EC50 or IC that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than a hepcidin reference compound in a ferroportin competitive binding assay. 50 has.

[0089] In certain embodiments, the hepcidin analogs of the present invention exhibit increased hepcidin activity compared to a hepcidin reference compound. In some embodiments, this activity is an in vitro or in vivo activity, such as an in vivo or in vitro activity described herein. In certain embodiments, the hepcidin analogs of the present invention exhibit 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 140-fold, 160-fold, 180-fold, or 200-fold higher hepcidin activity than a hepcidin reference compound. In certain embodiments, the hepcidin analogs of the invention exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% greater activity than a hepcidin reference compound.

[0090] In some embodiments, the peptide analogs of the invention exhibit in vitro activity in inducing degradation of human ferroportin protein that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% greater than a hepcidin reference compound, as measured according to the methods described herein.

[0091] In some embodiments, the peptides or peptide dimers of the invention exhibit in vivo activity in inducing a reduction in free plasma iron in an individual that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% greater than a hepcidin reference compound, as measured according to the methods described herein.

[0092] In some embodiments, the activity is an in vitro or in vivo activity, such as an in vivo or in vitro activity described herein. In certain embodiments, the hepcidin analogs of the invention have 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times greater activity, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, or 200 times greater activity than a hepcidin reference compound. exhibiting 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700% or 1000% greater activity, which may be in vitro activity for inducing degradation of ferroportin, e.g., as measured in accordance with the Examples herein, or which may be in vivo activity for reducing free plasma iron, e.g., as measured in accordance with the Examples herein.

[0093] In some embodiments, the hepcidin analog of the present invention mimics the hepcidin activity of the biologically active human 25 amino acid form, Hep25, and is referred to herein as "mini-hepcidin". As used herein, in certain embodiments, a compound (e.g., a hepcidin analog) with "hepcidin activity" means that the compound has the ability to reduce plasma iron concentration in a subject (e.g., a mouse or a human) when administered (e.g., parenterally) in a dose-dependent and time-dependent manner. See, for example, Rivera et al. (2005), Blood 106:2196-9. In some embodiments, the peptides of the present invention reduce plasma iron concentrations in a subject by at least about 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold, or by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

[0094] In some embodiments, the hepcidin analogs of the present invention have in vitro activity, as assayed by their ability to cause internalization 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 the dose-dependent loss of fluorescence of cells engineered to display ferroportin fused to green fluorescent protein, as seen in Nemeth et al. (2006) Blood 107:328-33. Aliquots of cells are incubated with graded concentrations of a reference preparation of Hep25 or minihepcidin for 24 hours. As provided herein, EC 50 Values ​​are provided as the concentration of a given compound (e.g., a hepcidin analog peptide or peptide dimer of the invention) that induces 50% of the maximal loss of fluorescence produced by a reference compound. EC 50 In certain embodiments, preferred hepcidin analogs of the invention have an EC of about 1,000 nM or less in an in vitro activity assay. 50 In certain embodiments, the hepcidin analogs of the invention have an EC value of less than any one of about 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 (e.g., as described in Nemeth et al. (2006) Blood 107:328-33 or in the Examples herein). 50 In some embodiments, the hepcidin analog or biotherapeutic composition (e.g., any one of the pharmaceutical compositions described herein) has an EC 50 Value or IC 50 It has a value.

[0095] Other methods known in the art for calculating the hepcidin activity and in vitro activity of the hepcidin analogs of the present invention may be used.For example, in certain embodiments, the in vitro activity of hepcidin analogs or reference peptides is measured by their ability to internalize cellular ferroportin, which is determined by immunohistochemistry or flow cytometry using an antibody that recognizes an extracellular epitope of ferroportin.Alternatively, in certain embodiments, the in vitro activity of hepcidin analogs or reference peptides is measured by their dose-dependent ability to inhibit iron efflux from ferroportin-expressing cells preloaded with radioisotopes or stable isotopes of iron, as seen in Nemeth et al. (2006) Blood 107:328-33.

[0096] In some embodiments, the hepcidin analogs of the invention exhibit increased stability (eg, as measured by half-life, proteolytic degradation rate) compared to a hepcidin reference compound. In certain embodiments, the stability of the hepcidin analogs of the invention is increased by at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 140-fold, 160-fold, 180-fold, or 200-fold, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% over a hepcidin reference compound. In some embodiments, the stability is as described herein. In some embodiments, the stability is plasma stability, optionally measured, for example, according to the methods described herein, In some embodiments, the stability is stability upon oral delivery.

[0097] In certain embodiments, the hepcidin analogs of the invention exhibit a longer half-life than a hepcidin reference compound. In certain embodiments, the hepcidin analogs of the invention exhibit a longer half-life under a given set of conditions (e.g., temperature, pH) 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 ...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 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, The half-life of the hepcidin analog of the present invention is extended due to its conjugation to one or more lipophilic substituents or half-life extending moieties, such as any of the lipophilic substituents or half-life extending moieties disclosed herein. In some embodiments, the half-life of the hepcidin analog of the present invention is extended due to its conjugation to one or more polymer moieties, such as any of the polymer moieties or half-life extending moieties disclosed herein. In certain embodiments, the hepcidin analog of the present invention has the above-mentioned half-life under a given set of conditions, where the temperature is about 25° C., about 4° C., or about 37° C., and the pH is physiological pH or about pH 7.4.

[0098] In certain embodiments, the hepcidin analogs of the invention comprising a conjugated half-life extending moiety have an increased serum half-life following oral, intravenous, or subcutaneous administration compared to the same analog but lacking the conjugated half-life extending moiety. In certain embodiments, the serum half-life of the hepcidin analogs of the invention following either 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 between 12-168 hours, 24-168 hours, 36-168 hours, or 48-168 hours.

[0099] In certain embodiments, the hepcidin analogs of the invention, e.g., hepcidin analogs comprising a conjugated half-life extending moiety, provide reduced serum iron concentrations following oral, intravenous, or subcutaneous administration to a subject. In certain embodiments, the subject's serum iron concentration is reduced to less than 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the serum iron concentration that would be present if the subject were not administered the hepcidin analog. In certain embodiments, the reduction in serum iron concentration is maintained for at least 1 hour, at least 4 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours following 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 subject's serum iron concentration decreases by less than 20% at about 4 hours or about 10 hours after administration (e.g., intravenously, orally, or subcutaneously) to the subject. In one embodiment, the subject's serum iron concentration decreases by less than 50% or less than 60% for about 24 to about 30 hours after administration (e.g., intravenously, orally, or subcutaneously).

[0100] In some embodiments, half-life is measured in vitro using any suitable method known in the art, for example, in some embodiments, the stability of the hepcidin analogs of the present invention is determined by incubating the hepcidin analogs 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 samples is analyzed by separating the hepcidin analog from serum proteins and then analyzing for the presence of the hepcidin analog of interest using LC-MS.

[0101] In some embodiments, the stability of hepcidin analogs is measured in vivo using any suitable method known in the art, for example, in some embodiments, the stability of hepcidin analogs is determined in vivo by administering a peptide or peptide dimer to a subject, such as a human or any mammal (e.g., a mouse), and then samples are taken from the subject by drawing blood at various time points, typically up to 24 hours.The samples are then analyzed as described above for the in vitro method of measuring half-life.In some embodiments, the in vivo stability of the hepcidin analogs of the present invention is determined by the method disclosed in the Examples herein.

[0102] In some embodiments, the present invention provides a hepcidin analog as described herein, which exhibits improved solubility or improved aggregation properties compared to a hepcidin reference compound. Solubility may be determined by any suitable method known in the art. In some embodiments, suitable methods known in the art for determining solubility include incubating the peptide (e.g., a hepcidin analog of the invention) in various buffers (acetate 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 pHs known in the art) and testing for aggregation or solubility using standard techniques. These include, but are not limited to, for example, visual precipitation, dynamic light scattering, circular dichroism, and fluorescent dyes to measure surface hydrophobicity and detect aggregation or fibrillation. In some embodiments, improved solubility means that the peptide (e.g., a hepcidin analog of the invention) is more soluble in a given liquid than a hepcidin reference compound.

[0103] In certain embodiments, the invention provides hepcidin analogs described herein that exhibit increased solubility in a particular solution or buffer, e.g., in water or in a buffer known in the art or disclosed herein, by 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, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% over a hepcidin reference compound.

[0104] In certain embodiments, the invention provides hepcidin analogs as described herein, which exhibit reduced aggregation, such that the peptide aggregates in solution at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times less, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% less than a hepcidin reference compound in a particular solution or buffer, such as water or a buffer known in the art or disclosed herein.

[0105] In some embodiments, the present invention provides hepcidin analogs described herein, which exhibit lower degradation (i.e., higher degradation stability) than a hepcidin reference compound, for example, greater than or about 10% lower, greater than or about 20% lower, greater than or about 30% lower, greater than or about 40% lower, or greater than or about 50% lower degradation. In some embodiments, degradation stability is determined by any suitable method known in the art. In some embodiments, suitable methods known in the art for determining degradation stability include the method described in Hawe et al J Pharm Sci, VOL.101, NO.3, 2012, p 895-913, which is incorporated herein in its entirety. Such methods are used in some embodiments to select potent sequences with extended shelf life.

[0106] In some embodiments, the hepcidin analogs of the present invention are synthetically produced, hi other embodiments, the hepcidin analogs of the present invention are recombinantly produced.

[0107] The various hepcidin analog monomer and dimer peptides of the present invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may include unnatural or non-natural amino acids, including, but not limited to, modified amino acids. In certain embodiments, modified amino acids include natural amino acids that have been chemically modified to include a group, groups, or chemical moiety that does not naturally occur on the amino acid. The hepcidin analogs of the present invention may additionally include D-amino acids. Still further, the hepcidin analog peptide monomers and dimers of the present invention may include amino acid analogs. In certain embodiments, the peptide analogs of the present invention include any of those described herein, where one or more natural amino acid residues of the peptide analogs are replaced with unnatural or non-natural amino acids, or D-amino acids.

[0108] In certain embodiments, hepcidin analogs of the invention include one or more modified or unnatural amino acids. For example, in certain embodiments, hepcidin analogs include 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-CF 3 ), 2-2-indan, 1-1-indan, cyclobutyl, βhPhe, hLeu, Gla, Phe(4-NH 2 ), 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 appreciate that other modified or unnatural amino acids, and various other substitutions of natural amino acids with modified or unnatural amino acids may be made to achieve similar desired results, and that such substitutions are within the teachings and spirit of the present invention.

[0109] The present invention includes any of the hepcidin analogs described herein, eg, in free form or in salt form.

[0110] The compounds described herein include isotopically labeled compounds that are identical to those listed in the various formulas and structures presented herein, but differ in the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, e.g., 2 H, 3 H, 1 3C, 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein include, for example, 3 H and 14 Compounds incorporating radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. Additionally, deuterium, i.e. 2 Substitution with an isotope such as H may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. In certain embodiments, the compounds are isotopically substituted with deuterium. In more particular embodiments, the most labile hydrogen is substituted with deuterium.

[0111] The hepcidin analogs of the present invention include any of the peptide monomers or dimers described herein linked to a linker moiety, including any of the specific linker moieties described herein.

[0112] Hepcidin analogs of the present invention include peptides, e.g., monomers or dimers, that include peptide monomer subunits having at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to a hepcidin analog peptide sequence described herein (e.g., any one of the peptides disclosed herein), including, but not limited to, any of the amino acid sequences shown in Tables 2 and 3.

[0113] In certain embodiments, a peptide analog of the invention, or a monomeric subunit of a dimeric peptide analog of the invention, comprises or consists of 7-35 amino acid residues, 8-35 amino acid residues, 9-35 amino acid residues, 10-35 amino acid residues, 7-25 amino acid residues, 8-25 amino acid residues, 9-25 amino acid residues, 10-25 amino acid residues, 7-18 amino acid residues, 8-18 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues, and optionally one or more additional non-amino acid moieties, e.g., conjugated chemical moieties, e.g., half-life extending moieties, PEG, or linker moieties. In certain embodiments, a monomeric subunit of a hepcidin analog comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues. In certain embodiments, a monomeric subunit of a hepcidin analog of the invention comprises or consists of 10-18 amino acid residues, and optionally one or more additional non-amino acid moieties, e.g., conjugated chemical moieties, e.g., PEG or linker moieties. In various embodiments, a monomeric subunit comprises or consists of 7-35 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues. In certain embodiments of any of the various formulas described herein, X comprises or consists of 7-35 amino acid residues, 8-35 amino acid residues, 9-35 amino acid residues, 10-35 amino acid residues, 7-25 amino acid residues, 8-25 amino acid residues, 9-25 amino acid residues, 10-25 amino acid residues, 7-18 amino acid residues, 8-18 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues.

[0114] In certain embodiments, the hepcidin analogs or dimers of the invention do not include any of the compounds described in PCT / US2014 / 030352 or PCT / US2015 / 038370.

[0115] Peptide Hepcidin Analogues In certain embodiments, the hepcidin analogs of the invention comprise a single peptide subunit optionally conjugated to an acid moiety, hi certain embodiments, the acid moiety is conjugated directly or via a linker.

[0116] In one embodiment, the present invention provides a method for the preparation of a compound of 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 But hydrogen, C 1 -C 6 Alkyl, C 6 -C 12 Aryl, C 6 -C 12 Aryl-C 1 -C 6 Alkyl, C 1 -C 20 Alkanoyl or C 1 -C 20 is cycloalkanoyl; R 2 But NH 2 , substituted amino, OH, or substituted hydroxy; X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1, or Tet2, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X2 is Ala, t-BuAla, Thr, substituted Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, t-BuAla, Gly, N-substituted Gly, His, or substituted His; X4 is Ala, t-BuAla, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Ala, t-BuAla, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X6 is absent or is any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen; X7 is absent or is Ala, t-BuAla, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X8 is absent or is Ala, t-BuAla, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, or 123 triazole; X9 is absent or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X10 is absent or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys, or (D)Lys; X12 to X14 are each absent or independently any amino acid; 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 or may be N-substituted; and iii) at least two of X1 to X14 are independently Ala or aMeAla, and each side chain methyl C of Ala is C 2 -C 12 Alkanyl or C2 -C 12 provided that it is cyclized via an alkenyl linker to form a macrocycle; alkanyl is an alkyl chain and alkenyl is an alkyl chain embedded with at least one double bond; Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor. leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and the substituted Phe is phenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted bhPhe is b-homophenylalanine, where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted Trp is N-methyl-L-tryptophan, a-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; the substituted bhTrp is N-methyl-L b-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid; 123 Triazole [ka] and Dab [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0117] In one embodiment, X1 and X6, X1 and X7, or X1 and X8 are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0118] In one embodiment, X4 and X6 or X4 and X8 are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0119] In one embodiment, X5 and X6 are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0120] In one embodiment, X and X or X and X are each Ala, and the side chain methyl C of each Ala is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0121] In one embodiment, C 2 -C 12 Alkanyl is -CH 2 -(CH 2 ) q -CH 2 -, wherein q is 2 to 10.

[0122] In one embodiment, C 2 -C 12 Alkenyl is -(CH 2 ) t1 -(CH=CH)-(CH 2 ) t2 In the formula, t1 and t2 each independently represent an integer of 0 to 9.

[0123] In one embodiment, the linker is -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 4 - or -(CH 2 ) 6 -It is.

[0124] In one embodiment, the linker is -(CH 2 ) t1 -(CH=CH)-(CH 2 ) t2 -, and t1 and t2 are each independently 0, 1, 2, or 3.

[0125] In one embodiment, the linker is -(CH 2 ) t1 -(CH=CH)-(CH 2 ) t2 -, and t1 and t2 are each independently 2.

[0126] In one embodiment, the linker is -(CH=CH)- or -(CH 2 )-(CH=CH)-(CH 2 )-.

[0127] In one embodiment, the linker is -(CH 2 ) 2 -(CH=CH)-(CH 2 ) 2 -It is.

[0128] 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 Ala or Pro; X6 is absent, Ala, Glu, or substituted Lys; X7 is absent or is Ala, Ile, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X8 is absent or is Ala, Ile, Glu, Asp, 123 triazole, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent or is bhPhe; X10 is absent or is Ala, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X11 is absent or is Pro, bhPhe, Lys, substituted Lys, or (D)Lys.

[0129] In one embodiment, X1 is Ala or Glu.

[0130] In one embodiment, X2 is Thr.

[0131] In one embodiment, X3 is His.

[0132] In one embodiment, X4 is Ala or Dpa.

[0133] In one embodiment, X5 is Ala or Pro.

[0134] In one embodiment, X6 is Ala or a substituted Lys.

[0135] In one embodiment, X7 is Ala, Ile, or a substituted Lys.

[0136] In one embodiment, X8 is Ala, Lys, or (D)Lys.

[0137] In one embodiment, X9 is absent or is bhF.

[0138] In one embodiment, X10 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0139] In one embodiment, X11 is absent, Arg, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0140] In one embodiment, X12, X13, and X14 are each absent.

[0141] In one embodiment, the peptide is according to formula II: R 1 -Ala'-Thr-His-[Dpa]-Pro-X6-X7-Ala'-X9-X10-X11-X12-X13-X14-R 2 (II) In the formula, R 1 , R 2 X6 to X7, and X9 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0142] In one embodiment, the peptide is according to formula II:

number

[0143] In one embodiment, X6 is Ala.

[0144] In one embodiment, X6 is Lys substituted with Ahx-Palm.

[0145] In one embodiment, X6 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0146] In one embodiment, X6 is absent.

[0147] In one embodiment, X6 is (D)Lys.

[0148] In one embodiment, X6 is Lys.

[0149] In one embodiment, X6 is Lys substituted with Ahx-Palm.

[0150] In one embodiment, X6 is Lys(Ahx_Palm).

[0151] In one embodiment, X6 is a conjugated amino acid.

[0152] In one embodiment, X6 is a conjugated Lys or (D)Lys.

[0153] In one embodiment, X6 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety.

[0154] 38. The hepcidin analog of claim 37, wherein L1 is a single bond.

[0155] In one embodiment, L1 is iso-Glu.

[0156] In one embodiment, L1 is Ahx.

[0157] In one embodiment, L1 is iso-Glu-Ahx.

[0158] In one embodiment, L1 is PEG.

[0159] 38. The hepcidin analog of claim 37, or a pharma- ceutically acceptable salt or solvate thereof, wherein L1 is PEG-Ahx.

[0160] In one embodiment, L1 is iso-Glu-PEG-Ahx.

[0161] 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.

[0162] In one embodiment, m is 1.

[0163] In one embodiment, m is 2.

[0164] In one embodiment, n is 2.

[0165] In one embodiment, n is 4.

[0166] In one embodiment, n is 8.

[0167] In one embodiment, n is 11.

[0168] In one embodiment, n is 12.

[0169] In one embodiment, n is 20K.

[0170] In one embodiment, PEG is 1Peg2, wherein 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0171] In one embodiment, PEG is 2Peg2, wherein 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0172] In one embodiment, PEG is 1Peg2-1Peg2, where each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0173] In one embodiment, the PEG is 1Peg2-1Peg2, where 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-.

[0174] In one embodiment, PEG is 2Peg4, wherein 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-.

[0175] In one embodiment, PEG is 1Peg8, and 1Peg8 is -C(O)-CH2-(Peg)8-N(H)- or -[C(O)-CH2-(OCH2CH2)8-NH]-.

[0176] In one embodiment, PEG is 2Peg8, wherein 2Peg8 is -C(O)-CH2-CH2-(Peg)8-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)8-NH]-.

[0177] In one embodiment, PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-.

[0178] 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]-.

[0179] 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]-.

[0180] In one embodiment, when PEG is attached to Lys, the -C(O)- of PEG is attached to the Ne of Lys.

[0181] In one embodiment, when PEG is attached to isoGlu, the -N(H)- of PEG is attached to the -C(O)- of isoGlu.

[0182] In one embodiment, when PEG is attached to Ahx, the -N(H)- of PEG is attached to the -C(O)- of Ahx.

[0183] In one embodiment, when PEG is attached to the Palm, the -N(H)- of the PEG is attached to the -C(O)- of the Palm.

[0184] In one embodiment, Z is Palm.

[0185] In one embodiment, the peptide is according to formula III: R 1 -Glu-Thr-His-Ala'-Pro-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (III) In the formula, R 1 , R 2 and X7 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0186] In one embodiment, the peptide is according to formula III:

number

[0187] In one embodiment, the peptide is according to formula IV: R 1 -Glu-Thr-His-[Dpa]-Ala'-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (IV) In the formula, R 1 , R 2 and X7 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0188] In one embodiment, the peptide is according to formula IV:

number

[0189] In one embodiment, the peptide is according to formula V: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala'-Ala'-X8-X9-X10-X11-X12-X13-X14-R 2 (V) In the formula, R 1 , R 2 and X8 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12 It is cyclized via an alkenyl linker to form a macrocycle.

[0190] In one embodiment, the peptide is according to formula V:

number

[0191] In one embodiment, X8 is Lys or (D)Lys.

[0192] In one embodiment, the peptide is according to formula VI: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala'-X7-Ala'-X9-X10-X11-X12-X13-X14-R 2 (VI) In the formula, R 1 , R 2 and X8 to X14 are as described for formula (I), Ala' is alanine, and each side chain methyl C of Ala' is C 2 -C 12 Alkanyl or C 2 -C 12It is cyclized via an alkenyl linker to form a macrocycle.

[0193] In one embodiment, the peptide is according to formula VI:

number

[0194] In one embodiment, with respect to formulas (II)-(VI), the linker is -CH=CH-, -CH-CH=CH-, -CH=CH-CH 2 -, -CH 2 -CH=CH-CH 2 -, -CH 2 -CH 2 -CH=CH-CH 2 -, -CH 2 -CH=CH-CH 2 -CH 2 -, -CH 2 -CH 2 -CH=CH-CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH=CH-CH 2 -, -CH 2 -CH 2 -CH 2 -CH=CH-CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH=CH-CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH=CH-CH 2 -CH 2 -CH 2 -, -CH 2 -CH=CH-CH 2 -CH 2 -CH2 - or -CH=CH-CH 2 -CH 2 -.CH 2 -It is.

[0195] In one embodiment, X9 is absent.

[0196] In one embodiment, X9 is bhF.

[0197] In one embodiment, X11 is absent.

[0198] In one embodiment, X11 is Arg.

[0199] In one embodiment, X11 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0200] In one embodiment, X11 is (D)Lys.

[0201] In one embodiment, X12, X13, and X14 are each independently absent or any amino acid.

[0202] In one embodiment, X12, X13, and X14 are each absent.

[0203] In one embodiment, X10 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0204] In one embodiment, X10 is absent.

[0205] In one embodiment, X10 is (D)Lys.

[0206] In one embodiment, X10 is Lys.

[0207] In one embodiment, X10 is Lys substituted with Ahx-Palm.

[0208] In one embodiment, X10 is Lys(Ahx_Palm).

[0209] In one embodiment, X10 is a conjugated amino acid.

[0210] In one embodiment, X10 is a conjugated Lys or (D)Lys.

[0211] In one embodiment, X10 is Lys(L1Z) or (D)Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety.

[0212] The hepcidin analog of claim 90, wherein L1 is a single bond.

[0213] In one embodiment, L1 is iso-Glu.

[0214] In one embodiment, L1 is Ahx.

[0215] In one embodiment, L1 is iso-Glu-Ahx.

[0216] In one embodiment, L1 is PEG.

[0217] 91. The hepcidin analog of claim 90, or a pharma- ceutically acceptable salt or solvate thereof, wherein L1 is PEG-Ahx.

[0218] In one embodiment, L1 is iso-Glu-PEG-Ahx.

[0219] 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.

[0220] In one embodiment, m is 1.

[0221] In one embodiment, m is 2.

[0222] In one embodiment, n is 2.

[0223] In one embodiment, n is 4.

[0224] In one embodiment, n is 8.

[0225] In one embodiment, n is 11.

[0226] In one embodiment, n is 12.

[0227] In one embodiment, n is 20K.

[0228] In some embodiments of the compound of formula I, the present invention provides a compound of formula (X): [ka] or a pharma- ceutically acceptable salt or solvate thereof, wherein R 1 , X2, X3, X4, X5, X6, X7, X9, X10, X11, and R 2 is as defined herein and in the corresponding amino acid residues in the specification, claims, and Table 6B. 5 is H or C 1-6 In one embodiment, R 5 is H. In another embodiment, R 5 is methyl. x is a linking moiety. In some embodiments, L x is C 1-8 In another embodiment, L is an alkylene. x is C 2-8 In one embodiment, R is alkenylene. 1 is an isovaleric acid residue, i.e., 3-methylbutanoyl. In some embodiments, R 2 is phenyl-C 1-6Alkylene-amino, OH, or NH 2 In one embodiment, R 2 is OH or NH 2 In another embodiment, R 2 is 4-phenylbutylamino. In another embodiment, R 2 NH 2 or 4-phenylbutylamino. In some embodiments, L x is (trans)-CH 2 CH=CHCH 2 -, (cis)-CH 2 CH=CHCH 2 -, (cis)-(CH 2 ) 2 CH=CH(CH 2 ) 2 -, (trans)-(CH 2 ) 2 CH=CH(CH 2 ) 2 -, -(CH 2 ) 2 C(=CH 2 )C(=CH 2 )(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 4 - or -CH 2 C(=CH 2 )C(=CH 2 )CH 2In some embodiments, X2 is Thr, (NMe)Thr, or Thr_psi, X3 is His or His_psi, X4 is DIP or DIP_psi, X5 is Pro, and X6 is Ala, Sar, Lys(Ahx_Palm), Lys_Ahx_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_Dap_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_Dap_C18_diacid, -NHCH 2 CH 2 N + (CH 3 ) 2 -CH 2 C(O)- or Lys_1PEG2_1PEG2_Ahx_Palm. X7 is Arg, Tba, Tle, Ile, Ala, or Lys(calcine). X9 is Dip, bhF, or NMe_Lys_Ahx_Palm. X10 is Arg, (D)Arg, Lys_Ahx_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1PEG2_1PEG2_Ahx_C18_diacid, dK_betaine, or (D)Lys. X11 is Arg, (D)Arg, (D)Lys, or Lys_carnitine. 1 is isovaleric acid. 2 NH 2 Or N-butyl-Phe.

[0229] In another embodiment of the peptide compound of formula I, the present invention provides a peptide compound of formula (XI): [ka] or a pharma- ceutically acceptable salt or solvate thereof, wherein R 1 , X2, X3, X4, X5, X6, X8, X9, X10, X11, and R 2 are as defined herein and in the corresponding amino acid residues in the specification, claims, and Table 6C. x is C 2-8 In one embodiment, R is alkenylene. 1 is an isovaleric acid residue, i.e., 3-methylbutanoyl. In some embodiments, R 2 is phenyl-C 1-6 Alkylene-amino, OH, or NH 2 In certain embodiments, R 2 is 4-phenylbutylamino, OH, or NH 2 In one embodiment, R 2 is OH or NH 2 In another embodiment, R 2 is 4-phenylbutylamino. In one embodiment, R 2 NH 2 or 4-phenylbutylamino. In some embodiments, L x is (trans)-CH 2 CH=CHCH 2 -, (cis)-CH 2 CH=CHCH 2 -, (cis)-(CH 2 ) 2 CH=CH(CH 2 ) 2 -, (trans)-(CH 2 ) 2 CH=CH(CH 2 ) 2 -, -(CH 2 ) 2 C(=CH 2 )C(=CH 2 )(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 4 - or -CH 2 C(=CH 2 )C(=CH2 )CH 2 In some embodiments, X2 is Thr, (NMe)Thr, or Thr_psi, X3 is His or His_psi, X4 is DIP or DIP_psi, X5 is Pro, and X6 is Ala, Sar, Lys(Ahx_Palm), Lys_Ahx_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_Dap_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_Dap_C18_diacid, -NHCH 2 CH 2 N + (CH 3 ) 2 -CH 2 C(O)- or Lys_1PEG2_1PEG2_Ahx_Palm. X8 is Ala, (a-Me)Ala, bhPhe, Lys, or (D)Lys. X9 is Dip, bhF, or NMe_Lys_Ahx_Palm. X10 is Arg, (D)Arg, Lys_Ahx_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1PEG2_1PEG2_Ahx_C18_diacid, dK_betaine, or (D)Lys. X11 is Arg, (D)Arg, (D)Lys, or Lys_carnitine. 1 is isovaleric acid. 2 NH 2 Or N-butyl-Phe.

[0230] In one embodiment, PEG is 1Peg2, wherein 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0231] In one embodiment, PEG is 2Peg2, wherein 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0232] In one embodiment, PEG is 1Peg2-1Peg2, where each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0233] In one embodiment, the PEG is 1Peg2-1Peg2, where 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-.

[0234] In one embodiment, PEG is 2Peg4, wherein 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-.

[0235] In one embodiment, PEG is 1Peg8, and 1Peg8 is -C(O)-CH2-(Peg)8-N(H)- or -[C(O)-CH2-(OCH2CH2)8-NH]-.

[0236] In one embodiment, PEG is 2Peg8, wherein 2Peg8 is -C(O)-CH2-CH2-(Peg)8-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)8-NH]-.

[0237] In one embodiment, PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-.

[0238] 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]-.

[0239] 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]-.

[0240] In one embodiment, when PEG is attached to Lys, the -C(O)- of PEG is attached to the Ne of Lys.

[0241] In one embodiment, when PEG is attached to isoGlu, the -N(H)- of PEG is attached to the -C(O)- of isoGlu.

[0242] In one embodiment, when PEG is attached to Ahx, the -N(H)- of PEG is attached to the -C(O)- of Ahx.

[0243] In one embodiment, when PEG is attached to the Palm, the -N(H)- of the PEG is attached to the -C(O)- of the Palm.

[0244] In one embodiment, Z is Palm.

[0245] 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 IsoGlu_Palm, -1PEG2_1PEG2_Dap_C18_diacid, -1PEG2_1PEG2_IsoGlu_C10_diacid, -1PEG2_1PEG2_IsoGlu_C12_diacid, -1PEG2_1PEG2_IsoGlu_C14_diacid, -1PEG2_1PEG2_IsoGlu_C16_diacid, -1PEG2_1PEG2_IsoGlu_C18_diacid, -1PEG2_1PEG2_IsoGlu_C22_diacid, -1PEG2_1PEG2_Ahx_C18_diacid, -1PEG2_1PEG2_C18_diacid, -1PEG8_IsoGlu_C18_diacid, -IsoGlu_C18_diacid, -PEG12_Ahx_C18_diacid, -PEG12_C16_diacid, -PEG12_C18_diacid, -1PEG2_1PEG2_1PEG2_C18_dioic acid, -1PEG2_1PEG2_1PEG2_IsoGlu_C18_diacid, -PEG12_IsoGlu_C18_diacid, -PEG4_IsoGlu_C18_diacid, or - PEG4_PEG4_IsoGlu_C18_diacid, Where: PEG11_OMe is -[C(O)-CH 2 -CH 2 -(OCH2 CH 2 ) 11 -OMe], 1PEG2 is -C(O)-CH 2 -(OCH 2 CH 2 ) 2 -NH-, PEG4 is -C(O)-CH 2 -CH 2 -(OCH 2 CH 2 ) 4 -NH-, PEG8 is -[C(O)-CH 2 -CH 2 -(OCH 2 CH 2 ) 8 -NH-, 1PEG8 is -[C(O)-CH 2 -(OCH 2 CH 2 ) 8 -NH-, PEG12 is -[C(O)-CH 2 -CH 2 -(OCH 2 CH 2 ) 12 -NH-, Ado is -[C(O)-(CH 2 ) 11 -NH]-, Cn acid is -C(O)(CH 2 ) n-2 -CH 3 and the C18 acid is -C(O)-(CH 2 ) 16 -Me, Palm is -C(O)-(CH 2 ) 14 -Me, isoGlu is isoglutamic acid, isoGlu_Palm [ka] and Ahx is -[C(O)-(CH 2 ) 5-NH]-, Cn_ diacid is -C(O)-(CH 2 ) n-2 -COOH, where n is 10, 12, 14, 16, 18, or 22.

[0246] In one embodiment, X6 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid), Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0247] In one embodiment, X6 or X10 is (D)Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid), (D) Lys(1PEG2_1PEG2_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0248] In one embodiment, X6 or X10 is Lys(1PEG8_IsoGlu_C n _ diacid), Lys(1PEG8_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0249] In one embodiment, X6 or X10 is (D)Lys(1PEG8_IsoGlu_C n _ diacid), (D) Lys(1PEG8_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0250] In one embodiment, X6 or X10 is Lys(1PEG2_1PEG2_Dap_C n _ diacid), Lys(1PEG2_1PEG2_Dap_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0251] In one embodiment, X6 or X10 is Lys(IsoGlu_C n _ diacid), Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0252] In one embodiment, X6 or X10 is (D)Lys(IsoGlu_C n _ diacid), (D) Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0253] In one embodiment, X6 or X10 is Lys(PEG12_IsoGlu_C n _ diacid), Lys(PEG12_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0254] In one embodiment, X6 or X10 is (D)Lys(PEG12_IsoGlu_C n _ diacid), (D) Lys(PEG12_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0255] In one embodiment, X6 or X10 is Lys(PEG4_IsoGlu_C n _ diacid), Lys(PEG4_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0256] In one embodiment, X6 or X10 is (D)Lys(PEG4_IsoGlu_C n _ diacid), (D) Lys(PEG4_IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0257] In one embodiment, X6 or X10 is Lys(PEG4_PEG4_IsoGlu_C n _ diacid), Lys(PEG4_PEG4_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0258] In one embodiment, X6 or X10 is (D)Lys(PEG4_PEG4_IsoGlu_C n _ diacid), (D) Lys(PEG4_PEG4_IsoGlu_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0259] In one embodiment, X6 or X10 is Lys(IsoGlu_C n _ diacid), Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0260] In one embodiment, X6 or X10 is (D)Lys(IsoGlu_C n _ diacid), (D) Lys(IsoGlu_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0261] In one embodiment, X6 or X10 is Lys(PEG12_Ahx_C n _ diacid), Lys(PEG12_Ahx_C n _ diacid) [ka] and n is 10, 12, 14, 16, or 18.

[0262] In one embodiment, X6 or X10 is Lys(PEG12_Ahx_C n _ diacid), Lys(PEG12_Ahx_Cn _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0263] In one embodiment, X6 or X10 is (D)Lys(PEG12_Ahx_C n _ diacid), (D) Lys(PEG12_Ahx_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0264] In one embodiment, X6 or X10 is Lys(PEG_C n _ diacid), Lys(PEG12_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0265] In one embodiment, X6 or X10 is (D)Lys(PEG_C n _ diacid), (D) Lys(PEG12_C n _ diacid) [ka] ; and n is 10, 12, 14, 16, or 18.

[0266] In one embodiment, X1 is Ala.

[0267] In certain embodiments, X2 is Thr, Ala, N-MeThr, or t-BuAla.

[0268] In one embodiment, X3 is His, Ala, N-MeHis, or t-BuAla. In certain embodiments, X2 is His.

[0269] In one embodiment, X4 is Dpa, Ala, N-MePhe, or t-BuAla. In certain embodiments, X4 is Dpa.

[0270] In one embodiment, X5 is Pro, Ala, or t-BuAla. In certain embodiments, X5 is Pro.

[0271] In one embodiment, X6 is Ala, substituted Lys, N-MeAla, or t-BuAla. In another embodiment, X6 is Cys. In another embodiment, X6 is Gaba.

[0272] In one embodiment, X7 is absent, Lys, substituted Lys, Ala, Ile, t-BuAla, or N-MeLeu. In a particular embodiment, X7 is Ala.

[0273] In one embodiment, X8 is absent, Ala, or (D)Lys. In a particular embodiment, X8 is Ala.

[0274] In one embodiment, X9 is absent, Ala, bhPhe, Phe, or substituted Phe. In one embodiment, X9 is Ala. In another embodiment, X9 is Phe. In another embodiment, X9 is N-MePhe. In certain embodiments, X9 is bhPhe.

[0275] In one embodiment, X10 is absent, Lys, (D)Lys, substituted Lys, substituted (D)Lys, or Ala. In another embodiment, X10 is substituted N-MeLys. In a particular embodiment, X10 is substituted Lys. In one embodiment, the Lys substitution is Ahx_Palm. In another embodiment, the Lys substitution is 1PEG2_1PEG2_Dap_C18_diacid.

[0276] In one embodiment, X11 is Ala, t-BuAla, Lys, (D)Lys, or N-Me(D)Lys. In certain embodiments, X11 is (D)Lys.

[0277] In one embodiment, X12 is Cys or absent. In certain embodiments, X12 is absent.

[0278] In certain embodiments, X13 is absent.

[0279] In certain embodiments, X14 is absent.

[0280] In one embodiment, R 2 NH 2 It is.

[0281] In one embodiment, R 2 is a substituted amino.

[0282] In one embodiment, R 2 is N-alkylamino.

[0283] In one embodiment, R 2 is N-alkylamino, where the alkyl is further substituted or unsubstituted.

[0284] In one embodiment, R 2 is N-alkylamino, where the alkyl is further substituted aryl or heteroaryl.

[0285] In one embodiment, R 2 is alkylamino, where alkyl is unsubstituted or substituted with aryl, and alkyl is ethyl, propyl, butyl, or pentyl.

[0286] In one embodiment, R 2is alkylamino, where alkyl is unsubstituted or substituted with phenyl, and alkyl is ethyl, propyl, butyl, or pentyl.

[0287] In one embodiment, R 2 is OH.

[0288] In one embodiment, R 1 is C 1 -C 20 It is an alkanoyl.

[0289] In one embodiment, R 1 is IVA or isovaleric acid.

[0290] In one embodiment, the peptide is a linear peptide.

[0291] In one embodiment, the peptide is a lactam.

[0292] In one embodiment, the peptide is a lactam, where any free -NH 2 Any free -C(O) 2 It is cyclized with H.

[0293] In one embodiment, the peptide is any one of the peptides listed in Table 6A or a variant thereof.

[0294] In another embodiment, the present invention provides a peptide as set forth in Table 6B or Table 6C, or a pharma- ceutically acceptable salt or solvate thereof.

[0295] In one embodiment, the peptide comprises: [ka] E or Z isomers, or [ka] It is either the E or Z isomer.

[0296] In one embodiment, the peptide is the E isomer. In another embodiment, the peptide is the Z isomer. In another embodiment, the peptide is a mixture of E and Z isomers.

[0297] In one embodiment, R 2 NH 2 In another embodiment, R 2 is a substituted amino. In another embodiment, R 2 is alkylamino or (substituted alkyl)amino. In another embodiment, R 2 is methylamino, ethylamino, propylamino, benzylamino, phenyl-C 1-6 It is alkylamino, 4-phenylbutylamino, or phenethylamino.

[0298] In one embodiment, R 2 is OH.

[0299] In one embodiment, R 1 is C 1 -C 20 It is an alkanoyl.

[0300] In one embodiment, R 1 is IVA or isovaleric acid.

[0301] In certain embodiments of any of the peptide analogs having any of the various formulas described herein, R 1 is selected from methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and the conjugated amides of lauric acid, hexadecanoic acid, and γ-Glu-hexadecanoic acid.

[0302] In certain embodiments, the substituted Lys is Ac, PEG, Ahx, isoGlu, C 10 -C 20 Alkanoyl, PEG-Ahx, PEG-isoGlu, Ahx-C 10 -C 20Alkanoyl, isoGlu-C 10 -C 20 Alkanoyl, PEG-Ahx-C 10 -C 20 Alkanoyl, PEG-isoGlu-C 10 -C 20 In one embodiment, Lys is substituted with alkanoyl, or any of the others described herein. ε is replaced by.

[0303] In certain embodiments, the substituted (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 In one embodiment, (D)Lys is substituted with alkanoyl, or any of the others described herein. In one embodiment, (D)Lys is substituted with the N of (D)Lys. ε is replaced by.

[0304] In certain embodiments, C 10 -C 20 Alkanoyl is Palm.

[0305] In certain embodiments, the invention includes polypeptides having an amino acid sequence set forth in Table 6A, Table 6B, or Table 6C, or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity to any of these amino acid sequences.

[0306] In certain embodiments, the invention includes hepcidin analogs having the structure set forth below or comprising the amino acid sequence set forth below. Isopropyl oxalate-ETH-[Ala(1)]-P-[Ala(1)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Ala(2)]-P-[Ala(1)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-P-[Ala(1)]-I-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Ala(1)]-P-[Ala(2)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Ala(2)]-P-[Ala(2)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-P-[Ala(2)]-I-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Yoshiki oxalic acid-ETH-[Ala(1)]-P-[Ala(2)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2(returning リンカー), Isopropyl oxalate-ETH-[Dpa]-[Ala(1)]-[Ala(1)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-[Ala(1)]-[Ala(2)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-[Ala(2)]-[Ala(1)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-[Ala(2)]-[Ala(2)]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-P-[Ala(1)]-[Ala(1)]-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-P-[Ala(1)]-[Ala(2)]-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-P-[Ala(2)]-[Ala(1)]-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-ETH-[Dpa]-P-[Ala(2)]-[Ala(2)]-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PAI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PAI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PAI-[Ala(2)]-[Lys(Ahx_Palm)]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-P-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-P-[Ala(2)]-[Lys(Ahx_Palm)]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PAI-[Ala(1)]-[bhPhe]-[Lys(Ahx_Palm)]-R-NH2, Isopropyl oxalate-[Ala(1)]-TH-[Dpa]-PAI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-R-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-P-[Lys(Ahx_Palm)]-I-[Ala(2)]-[bhPhe]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PA-[Lys(Ahx_Palm)]-[Ala(2)]-[bhPhe]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PAI-[Ala(1)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(1)]-TH-[Dpa]-PAI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(3)]-TH-[Dpa]-PAI-[Ala(3)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(3)]-TH-[Dpa]-PAI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(3)]-TH-[Dpa]-PAI-[Ala(1)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(2)]-TH-[Dpa]-PAI-[Ala(3)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isopropyl oxalate-[Ala(1)]-TH-[Dpa]-PAI-[Ala(3)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, Isovaleric acid-[Ala(3)]-TH-[Dpa]-P-[Lys(Ahx_Palm)]-I-[Ala(3)]-[bhPhe]-[(D)Lys]-NH2, Isovaleric acid-[Ala(3)]-TH-[Dpa]-PA-[Lys(Ahx_Palm)]-[Ala(3)]-[bhPhe]-[(D)Lys]-NH2, Isovaleric acid-[Ala(3)]-TH-[Dpa]-P-[Lys(Ahx_Palm)]-[Ala(3)]-[bhPhe]-[(D)Lys]-NH2, Isovaleric acid-[Ala(2)]-TH-[Dpa]-P-[Lys(Ahx_Palm)]-I-[Ala(2)]-[bhPhe]-[(D)Lys]-NH2, Isovaleric acid-[Ala(2)]-TH-[Dpa]-PA-[Lys(Ahx_Palm)]-[Ala(2)]-[bhPhe]-[(D)Lys]-NH2, or Isovaleric acid-[Ala(2)]-TH-[Dpa]-P-[Lys(Ahx_Palm)]-[Ala(2)]-[bhPhe]-[(D)Lys]-NH2.

[0307] In one embodiment, the invention provides a peptide as set forth in Table 7, or a pharmaceutical sat or solvate thereof. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

[0308] In a particular embodiment, the peptide is any one of the peptides with FPN activity less than 100nM. In another embodiment, the peptide is any one of the peptides with FPN activity less than 50nM. In another embodiment, the peptide is any one of the peptides with FPN activity less than 20nM. In another embodiment, the peptide is any one of the peptides with FPN activity less than 10nM. In a more particular embodiment, the peptide is any one of the peptides with FPN activity less than 5nM.

[0309] Peptide Analog Conjugates In certain embodiments, the hepcidin analogs of the present invention, including both monomers and dimers, comprise one or more conjugated chemical substituents, such as lipophilic substituents and polymeric moieties, collectively referred to herein as half-life extending moieties.Without wishing to be bound by any particular theory, it is believed that the lipophilic substituents bind to albumin in the bloodstream, thereby protecting the hepcidin analogs from enzymatic degradation, and therefore extending their half-life.In addition, it is believed that the polymeric moieties extend half-life, reduce clearance in the bloodstream, and in some cases, enhance permeability through epithelia and retention in the lamina propria.Furthermore, it is also speculated that these substituents may in some cases enhance permeability through epithelia and retention in the lamina propria.Those skilled in the art will be well aware of suitable techniques for preparing the compounds used in the context of the present invention. For non-limiting examples of suitable chemistries, see, e.g., 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).

[0310] 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 linked) to a lipophilic substituent or other half-life extending moiety. The lipophilic substituent may be covalently linked to an atom in the amino acid side chain, or alternatively, may be conjugated to the amino acid side chain via one or more spacer or linker moieties. The spacer or linker moiety, if present, may provide spacing between the hepcidin analog and the lipophilic substituent.

[0311] In certain embodiments, the lipophilic substituent or half-life extending moiety comprises a hydrocarbon chain having 4 to 30 C atoms, for example at least 8 or 12 C atoms, preferably 24 or less C atoms, or 20 or less C atoms. The hydrocarbon chain may be linear or branched, saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted with a moiety that forms 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 thus the hydrocarbon chain may form part of an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0312] Lipophilic substituents can be conjugated to any amino acid side chain in the hepcidin analog of the present invention. In certain embodiments, the amino acid side chain comprises a carboxyl group, a hydroxyl group, a thiol group, an amide group, or an amine group to form an ester, a sulfonyl ester, a thioester, an amide, or a sulfonamide with a spacer or a lipophilic substituent. For example, lipophilic substituents can be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys, or Dbu, Dpr, or Orn. ​​In certain embodiments, lipophilic substituents are conjugated to Lys. The amino acid shown as Lys in any of the formulas provided herein can be replaced by, for example, Dbu, Dpr, or Orn to which a lipophilic substituent is added.

[0313] In further embodiments of the invention, alternatively or additionally, the side chains of one or more amino acid residues in the hepcidin analogs of the invention may be conjugated to polymeric or other half-life extending moieties, for example, to increase solubility and / or half-life in vivo (e.g., plasma) and / or bioavailability. Such modifications are also known to decrease clearance (e.g., renal clearance) of therapeutic proteins and peptides.

[0314] As used herein, "polyethylene glycol" or "PEG" refers to a compound having the general formula H-(O-CH 2 -CH 2 ) n-OH polyether compound. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), and depending on their molecular weight, PEO, PEE, or POG as used herein refer to oligomers or polymers of ethylene oxide. Although these three names are chemically synonymous, PEG tends to refer to oligomers and polymers with molecular weights less than 20,000 g / mol, PEO refers to polymers with molecular weights greater than 20,000 g / mol, and POE refers to polymers of any molecular weight. Depending on their molecular weight, PEG and PEO are liquids or low melting solids. Throughout this disclosure, these three names are used interchangeably. 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 utilized 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 moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharma- ceutically inert. Suitable polymer moieties 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 half-life extension, such as mono-activated alkoxy-terminated polyalkylene oxides (POA), such as mono-methoxy-terminated polyethylene glycol (mPEG), are also included, and bis-activated polyethylene oxides (glycols) or other PEG derivatives are also contemplated. Suitable polymers vary substantially by weight, with a range of about 200 to about 40,000 typically being selected for purposes of the present invention. In certain embodiments, PEG having a molecular weight of 200-2,000 daltons or 200-500 daltons is used.Depending on the initiator used in the polymerization process, different forms of PEG may be used, for example, a common initiator is monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), or mPEG for short. Other suitable initiators are known in the art and are suitable for use in the present invention.

[0315] Low molecular weight PEGs are also available as pure oligomers, referred to as monodisperse, uniform, or discrete, which find use in certain embodiments of the invention.

[0316] PEGs are also available in different shapes; branched PEGs have 3-10 PEG chains emanating from a central core group, star PEGs have 10-100 PEG chains emanating from a central core group, and comb PEGs have multiple PEG chains usually grafted onto a polymer backbone. PEGs can also be linear. A number often included in the name of PEGs indicates their average molecular weight (e.g., a PEG with n=9 has an average molecular weight of approximately 400 daltons and would be labeled PEG 400).

[0317] As used herein, "PEGylation" refers to the act of coupling (e.g., covalently) a PEG structure to a hepcidin analog of the invention, and in certain embodiments, is referred to as a "PEGylated hepcidin analog." In certain embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG portion of the conjugated half-life extending moiety is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, it is PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8. In some embodiments, the spacer is PEGylated. In certain embodiments, the PEG of the PEGylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8.

[0318] In some embodiments, the present invention includes hepcidin analog peptides (or dimers thereof) conjugated to PEG, for example, covalently linked via amide, thiol, by click chemistry, or by any other suitable means known in the art. In certain embodiments, PEG is linked via an amide bond, and therefore certain PEG derivatives used are appropriately functionalized. For example, in certain embodiments, PEG11, O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol, has both amine and carboxylic acid for coupling to the peptides of the present invention. In certain embodiments, PEG25 has a diacid moiety and 25 glycol moieties.

[0319] Other suitable polymer moieties include poly-amino acids, such as poly-lysine, poly-aspartic acid, and poly-glutamic acid (see, e.g., Gombotz, et al. (1995), Bioconjugate Chem., vol. 6:332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57; and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73,:721-729). The polymer moiety may be linear or branched. In some embodiments, it has a molecular weight of 500 to 40,000 Da, e.g., 500 to 10,000 Da, 1000 to 5000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da.

[0320] In some embodiments, the hepcidin analogs of the invention may include two or more such polymeric moieties, in which case the total molecular weight of all such moieties will generally be within the ranges set forth above.

[0321] In some embodiments, the polymer moiety may be (covalently) coupled to the amino, carboxyl, or thiol groups of the amino acid side chains. Certain examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, and the carboxyl groups of Asp and Glu residues may also be involved.

[0322] Those skilled in the art will be fully aware of suitable techniques that can be used to carry out coupling reactions.For example, PEG moieties with methoxy groups can be coupled to Cys thiol groups via maleimide bonds using commercially available reagents from Nektar Therapeutics AL.For details of suitable chemistry, see also WO2008 / 101017 and the above references.Maleimide-functionalized PEG can also be conjugated to the side chain sulfhydryl groups of Cys residues.

[0323] As used herein, disulfide bond oxidation can occur in a single step or is a two-step process. As used herein, in the case of a single oxidation step, a trityl protecting group is often used during construction to allow deprotection during cleavage followed by solution oxidation. If a second disulfide bond is required, there is a choice between natural oxidation or selective oxidation. In the case of selective oxidation, which requires orthogonal protecting groups, Acm and trityl are used as protecting groups for cysteines. Cleavage results in the removal of one protecting pair of cysteines, allowing oxidation of this pair. This is followed by a second oxidative deprotection step of the cysteine ​​protecting Acm group. In the case of natural oxidation, trityl protecting groups are used for all cysteines, allowing native folding of the peptide.

[0324] Those skilled in the art will be well aware of suitable techniques that can be used to carry out the oxidation step.

[0325] In certain embodiments, the hepcidin analogs of the present invention comprise a half-life extending moiety which may be selected from, but is not limited to, Ahx-Palm, PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-lauric acid, isoGlu-mysteric acid, and isoGlu-isovaleric acid.

[0326] In certain embodiments, the hepcidin analog comprises a half-life extending moiety having the structure shown below, where n is 0-24 or n is 14-24.

number

[0327] In certain embodiments, the hepcidin analogs of the invention comprise a conjugated half-life extending moiety as shown in Table 2. [Table 2-1] [Table 2-2]

[0328] In certain embodiments, the half-life extending moiety is directly conjugated to the hepcidin analog, while in other embodiments, the half-life extending moiety is conjugated to the hepcidin analog peptide via a linker moiety, e.g., any of those shown in Table 3. [Table 3-1] [Table 3-2]

[0329] With reference to the linker structures shown in Table 3, a reference to n=1-24 or n=1-25, etc. (e.g., in L4 or L5) indicates that n can be any integer within the recited range. Additional linker moieties can be used and are indicated in the "Abbreviations" table.

[0330] In certain embodiments, the hepcidin analogs of the invention comprise any of the linker moieties shown in Table 3 and any of the half-life extending moieties shown in Table 2, including any of the following combinations shown in Table 4: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0331] In certain embodiments, the hepcidin analog comprises two or more linkers. In certain embodiments, the two or more linkers are concatamerized, i.e., linked to one another.

[0332] In related embodiments, the invention includes a polynucleotide that encodes a polypeptide having a peptide sequence present in any of the hepcidin analogs described herein.

[0333] In addition, the present invention includes vectors, such as expression vectors, which comprise the polynucleotides of the present invention.

[0334] 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, comprising administering to 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 activity or expression of ferroportin, comprising administering to the individual a hepcidin analog or composition of the present invention in an amount sufficient to bind to and stimulate ferroportin or mimic hepcidin in the subject. In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by iron metabolism dysregulation, comprising administering to the subject a hepcidin analog or composition of the present invention.

[0335] In some embodiments, the methods of the invention include providing a hepcidin analog or a composition of the invention to a subject in need thereof. In certain embodiments, the subject in need thereof has been diagnosed with or has been determined to be at risk for developing a disease or disorder characterized by dysregulated iron levels (e.g., an iron metabolism disease or disorder, a disease or disorder associated with iron overload, and a disease or disorder associated with abnormal hepcidin activity or expression). In certain embodiments, the subject is a mammal (e.g., a human).

[0336] In certain embodiments, the disease or disorder is an iron metabolism disorder, such as an iron overload disorder, an iron deficiency disorder, an iron biodistribution disorder, or another iron metabolism disorder, and other disorders potentially related to iron metabolism. In certain embodiments, the iron metabolism disorder is hemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, beta thalassemia, sideroblastic anemia, porphyria, porphyria cutanea tarda, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital dyserythroplastic anemia, hypochromic microcytic anemia, sickle cell disease, polycythemia vera (primary and secondary), secondary erythrocytosis, Hepcidin hyperplasia, e.g. chronic obstructive pulmonary disease (COPD), post-kidney transplant, Chuvash, 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 hepcidin or induce its overproduction, conditions with hepcidin excess, Friedreich's ataxia, Greisl's syndrome, Hallervorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer (e.g. liver cancer), hepatitis, liver cirrhosis, pica, chronic renal failure, insulin resistance, diabetes, atherosclerosis, neurodegenerative disorders, dementia, multiple sclerosis, Parkinson's disease, Huntington's disease, or Alzheimer's disease.

[0337] In certain embodiments, the disease or disorder is associated with an iron overload disease, such as ferrohemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0338] 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 (type I or type II), insulin resistance, glucose intolerance, and other disorders may be improved by treating the underlying iron metabolism disorder. 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 using methods known in the art, such as assays known in the art that monitor hepcidin, hemojuvelin, or iron concentration and expression, such as the assays in WO2004 / 092405, incorporated herein by reference, and the assays described in U.S. Patent No. 7,534,764, incorporated herein by reference.

[0339] In certain embodiments, the disease or disorder is postmenopausal osteoporosis.

[0340] In certain embodiments of the invention, the iron metabolism disorder is an iron overload disorder, including hereditary hemochromatosis, iron-loading anemia, alcoholic liver disease, heart disease and / or heart failure, cardiomyopathy, and chronic hepatitis C.

[0341] In certain embodiments, any of these diseases, disorders, or indications is caused by or associated with a hepcidin deficiency or iron overload.

[0342] In some embodiments, the methods of the invention include providing a hepcidin analog of the invention (i.e., a first therapeutic agent) to a subject in need thereof 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 pharmaceutical composition is administered to the subject. In certain embodiments, the second therapeutic agent is an iron chelator. In certain embodiments, the second therapeutic agent is selected from the iron chelators deferoxamine and deferasirox (Exjade™). In another embodiment, the method includes administering a third therapeutic agent to the subject.

[0343] The present invention provides compositions (e.g., pharmaceutical compositions) comprising one or more hepcidin analogs of the present invention and a pharma- ceutically acceptable carrier, excipient, or diluent. A pharma-ceutically acceptable carrier, excipient, or diluent refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like.

[0344] The term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use 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, sterile saline and phosphate buffered saline at slightly acidic or physiological pH can be used. Suitable pH buffers can 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. This term further includes any carrier agent listed in the United States Pharmacopoeia for use in animals, including humans.

[0345] In certain embodiments, the composition comprises two or more hepcidin analogs disclosed herein. In certain embodiments, the combination is selected from one of: (i) any two or more of the hepcidin analog peptide monomers disclosed herein, (ii) any two or more of the hepcidin analog peptide dimers disclosed herein, (iii) any one or more of the hepcidin analog peptide monomers disclosed herein, and any one or more of the hepcidin analog peptide dimers disclosed herein.

[0346] It is understood that the inclusion of a hepcidin analog of the invention (i.e., one or more hepcidin analog peptide monomers of the invention or one or more hepcidin analog peptide dimers of the invention) in a pharmaceutical composition also encompasses the inclusion of a pharma- ceutically acceptable salt or solvate of the hepcidin analog of the invention. In certain embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, excipients, or vehicles.

[0347] In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog, or a pharma- ceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders disclosed herein or elsewhere (see, e.g., methods of treatment herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide monomer, or a pharma- ceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders disclosed herein or elsewhere (see, e.g., methods of treatment herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide dimer, or a pharma- ceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders disclosed herein or elsewhere (see, e.g., methods of treatment herein).

[0348] The hepcidin analogs of the present invention are suitable for administration with or without storage, and can be formulated as pharmaceutical compositions that typically include a therapeutically effective amount of at least one hepcidin analog of the present invention together with a pharma- ceutically acceptable carrier, excipient, or vehicle.

[0349] In some embodiments, the hepcidin analog pharmaceutical composition of the present invention is in unit dosage form. In such dosage form, the composition is divided into unit doses containing appropriate amounts of active ingredient. The unit dosage form may be presented as a packaged preparation, the package containing discrete amounts of preparation, such as packaged tablets, capsules, or powders in, for example, vials or ampoules. The unit dosage form may be, for example, a capsule, cachet, or tablet itself, or an appropriate number of any of these packaged forms. The unit dosage form may also be provided in a single-dose injectable form, for example, in the form of a pen device containing a liquid phase (typically aqueous) composition. The composition may be formulated for any suitable route and means of administration, for example, any one of the routes and means of administration disclosed herein.

[0350] In certain embodiments, the hepcidin analog or the pharmaceutical composition comprising the hepcidin analog is suspended in a sustained release matrix. A sustained release matrix as used herein is a matrix made of a material, usually a polymer, that can be degraded by enzymatic or acid-base hydrolysis or by dissolution. When inserted into the body, the matrix is ​​acted upon by enzymes and body fluids. The sustained release matrix is ​​selected from biocompatible materials such as liposomes, polylactides (polylactic acids), polyglycolides (glycolic acid polymers), polylactide-co-glycolides (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, polyvinyl propylene, polyvinyl pyrrolidone, and silicones. One embodiment of a biodegradable matrix is ​​a matrix of any polylactide, polyglycolide, or polylactide-co-glycolide (lactic acid / glycolic acid copolymer).

[0351] In certain embodiments, the composition is administered parenterally, subcutaneously, or orally. In certain embodiments, the composition is administered orally, intravesically, intravaginally, intraperitoneally, intrarectally, topically (by powder, ointment, drop, suppository, or transdermal patch, including intravitreal delivery, intranasal delivery, and delivery by inhalation), or bucally. As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intraarticular injection and infusion. Thus, in certain embodiments, the composition is formulated for delivery by any of these administration routes.

[0352] In certain embodiments, pharmaceutical compositions for parenteral injection include sterilized pharma- ceutical acceptable aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders for reconstitution immediately before use into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, betacyclodextrin, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. For example, proper fluidity can be maintained by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also include adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0353] Injectable depot forms include those made by forming microencapsule matrices of the hepcidin analog in one or more biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), poly(anhydrides), and (poly)glycols, e.g., PEG. Depending on the ratio of peptide to polymer and the nature of the particular polymer used, the release rate of the hepcidin analog can be controlled. Injectable depot formulations are also prepared by entrapping the hepcidin analog in liposomes or microemulsions that are compatible with body tissues.

[0354] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water, or other sterile injectable medium immediately before use.

[0355] The hepcidin analogs of the present invention may also be administered in liposomes or other lipid-based carriers. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, metabolizable lipid capable of forming liposomes can be used. The present composition in liposomal form can include stabilizers, preservatives, excipients, and the like, in addition to the hepcidin analogs of the present invention. In certain embodiments, lipids include phospholipids, including both natural and synthetic phosphatidylcholines (lecithins) and serine. Methods for forming liposomes are known in the art.

[0356] Pharmaceutical compositions used in the present invention suitable for parenteral administration may comprise sterile aqueous solutions and / or suspensions of the peptide inhibitor, generally made isotonic with the blood of the recipient using sodium chloride, glycerin, glucose, mannitol, sorbitol, or the like.

[0357] In some aspects, the present invention provides pharmaceutical compositions for oral delivery.The compositions and hepcidin analogs of the present invention can be prepared for oral administration according to any of the methods, techniques, and / or delivery vehicles described herein.Furthermore, those skilled in the art will understand that the hepcidin analogs of the present invention can be modified or integrated into systems or delivery vehicles not disclosed herein but well known in the art and compatible for use in oral delivery of peptides.

[0358] In certain embodiments, formulations for oral administration may include adjuvants (e.g., resorcinol and / or non-ionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) for artificially increasing the permeability of the intestinal wall, and / or enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF), or trasylol) for inhibiting enzymatic degradation. In certain embodiments, the hepcidin analog in a solid-type dosage form for oral administration may be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, or glyceride. These dosage forms may also contain other types of additives, for example, inert diluents, lubricants such as magnesium stearate, preservatives such as parabens, sorbic acid, antioxidants such as ascorbic acid, alpha-tocopherol, cysteine, disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavorings, or perfusants.

[0359] In certain embodiments, oral dosage forms or unit doses compatible with the hepcidin analogues of the present invention may include a mixture of hepcidin analogues and non-drug ingredients or excipients, as well as other non-recyclable materials that may be considered as either ingredients or packaging. Oral compositions may include at least one of liquid dosage forms, solid dosage forms, and semi-solid dosage forms. In some embodiments, oral dosage forms are provided that include an effective amount of hepcidin analogues, and the dosage forms include at least one of pills, tablets, capsules, gels, pastes, beverages, syrups, ointments, and suppositories. In some cases, oral dosage forms are provided that are designed and configured to achieve delayed release of the hepcidin analogues in the small intestine and / or colon of a subject.

[0360] In one embodiment, the 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. At least some embodiments provide a pharmaceutical composition 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 that comprises an enteric coating that comprises a polymer with a dissociable carboxylic acid group, such as a cellulose derivative, for example, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate, and similar cellulose derivatives, as well as other carbohydrate polymers.

[0361] 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 in the lower digestive system of a subject 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 with any compatible oral drug delivery system or component.For example, in some embodiments, the hepcidin analog of the present invention is provided in a lipid carrier system, which includes at least one of polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0362] To overcome peptide degradation in the small intestine, some embodiments of the present invention include a hydrogel polymer carrier system in which the hepcidin analog of the present invention is included, whereby the hydrogel polymer protects the hepcidin analog from proteolytic degradation in the small intestine and / or colon. The hepcidin analog of the present invention can be further formulated for compatible use with carrier systems designed to increase dissolution kinetics and enhance intestinal absorption of peptides. These methods include the use of liposomes, micelles, and nanoparticles to increase gastrointestinal permeation of peptides.

[0363] Various bioresponsive systems may be combined with one or more hepcidin analogs of the present invention to provide pharmaceuticals for oral delivery. In some embodiments, the hepcidin analogs of the present invention are used in combination with bioresponsive systems such as hydrogels and mucoadhesive polymers with hydrogen-bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan, and alginate) to provide therapeutic agents for oral administration. Other embodiments include methods for optimizing or extending the drug residence time of the hepcidin analogs disclosed herein, where the surface of the hepcidin analog surface is modified to include mucoadhesive properties through hydrogen bonds, polymers with linked mucin, or / and hydrophobic interactions. These modified peptide molecules may exhibit increased drug residence time in a subject, according to a desired feature of the present invention. Furthermore, targeted mucoadhesive systems may specifically bind to receptors on the surface of enterocytes and M-cells, thereby further increasing the uptake of particles containing the present hepcidin analogs.

[0364] Other embodiments include a method for oral delivery of the hepcidin analog of the present invention, wherein the hepcidin analog is provided to a subject in combination with a permeation enhancer that enhances the transport of peptides across the intestinal mucosa by increasing paracellular or transcellular permeation.For example, in one embodiment, a permeation enhancer is combined with the hepcidin analog, wherein the permeation enhancer comprises at least one of a long-chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent.In one embodiment, a permeation enhancer is used that comprises sodium N-[hydroxybenzoyl)amino]caprylate to form a weak non-covalent bond with the hepcidin analog of the present invention, wherein the permeation enhancer favors membrane transport and further dissociation upon reaching the blood circulation.In another embodiment, the hepcidin analog of the present invention is conjugated to oligoarginine, thereby increasing the cellular penetration of the peptide into various cell types. Additionally, in at least one embodiment, a non-covalent bond is provided between the peptide inhibitors of the present invention and a permeation enhancer selected from the group consisting of cyclodextrins (CDs) and dendrimers, where the permeation enhancer reduces peptide aggregation and increases the stability and solubility of the hepcidin analog molecule.

[0365] Other embodiments of the invention provide methods for treating a subject with a hepcidin analog of the invention having an increased half-life. In one aspect, the invention provides a hepcidin analog having a half-life of at least several hours to a day in vitro or in vivo sufficient for once-daily (qd) or twice-daily (bid) administration in a therapeutically effective amount (e.g., when administered to a human subject). In another embodiment, the hepcidin analog has a half-life of 3 days or more sufficient for once-weekly (qw) administration in a therapeutically effective amount. In yet another embodiment, the hepcidin analog has a half-life of 8 days or more sufficient for biweekly (biw) or monthly administration in a therapeutically effective amount. In another embodiment, the hepcidin analog is derivatized or modified to have a longer half-life compared to an underivatized or unmodified hepcidin analog. In another embodiment, the hepcidin analog includes one or more chemical modifications to increase serum half-life.

[0366] When used in at least one of the treatment or delivery systems described herein, the hepcidin analogs of the present invention may be used in pure form or, if such forms exist, in a pharma- ceutically acceptable salt form.

[0367] Dosage The total daily usage amount of the hepcidin analogs and compositions of the present invention can be determined by the attending physician within the scope 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 the severity of the disorder, b) the activity of the specific compound used, c) the specific composition used, the age, weight, general health, sex, and diet of the patient, 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) the drugs used in combination or simultaneously with the specific hepcidin analog used, and similar factors well known in the medical arts.

[0368] In certain embodiments, the total daily dose of the hepcidin analogs of the invention administered to a human or other mammalian host in a single or divided dose can be, for example, an amount of 0.0001 to 300 mg / kg body weight or 1 to 300 mg / kg body weight. In certain embodiments, the dosage of the hepcidin analogs of the invention ranges from about 0.0001 to about 100 mg / kg body weight per day, e.g., from about 0.0005 to about 50 mg / kg body weight per day, e.g., from about 0.001 to about 10 mg / kg body weight per day, e.g., from about 0.01 to about 1 mg / kg body weight per day, administered in one or more doses, such as 1 to 3 doses. In certain embodiments, the total dosage is, for example, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg for a human patient, about once or twice weekly. In certain embodiments, the total dosage ranges from about 1 mg to about 5 mg, or from about 1 mg to about 3 mg, or from about 2 mg to about 3 mg per human patient, for example, once weekly.

[0369] In various embodiments, the hepcidin analogs of the invention can be administered to a subject continuously (e.g., by intravenous administration or another continuous drug administration method) or at intervals, typically at regular time intervals, including, for example, once daily, twice daily, once every 2, 3, 4, 5, or 6 days, once or twice weekly, once or twice monthly, etc., depending on the dosage and pharmaceutical composition selected by one of skill in the art that is desired for a particular subject.

[0370] Such periodic hepcidin analog dosing regimens of the present invention may be advantageously interrupted for a period of time under certain circumstances, such as, for example, during long-term chronic administration, to allow the medicated subject to reduce the level of the drug or to stop taking the drug (often referred to as taking a "drug holiday"). Drug holidays are useful, for example, to maintain or restore sensitivity to the drug, particularly during long-term chronic treatment, or to reduce undesirable side effects of long-term chronic treatment of the subject with the drug. The timing of the drug holiday depends on the timing of the periodic dosing regimen and the purpose of taking the drug holiday (e.g., to restore drug sensitivity and / or to reduce undesirable side effects of continuous long-term administration). In some embodiments, the drug holiday may be a reduction in the dosage of the drug (e.g., to fall below the therapeutically effective amount for a certain time interval). In other embodiments, administration of the drug is suspended for a certain time interval before administration is resumed using the same or a different dosing regimen (e.g., at a lower or higher dose and / or dosing frequency). Thus, the drug holiday of the present invention may be selected from a wide range of durations and dosing regimens. Exemplary rest periods are 2 days or more, 1 week or more, or 1 month or more, up to about 24 months.Thus, for example, a regular once-daily dosing regimen using the peptide, peptide analog, or dimer of the present invention may be interrupted by a rest period of, for example, 1 week, or 2 weeks, or 4 weeks, after which the aforementioned regular dosing regimen (e.g., a once-daily or once-weekly dosing regimen) is resumed.It is envisioned that various other rest regimens are useful for administering the hepcidin analog of the present invention.

[0371] Thus, the hepcidin analogs can be delivered by a dosing regimen that includes two or more dosing phases separated by respective drug holiday phases.

[0372] During each administration phase, the hepcidin analog is administered to the recipient subject in a therapeutically effective amount according to a predetermined administration pattern. The administration pattern may include continuous administration of the drug to the recipient subject for the duration of the administration phase. Alternatively, the administration pattern may include administration of multiple doses of the hepcidin analog to the recipient subject, the doses being spaced apart by administration intervals.

[0373] The dosing pattern may include at least 2 doses per dosing phase, at least 5 doses per dosing phase, at least 10 doses per dosing phase, at least 20 doses per dosing phase, at least 30 doses per dosing phase, or more.

[0374] The dosing intervals can be regular dosing intervals, which can be once daily, twice daily, once every 2, 3, 4, 5 or 6 days, weekly or twice weekly, monthly or twice monthly, or regular less frequent dosing intervals, depending on the particular dosage formulation, bioavailability, and pharmacokinetic profile of the hepcidin analogs of the present invention.

[0375] 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.

[0376] If the dosing pattern includes multiple doses, the duration of the following drug holiday phase is longer than the dosing interval used in that dosing pattern. If the dosing interval is irregular, the duration of the drug holiday phase may be longer than the average interval between doses over the course of the dosing phase. Alternatively, the duration of the drug holiday may be longer than the longest interval between successive doses during the dosing phase.

[0377] The duration of the drug holiday may be at least twice the relevant dosing interval (or the average thereof), at least three times, at least four times, at least five times, at least ten times, or at least 20 times the relevant dosing interval or the average thereof.

[0378] Within these constraints, the drug holiday 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 administration pattern during the previous administration phase.

[0379] The dosing regimen includes at least two dosing phases. The consecutive dosing phases are separated by respective drug-free phases. Thus, the dosing regimen can 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 dosing phases, each separated by a respective drug-free phase.

[0380] The sequential administration phase may utilize the same administration pattern, although this may not necessarily be desirable or necessary. However, when other drugs or active agents are administered in combination with the hepcidin analogs of the present invention, typically the same drug or active agent combination is administered in the sequential administration phase. In certain embodiments, the recipient subject is a human.

[0381] In some embodiments, the present invention provides compositions and medicaments comprising at least one hepcidin analog disclosed herein. In some embodiments, the present invention provides methods of manufacturing medicaments comprising at least one hepcidin analog disclosed herein for the treatment of iron metabolism disorders, such as iron overload disorders. In some embodiments, the present invention provides methods of manufacturing medicaments comprising at least one hepcidin analog disclosed herein for the treatment of diabetes (type I or type II), insulin resistance, or glucose intolerance. Also provided are methods of treating iron metabolism disorders in a subject, e.g., a mammalian subject, preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount. Also provided are methods of treating diabetes (type I or type II), insulin resistance, or glucose intolerance in a subject, e.g., a mammalian subject, preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount.

[0382] In some embodiments, the present invention provides processes for producing the hepcidin analogs or hepcidin analog compositions (eg, pharmaceutical compositions) disclosed herein.

[0383] In some embodiments, the present invention provides a device comprising at least one hepcidin analog of the present invention, or a pharma- ceutically acceptable salt or solvate thereof, for delivering the hepcidin analog to a subject.

[0384] In some embodiments, the present invention provides methods of binding to or inducing internalization and degradation of ferroportin comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0385] In some embodiments, the present invention provides methods of binding to ferroportin and blocking pore and efflux transporter function without causing ferroportin internalization, comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0386] In some embodiments, the invention provides kits comprising at least one hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) disclosed herein packaged together with reagents, devices, instructions, or combinations thereof.

[0387] In some embodiments, the present invention provides methods of administering a 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 well known in the art and understood by one of skill in the art. In some embodiments, the present invention provides a complex comprising at least one hepcidin analog disclosed herein bound to ferroportin, preferably human ferroportin, or an antibody, e.g., an antibody that specifically binds to a hepcidin analog disclosed herein, Hep25, or a combination thereof.

[0388] In some embodiments, the hepcidin analogs of the invention have a measured EC value (e.g., EC ) of less than 500 nM in an FPN internalization assay. 50). As one of skill in the art would understand, the function of the hepcidin analogs depends on the tertiary structure of the hepcidin analogs and the binding surface presented. Thus, minor changes can be made to the sequence encoding the hepcidin analogs that do not affect the fold or are not present on the binding surface and still maintain functionality. In other embodiments, the invention provides hepcidin analogs that exhibit activity (e.g., hepcidin activity) or that have 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 that exhibit activity (e.g., hepcidin activity) or that alleviate symptoms of a disease or indication in which hepcidin is implicated.

[0389] In other embodiments, the invention provides hepcidin analogs having 85% or greater (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity or homology to the amino acid sequence of any of the hepcidin analogs presented herein, or to a peptide according to any one of the formulas or hepcidin analogs described herein.

[0390] In some embodiments, the hepcidin analogs of the present invention may include functional fragments or variants thereof having up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to one or more of the specific peptide analog sequences listed herein.

[0391] In addition to the methods described in the examples herein, the hepcidin analogs of the present invention can be produced using methods known in the art, including chemical synthesis, biosynthesis, or in vitro synthesis using recombinant DNA methods, and solid-phase synthesis.See, for example, Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol.12, JK Setlow ed., Plenum Press, NY, pp.1-19, Merrifield (1964) J Amer Chem Soc 85:2149, Houghten (1985) PNAS USA 82:5131-5135, and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2 ed. Pierce, Rockford, IL, which are incorporated herein by reference.The hepcidin 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, which are incorporated herein by reference. Alternatively, the hepcidin analogs of the present invention can be produced by recombinant DNA techniques known in the art. Thus, polynucleotides encoding the polypeptides of the present invention are contemplated herein. In certain preferred embodiments, the polynucleotide is isolated. As used herein, an "isolated polynucleotide" refers to a polynucleotide that is in an environment different from the environment in which the polynucleotide naturally occurs. EXAMPLES

[0392] The following examples demonstrate certain specific embodiments of the present invention. Unless otherwise specified in detail, the following examples were carried out 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 do not claim to be completely definitive with respect to the conditions or scope of the present invention. Therefore, they should not be interpreted as limiting the scope of the present invention in any way. Abbreviation: DCM: dichloromethane DMF: N,N-dimethylformamide NMP: N-methylpyrrolidone HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU: 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate DCC: dicyclohexylcarbodiimide NHS: N-hydroxysuccinimide DIPEA: Diisopropylethylamine EtOH: Ethanol Et2O: Diethyl ether Hy: Hydrogen TFA: Trifluoroacetic acid TIS: Triisopropylsilane ACN: Acetonitrile HPLC: High-performance liquid chromatography ESI-MS: Electrospray ionization mass spectrometry PBS: Phosphate-buffered saline Boc: t-butoxycarbonyl Fmoc: Fluorenylmethyloxycarbonyl Acm: acetamidomethyl IVA: Isovaleric acid (or isovaleryl)

[0393] K(): In the peptide sequences provided herein where a compound or chemical group is presented in parentheses immediately following a lysine residue, it is understood that the compound or chemical group within the parentheses is the side chain conjugated to the lysine residue. Thus, by way of example and not by way of limitation, K-[(PEG8)]- indicates that a PEG8 moiety is conjugated to the side chain of this lysine.

[0394] Palm: indicates the conjugation of palmitic acid (palmitoyl).

[0395] Synthesis Protocol-1 Synthesis of peptide monomers Peptide monomers of the invention were synthesized using Merrifield solid phase synthesis techniques on a Protein Technology Symphony multichannel synthesizer. Peptides were assembled using HBTU (O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate), diisopropylethylamine (DIEA) coupling conditions. For some amino acid couplings, PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) and DIEA conditions were used. Rink Amide MBHA resin (100-200 mesh, 0.57 mmol / g) was used for peptides with C-terminal amides, and Wang resin preloaded with N-α-Fmoc protected amino acids was used for peptides with C-terminal acids. Coupling reagents (premix 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 based on medicinal chemistry optimization and / or phage display and screened to identify those with superior binding and / or inhibitory properties.

[0396] construction Peptides were assembled using standard Symphony protocols. Peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4 mL DMF, followed by treatment with 2.5 mL 20% 4-methylpiperidine (Fmoc deprotection) for 10 min. The resin was then filtered, washed twice with DMF (4 ml), and treated again with piperidine for another 30 min. The resin was again washed three times with DMF (4 ml), followed by addition of 2.5 ml of amino acid and 2.5 ml of HBTU-DIEA mixture. After frequent stirring for 45 min, the resin was filtered and washed three times with DMF (4 ml each). For a typical peptide of the invention, a double coupling was performed. After the coupling reaction was completed, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling.

[0397] Disconnect After completion of the peptide construction, the peptide was cleaved from the resin by treatment with a cleavage reagent such as Reagent K (82.5% trifluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, 2.5% 1,2-ethanedithiol), which was able to successfully cleave the peptide from the resin as well as from all remaining side chain protecting groups.

[0398] The cleaved peptide was precipitated in cold diethyl ether, followed by two washes with ethyl ether. The filtrate was poured off, a second aliquot of cold ether was added, and the procedure was repeated. The crude peptide was dissolved in an acetonitrile / water (7:3 with 1% TFA) solution and filtered. The quality of the linear peptide was then verified using electrospray ionization mass spectrometry (ESI-MS) (Micromass / Waters ZQ) before being purified.

[0399] purification Analytical reversed-phase high performance liquid chromatography (HPLC) was performed on a Gemini C18 column (4.6 mm x 250 mm) (Phenomenex). Semi-preparative reversed-phase HPLC was performed on a Gemini 10 μm C18 column (22 mm x 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 (analytical) and 20 mL / min (preparative). 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 (analytical) and 15 mL / min (preparative). Synthesis Protocol-2

[0400] Synthesis of peptide monomers Peptide monomers of the invention were synthesized using standard Fmoc solid phase synthesis techniques on a CEM Liberty Blue™ microwave peptide synthesizer. Peptides were assembled using Oxyma / DIC (ethyl cyanohydroxyiminoacetate / diisopropylcarbodiimide) with microwave heating. Rink Amide-MBHA resin (100-200 mesh, 0.66 mmol / g) was used for peptides with C-terminal amides and Wang resin preloaded with N-α-Fmoc protected amino acids was used for peptides with C-terminal acids. Oxyma was prepared as a 1 M solution in DMF with 0.1 M DIEA. DIC was prepared as a 0.5 M solution in DMF. Amino acids were prepared at 200 mM. Peptide inhibitors of the invention were identified based on medicinal chemistry optimization and / or phage display and screened to identify those with superior binding and / or inhibitory properties.

[0401] construction Peptides were made using the standard CEM Liberty Blue™ protocol. Peptide sequences were assembled as follows: Resin (400 mg, 0.25 mmol) was suspended in 10 ml of 50 / 50 DMF / DCM. The resin was then transferred to a reaction vessel in a microwave cavity. Peptides were assembled using repeated cycles of Fmoc deprotection and Oxyma / DIC coupling. For deprotection, a 20% solution of 4-methylpiperidine in DMF was added to the reaction vessel and heated to 90° C. for 65 seconds. The deprotection solution was drained and the resin was washed three times with DMF. Then, for most amino acids, 5 equivalents of amino acid, Oxyma, and DIC were added to the reaction vessel and the reaction mixture was rapidly heated to 90° C. for 4 minutes by microwave irradiation. For arginine and histidine residues, milder conditions were used using temperatures of 75° C. and 50° C. for 10 minutes, respectively, to prevent racemization. Rare and expensive amino acids were often coupled manually overnight at room temperature using only 1.5-2 equivalents of reagent. Difficult couplings were often double-coupled at 90 °C for 4 min twice. After coupling, the resin was washed with DMF and the entire cycle was repeated until the desired peptide construction was completed.

[0402] Disconnect After completion of peptide assembly, 91:5:2:2 TFA / H 2 The peptide was cleaved from the resin by treatment with a standard cleavage cocktail of O / TIPS / DODT for 2 h. If more than one Arg(Pbf) residue was present, cleavage was allowed to proceed for an additional hour.

[0403] The cleaved peptide was precipitated in cold diethyl ether. The filtrate was decanted off, a second aliquot of cold ether was added, and the procedure was repeated. The quality of the linear peptide was then verified using electrospray ionization mass spectrometry (ESI-MS) (Waters® Micromass® ZQ™) before purification.

[0404] purification Analytical reversed-phase high performance liquid chromatography (HPLC) was performed on a Gemini® C18 column (4.6 mm x 250 mm) (Phenomenex). Semi-preparative reversed-phase HPLC was performed on a Gemini® 10 μm C18 column (22 mm x 250 mm) (Phenomenex) or a Jupiter® 10 μm, 300 Å C18 column (21.2 mm x 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 (analytical) and 20 mL / min (preparative).

[0405] Example 1A Synthesis of peptide analogues Unless otherwise specified, reagents and solvents used below were commercially available of standard laboratory reagent or analytical grade and were used without further purification.

[0406] Peptide Solid Phase Synthesis Procedure Method A The peptide analogs of the present invention were chemically synthesized using an optimized 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol. For C-terminal amides, Rink amide resin was used, but Wang and trityl resins were also used to generate C-terminal acids. 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. Acm (acetamidomethyl) was also used as a Cys protecting group for selective disulfide bridge formation. For coupling, a 4-10-fold excess of DMF solution containing Fmoc amino acid, HBTU, and DIEA (1:1:1.1) was added to the swollen resin [HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIEA: diisopropylethylamine, DMF: dimethylformamide]. HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate) was used instead of HBTU to improve coupling efficiency in difficult regions. Removal of the Fmoc protecting group was achieved by treatment with DMF / piperidine (2:1) solution.

[0407] Method B Alternatively, peptides were synthesized using a CEM liberty Blue microwave assisted peptide synthesizer. Using the Liberty Blue, FMOC deprotection was performed by adding 20% ​​4-methylpiperdine in DMF with 0.1M Oxyma in DMF, followed by heating to 90°C for 4 min using microwave irradiation. After a DMF wash, the FMOC-amino acid was coupled by adding 0.2M amino acid (4-6 eq), 0.5M DIC (4-6 eq), and 4-6 eq of 1M Oxyma (with 0.1M DIEA), all in DMF. The coupling solution is heated to 90°C for 4 min using microwave radiation. If Arg or other sterically hindered amino acids are coupled, a second coupling is used. If coupling with histidine, the reaction is heated to 50°C for 10 min. This cycle is repeated until the full-length peptide is obtained.

[0408] Peptide cleavage procedure from the resin Side chain deprotection and cleavage of peptide analogs of the invention (e.g., compound 2) was achieved by stirring the dried resin in a solution containing trifluoroacetic acid, water, ethanedithiol, and tri-isopropylsilane (90:5:2.5:2.5) for 2-4 hours. After removing the TFA, the peptide was precipitated using ice-cold diethyl ether. The solution was centrifuged and the ether was decanted, followed by a second diethyl ether wash. The peptide was dissolved in 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 assessed using electrospray ionization mass spectrometry (ESI-MS).

[0409] Peptide purification procedure Purification of the peptides of the invention (e.g., compound 2) was achieved using reversed-phase high performance liquid chromatography (RP-HPLC). Analysis was performed using a C18 column (3 μm, 50×2 mm) at a flow rate of 1 mL / min. Purification of linear peptides was 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 in water, buffer B: 0.043% TFA, 90% acetonitrile in water).

[0410] Those skilled in the art will appreciate that standard peptide synthesis techniques can be used to produce the compounds of the invention.

[0411] Conjugation of half-life extending moieties Peptide conjugation was performed on the resin. Lys(ivDde) ​​was used as the major amino acid. After assembly of the peptide on the resin, selective deprotection of the ivDde group was performed using 2% hydrazine in DMF for 5 min three times. Activation and acylation of the linker using HBTU, 1-2 equivalents of DIEA for 3 h and Fmoc removal followed by a second acylation with a lipid acid afforded the conjugated peptide.

[0412] Example 1B Synthesis of peptide ID number 16: Isovaleric acid-[Ala(2)]-TH-[Dpa]-PAI-[Ala(2)]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2, and the side chain C of Ala(2) and the side chain C of Ala(2) are cyclized via -CH2-CH=CH-CH2- (S / S isomer). The TFA salt of peptide ID number 16 was synthesized on Rink amide resin. Upon completion, 269 mg of 93.5% ultra-pure peptide ID number 16 was isolated as a white powder. Peptide ID number 16 was assembled on Rink Amide MBHA (100-200 mesh, 0.27 mmol / g) resin using standard Fmoc-protected synthesis conditions. The assembled peptide was isolated from the resin and protecting groups by strong acid cleavage followed by precipitation. The crude precipitate was then purified by RP-HPLC. The pure fractions were lyophilized to give the final product, peptide ID number 16.

[0413] Peptide construction Swollen resin: 3703 mg of Rink Amide MBHA solid phase resin (0.27 mmol / g loading) was transferred to a 250 mL reaction vessel. The resin was swelled with 60 mL of DMF (2 h).

[0414] Step 1: Coupling of FMOC-(D)Lys(Boc)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid FMOC-(D)Lys(Boc)-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before the next deprotection / coupling cycle was started.

[0415] Step 2: Coupling of FMOC-L-Lys(Dde)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid FMOC-L-Lys(Dde)-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0416] Step 3: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid FMOC-βhomo-L-Phe-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before the next deprotection / coupling cycle was started.

[0417] Step 4: Coupling of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-5-hexenoic acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-5-hexenoic acid in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0418] Step 5: Coupling of FMOC-Ile-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (5 washes for 0.1 min) followed by the addition of 7.5 mL of amino acid FMOC-Ile-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (3 washes for 0.1 min) before starting the next deprotection / coupling cycle.

[0419] Step 6: Coupling of FMOC-Ala-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (5 washes for 0.1 min) followed by the addition of 7.5 mL of amino acid FMOC-Ala-OH in DMF (400 mM) and 7.5 mL of a mixed solution of coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (3 washes for 0.1 min) before starting the next deprotection / coupling cycle.

[0420] Step 6': Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (5 washes for 0.1 min) followed by the addition of 7.5 mL of a solution of the amino acid FMOC-Pro-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (3 washes for 0.1 min) before starting the next deprotection / coupling cycle.

[0421] Step 7: Coupling of FMOC-L-DIP-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (5 washes for 0.1 min) followed by the addition of 7.5 mL of amino acid FMOC-L-DIP-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (3 washes for 0.1 min) before starting the next deprotection / coupling cycle.

[0422] Step 8: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid FMOC-L-His(Trt)-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0423] Step 9: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid FMOC-L-Thr(tBu)-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0424] Step 10: Coupling of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-5-hexenoic acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of amino acid (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-5-hexenoic acid in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0425] Step 11: Coupling of isovaleric acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of a solution of amino acid isovaleric acid in DMF (400 mM) and 7.5 mL of a mixed solution of coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0426] Step 12: Dde removal and coupling of Fmoc-Ahx-OH: Dde was removed from the Lys C-terminus of the resin-bound peptide using 3% hydrazine in DMF (three times for 20 min) followed by DMF washes. After deprotection, the resin was washed with 60 mL of DMF (five times for 0.1 min washes) followed by the addition of 7.5 mL of amino acid Fmoc-Ahx-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three times for 0.1 min washes) before starting the next deprotection / coupling cycle.

[0427] Step 13: Coupling of palmitic acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 mL of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (5 washes for 0.1 min) followed by the addition of 7.5 mL of a solution of the amino acid palmitic acid (400 mM) in DMF and 7.5 mL of a mixed solution of the coupling reagent HATU-DIEA (400 mM and 800 mM) in DMF. The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (3 washes for 0.1 min) before starting the next deprotection / coupling cycle.

[0428] Step 14: Ring-closing metathesis. The resin was washed with 60 ml of DCM (3 x 1 min washes) and then treated with 50 ml of a 6 mM solution of Grubbs' catalyst 1st generation in DCM (5.01 mg ml-1, 30 mol% with respect to resin substitution). The solution was reacted twice under nitrogen under microwave conditions at 60°C (2 h) and then drained. The resin was washed 3 times with DMF (60 ml each) and DCM (60 ml) before being dried and cleaved.

[0429] Step 15: TFA cleavage and isopropyl ether precipitation: 60 ml of cleavage cocktail [TFA cleavage cocktail (90 / 2.5 / 2.5 / 5 TFA / water / Tips / DTT) was added to the protected resin-bound peptide and shaken for 3 hours. Cold isopropyl ether was added, resulting in a white precipitate, which was then centrifuged. The isopropyl ether was decanted and discarded, and the precipitate was washed two more times with isopropyl ether. The resulting white precipitate cake was dissolved in acetonitrile / water (1:1), filtered, and then purified.

[0430] Step 16: RP-HPLC purification: Semi-preparative reversed-phase HPLC was performed on an Xtimate 10 μm C18 column (50 mm × 250 mm) (SHIMADZU LC-8A). Separation was achieved using a linear gradient of buffer B in buffer A (mobile phase A: water containing 0.075% TFA, mobile phase B: acetonitrile (ACN)) at a flow rate of 80 mL / min (preparative).

[0431] Step 17: Final lyophilization and analysis: The collected fractions were analyzed by analytical RP-HPLC and all fractions with purity above 95% were combined. The combined fractions were lyophilized to give peptide no. 16 as a white powder with a purity of 93.5%. Low resolution LC / MS of the purified no. 16 gave one charge state of the peptide, M+2 / 2 904.9 and molecular ion [M+1] 1808.4. The experimental mass is consistent with the theoretical mass of 1808.4 Da [M+1].

[0432] Example 1c Synthesis of peptide ID number 33: Isovaleric acid-[Ala(3)]-TH-[Dpa]-P-[Lys(Ahx_Palm)]-I-[Ala(3)]-[bhPhe]-NH2, and the side chain C of Ala(3) and the side chain C of Ala(3) are cyclized via -CH2-CH=CH-CH2- (S / S isomer). The TFA salt of peptide ID number 33 was synthesized on Rink amide resin. Upon completion, 17.2 mg of 87.1% ultra pure peptide ID number 33 was isolated as a white powder. Peptide ID number 33 was assembled on Rink Amide MBHA (100-200 mesh, 0.27 mmol / g) resin using standard Fmoc-protected synthesis conditions. The assembled peptide was isolated from the resin and protecting groups by strong acid cleavage followed by precipitation. The crude precipitate was then purified by RP-HPLC. The pure fractions were lyophilized to give the final product, peptide ID number 33.

[0433] Peptide construction Swollen resin: 200 mg of Rink Amide MBHA solid phase resin (0.27 mmol / g loading) was transferred to a 250 mL reaction vessel. The resin was swelled in 60 mL of DMF (2 h).

[0434] Step 1: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 60 ml of 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with 60 mL of DMF (five 0.1 min washes) followed by the addition of 7.5 mL of the amino acid FMOC-βhomo-L-Phe-OH in DMF (400 mM) and 7.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (400 mM and 800 mM). The coupling reaction was mixed for 1 h and filtered. After the coupling reaction was complete, the resin was washed with 60 mL of DMF (three 0.1 min washes) before starting the next deprotection / coupling cycle.

[0435] Step 2: Coupling of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-6-heptanoic acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of the amino acid (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-6-heptanoic acid in DMF (200 mM) and 2.5 mL of a mixture of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0436] Step 3: Coupling of FMOC-Ile-OH: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid FMOC-Ile-OH in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0437] Step 4: 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 in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid FMOC-L-Lys(IvDde)-OH in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0438] Step 5: 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 in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid FMOC-Pro-OH in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0439] Step 6: 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 in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid FMOC-L-DIP-OH in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0440] Step 7: 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 in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid FMOC-L-His(Trt)-OH in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0441] Step 8: 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 in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid FMOC-L-Thr(tBu)-OH in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0442] Step 9: Coupling of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-5-heptanoic acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 ml of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of the amino acid (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-6-heptanoic acid in DMF (200 mM) and 2.5 mL of a mixture of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0443] Step 10: Coupling of isovaleric acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of isovaleric acid in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0444] Step 11: IvDde removal and coupling of Fmoc-Ahx-OH: IvDde was removed from the Lys C-terminus of the resin-bound peptide using 2-5% hydrazine in DMF (four 30 min applications), followed by DMF washes. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of amino acid Fmoc-Ahx-OH in DMF (200 mM) and 2.0 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0445] Step 12: Coupling of palmitic acid: Deprotection of the Fmoc group was achieved by treating the swollen Rink Amide resin with 2.5 mL of 20% piperidine in DMF twice for 5 and 10 min, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (three 0.1 min washes), followed by the addition of 2.5 mL of isovaleric acid in DMF (200 mM) and 2.5 mL of a mixed solution of the coupling reagent HBTU-DIEA in DMF (200 mM and 220 mM). The coupling reaction was mixed for 1 h, 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 min washes) before starting the next deprotection / coupling cycle.

[0446] Step 13: Ring-closing metathesis. The resin was washed with 2 ml DCM (3 x 1 min washes) and then with 2 ml DCE (3 x 1 min washes) before being treated with 2 ml of a 6 mM solution of Grubbs' catalyst 1st generation in DCE (4.94 mg ml-1, 20 mol% with respect to resin substitution). The solution was refluxed under nitrogen overnight (12 h) and then drained. The resin was washed 3 times (4 ml each) with DMF and DCM (4 ml) before being dried and cleaved.

[0447] Step 14: TFA cleavage and ether precipitation: 10 ml of cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT) was added to the protected resin-bound peptide and shaken for 2 h. Cold diethyl ether was added, resulting in a white precipitate, followed by centrifugation. The ether was decanted and discarded, and the precipitate was washed two more times with ether. The resulting white precipitate cake was dissolved in acetonitrile / water (7:3), filtered, and then purified.

[0448] Step 15: RP-HPLC purification: Semi-preparative reversed-phase HPLC was performed on 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 (preparative).

[0449] Step 16: Final lyophilization and analysis: The collected fractions were analyzed by analytical RP-HPLC and all fractions with purity above 95% were combined. The combined fractions were lyophilized to give peptide ID number 33 as a white powder with a purity of 87.1%. Low resolution LC / MS of the purified ID number 33 gave the two charge states of the peptide, M+2 / 2 883.2 and molecular ion [M+1] 1765.4. The experimental mass is consistent with the theoretical mass of 1765.4 Da [M+1].

[0450] Example 2 Peptide analogue activity The peptide analogs were tested in vitro for induction of internalization of the human ferroportin protein. After internalization, the ferroporin protein is degraded. The assay used (FPN activity assay) measures the decrease in receptor fluorescence.

[0451] The cDNA encoding human ferroportin (SLC40A1) was cloned from Origene's cDNA clone (NM_014585). The DNA encoding ferroportin was amplified by PCR using primers that also encoded terminal restriction sites for subcloning, but did not contain a stop codon. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker such that the reading frame of ferroportin 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 of the ferroportin-GFP receptor expression plasmid. These cells were grown in growth medium according to standard protocols and transfected with the plasmid using Lipofectamine (manufacturer's protocol, Invitrogen). Cells stably expressing ferroportin-GFP were selected using G418 in the growth medium (in that only cells that had taken up and integrated the cDNA expression plasmid would survive) and sorted several times on a Cytomation MoFlo™ cell sorter to obtain GFP positive cells (488 nm / 530 nm). Cells were expanded and frozen in aliquots.

[0452] 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. Compounds were added to the incubator for at least 18 hours to the desired final concentration. After incubation, the remaining GFP fluorescence was determined by either total cell GFP fluorescence (Envision plate reader, 485 / 535 filter pair) or Beckman Coulter Quanta™ flow cytometer (expressed as the geometric mean of the fluorescence intensity at 485 nm / 525 nm). Compounds were added to the incubator for at least 18 hours and up to 24 hours to the desired final concentration.

[0453] In one particular experiment, the reference compounds included native hepcidin, minihepcidin, and an analog of minihepcidin, R1-minihepcidin. The "RI" in RI-minihepcidin refers to retroinverse. A retroinverse peptide is a peptide with the reverse sequence for all D amino acids. One example is Hy-Glu-Thr-His-NH 2 Hy-DHis-DThr-DGlu-NH 2 The EC values ​​of these reference compounds for ferroportin internalization / degradation were 50 was determined according to the FPN activity assay described above. These peptides served as control standards. [Table 5] Potency IC determined for various peptide analogs of the invention 50 or EC 50 Values ​​(nM) are provided in Tables 6A-6C. These values ​​were determined as described herein. Compound ID numbers are indicated as "Compound ID" and reference compounds are indicated as "Reference Compound." FPN EC 50 Values ​​are shown in Tables 6A-6C. If not shown, data was not determined. [Table 6A-1] [Table 6A-2] [Table 6A-3] [Table 6A-4] [Table 6A-5] [Table 6A-6] [Table 6A-7] [Table 6A-8] [Table 6A-9] [Table 6A-10] [Table 6A-11] [Table 6A-12] [Table 6A-13] [Table 6A-14] [Table 6A-15] [Table 6A-16] [Table 6A-17] @ Ala(1) - CH between the side chain carbon of alanine and the carbon of the vinyl group 2 - does not exist, and there is one -CH between the side chain carbon of Ala(2)-alanine and the carbon of the vinyl group. 2 -, there are two -CH groups between the side chain carbon and the vinyl group carbon of Ala(3)-alanine. 2 -exists.

[0454] In Tables 6B and 6C, for FPN and T47D internalization assays, IC 50 The symbols representing values ​​have the following meaning:****=IC 50 is 1 nM or more and 10 nM or less, ***=IC 50 is greater than 10 nM and less than or equal to 100 nM, **=IC 50is >100nM <500nM, *=>500NM. If not indicated, data was not yet available. [Table 6B-1] [Table 6B-2] [Table 6B-3] [Table 6C]

[0455] Example 2C Peptide analogue activity The potency of peptides in triggering ferroportin internalization was evaluated in 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 potency of test peptides was evaluated in the presence of serum albumin, a major protein component in blood. T47D cells were maintained in RPMI medium (containing the required amount of fetal bovine serum) and subcultured periodically. In preparation for the assay, cells were seeded in 96-well plates at a density of 80-100k cells / well in a volume of 100ul and allowed to sit overnight. The following day, test peptides were first prepared in a dilution series (10-point series, starting concentration approximately 5μM, typically 3-4 fold dilution steps), all prepared in 0.5% mouse serum albumin (MSA purified from mouse serum, Sigma, A3139). The test peptide dilution series was allowed to incubate at room temperature for 30 minutes. The medium was then aspirated from the 96-well cell plates and a test peptide dilution series was added. After 1 hour of incubation, the medium containing the test peptide was aspirated and the AF647-conjugated detection peptide was added at a fixed concentration of 200 nM. The AF647-conjugated detection peptide was previously validated to bind to ferroportin and cause its internalization. In preparation for flow cytometry analysis, the cells were incubated for 2 hours and then washed again. The median fluorescence intensity (MFI) of the AF647-positive population was measured (after removing dead cells and non-singlets from the analysis). The MFI values ​​were used to generate dose-response curves and obtain the IC50 potency of the test peptide. The IC50 potency was calculated using a four-parameter nonlinear fit function in Graphpad Prism (Table 6A).

[0456] Example 2D LAD2 activity of peptide analogues In anaphylactoid reactions, the main mechanism requires direct stimulation of mast cells or basophils, resulting in the release of anaphylactic mediators such as histamine and β-hexosaminidase. A recent study by McNeil et al. (McNeil BD et al., 2015) reported that a specific membrane receptor, MrgprX2, on human mast cells induces anaphylactoid reactions. The LAD2 (Laboratory of Allergic Diseases 2) human mast cell line, derived from human mast cell sarcoma / leukemia (Kirshenbaum et al., 2003), is commonly used to study anaphylactoid reactions because its biological properties are identical to those of primary human mast cells, including overexpression of the MrgprX2 receptor and sensitivity to degranulation peptides (Kulka et al., 2008). The release of anaphylactic mediators such as β-hexosaminidase is assessed by fluorimetry.

[0457] The degranulation potential of hepcidin mimetics was evaluated in LAD2 cells. On the day of the assay, serial dilutions of compounds were added to LAD2 cells seeded at 20,000 cells / well in a 96-well plate. After 30 minutes of incubation, the amount of β-hexosaminidase released in the supernatant and cell lysate was quantified using the fluorescent substrate 4-methylumbelliferyl-N-acetyl-bD-glucosaminide. Dose-response curves were generated by plotting the % β-hexosaminidase release (y-axis) against the concentration of the peptide tested (x-axis). EC 50 Values ​​and standard errors were calculated using XLfit 5.5.0.5 based on the following equation: 4 parameter sigmoidal model: f=(A+((BA) / (1+((C / x)^D)))) where A=Emin, B=Emax, C=EC50, and D=slope. References: McNeil BD et al., Nature, 12, 519 (2015), Kirshenbaum et al. Leukemia Res. 27, 677 (2003), Kulka et al. Immunology 123, 398 (2008).

[0458] Example 3 In vivo validation of peptide analogues Hepcidin analogs of the present invention were tested for in vivo activity to determine their ability to reduce free Fe2+ in serum.

[0459] Hepcidin analogs or vehicle control were administered either intravenously or subcutaneously at 1000 nmol / kg to mice (n=3 / group). Serum samples were collected from the hepcidin analog administered mice groups at 30 min, 1 h, 2 h, 4 h, 10 h, 24 h, 30 h, 36 h, and 48 h after administration. Iron content in plasma / serum was measured using a colorimetric assay in a Cobas c 111 according to the manufacturer's instructions for the assay (assay:IRON2:ACN 661).

[0460] In another experiment, various hepcidin analogs or vehicle control were administered subcutaneously at 1000 nmol / kg to mice (n=3 / group). Serum samples were collected from groups of mice administered vehicle or hepcidin analogs 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).

[0461] These studies demonstrate that the hepcidin analogs of the present invention lower serum iron levels for at least 30 hours, thus demonstrating increased serum stability.

[0462] Example 4 In vitro validation of peptide analogues Based in part on the structure-activity relationships (SAR) determined from the results of the experiments described herein, various hepcidin-like peptides of the invention were synthesized using the methods described in Example 1 and tested for in vitro activity as described in Example 2. Reference compounds included native hepcidin, minihepcidin, R1-minihepcidin, Reference Compound 1, and Reference Compound 2. The IC of the peptides 50 Value or EC 50 The values ​​are summarized in Tables 6A-6C.

[0463] Example 5 plasma stability To complement the in vivo results and aid in the design of potent, stable ferroportin agonists, plasma stability experiments were performed. Ex vivo stability studies were first performed in rat and mouse plasma to predict stability in these matrices.

[0464] Peptides of interest (20 μM) were incubated with pre-warmed plasma (BioreclamationIVT) at 37°C. Aliquots were taken at various time points (e.g., 0, 0.25, 1, 3, 6, and 24 h) up to 24 h and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid with 1 μM internal standard). Quenched samples were stored at 4°C until the end of the experiment and centrifuged at 17,000g for 15 min. Supernatants were diluted 1:1 with deionized water and analyzed using LC-MS. The percentage remaining at each time point was calculated based on the peak area ratio (analyte to internal standard) relative to the initial level at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

[0465] Example 6 Reduction of serum iron in mice A hepcidin mimetic compound designed for oral stability was tested for systemic absorption by PO administration in a wild-type mouse model, C57BL / 6. The animals were acclimated to normal rodent chow for 4-5 days and fasted overnight before the start of the study. Each group of 4 animals was administered either vehicle or compound. Compounds were formulated in saline at a concentration of 5 mg / mL. Mice were administered dosing solutions by oral gavage in a volume of 200 μl per animal weighing 20 g. Each group was administered a single dose of compound at 50 mg / kg / dose. The group marked as vehicle received the formulation only. Blood was collected at 4 hours post-dose and serum was prepared for PK and PD measurements. Compound concentrations were measured by mass spectrometry and iron concentrations in the samples were measured using a colorimetric method on a Roche cobas c system.

[0466] Example 7 Reduction of serum iron in mice In a separate experiment, a new set of compounds was tested for systemic absorption by PO administration in a wild-type mouse model, C57BL / 6. The animals were acclimated 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 maintained on this diet for the remainder of the study. Each group of 5 animals was administered either vehicle or compound. The concentration of the compound was 30 mg / mL and was formulated in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withdrawn approximately 2 hours prior to each dose to ensure that there were no food particles in the stomach prior to PO administration. Mice were administered the dosing solution by oral gavage in a volume of 200 μl per animal weighing 20 g. Each group was administered 2 daily doses of 300 mg / kg / dose of compound. The group marked vehicle received the formulation alone. Blood was collected 4.5 hours after the last dose and serum was prepared for PD measurements. Serum iron concentrations were measured using a colorimetric method on a Roche cobas c system.

[0467] Example 8 Pharmacodynamic effects of representative compounds on serum iron-reducing ability in mice In a second in vivo study, representative compounds were tested for pharmacodynamic effects at a single dose of 300 mg / kg / dose versus two doses of 300 mg / kg QD (once a day) for 2 days. C57BL / 6 mice were acclimated to normal rodent chow for 4-5 days prior to the start of the study. The night before the first dose, mice were switched to a low iron diet (containing 2 ppm iron) and maintained on this diet for the remainder of the study. Groups of 5 animals were dosed with either vehicle or compound. Compounds were formulated in 0.7% NaCl + 10 mM sodium acetate buffer at a concentration of 30 mg / mL. Food was withdrawn approximately 2 hours prior to each dose to ensure that there were no food particles in the stomach prior to PO dosing. Mice were administered dosing solutions by oral gavage in a volume of 200 μl per animal weighing 20 g.

[0468] Example 9 PK / PD Effects of Oral Administration of Representative Compounds of the Invention in Mice In another in vivo study using a healthy wild-type mouse model C57 / BL6, representative compounds were tested for PK and PD effects with multiple doses over a 3-day period. Mice were maintained on normal rodent diet during acclimation and switched to an iron-deficient diet (containing approximately 2 ppm iron) the night before the first dose. Each group of 5 mice was administered either the vehicle or representative compounds of the present invention in a BID format over a 3-day period, for a total of 6 doses at different dose strengths. Mice were administered representative compounds formulated in 0.7% saline and 10 mM sodium acetate by oral gavage. Different groups were administered either vehicle, 150 mg / kg / dose BID, 75 mg / kg / dose BID, 37.5 mg / kg / dose BID, or 18.75 mg / kg / dose BID. Additional groups were administered 100 mg / kg / dose BID in addition to a total of 100 mg / kg / day of compound / drinking water (DW) solution, resulting in a total dose of 300 g / kg / day. Three hours after the last dose, the vehicle group marked as iron loading and all compound-treated groups were administered iron solution by oral gavage at 4 mg / kg iron per animal. 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 samples were measured using colorimetry on a Roche cobas c system.

[0469] Example 10 Reduction of serum iron in mice In a separate triage, a new set of compounds was tested for pharmacodynamic effects when administered orally to a wild-type mouse model, C57BL / 6. The animals were acclimated to normal rodent chow for 4-5 days prior to the start of the study. A group of five animals designated to receive two doses of a representative compound were administered an iron-deficient diet (containing 2 ppm iron) the night before the first dose, while all other groups designated to receive a single dose of a different compound were treated with the iron-deficient diet for two nights prior to compound administration. The concentration of the compound in the dosing solution was 30 mg / mL and was formulated in 0.7% NaCl + 10 mM sodium acetate buffer. Food was withdrawn approximately 2 hours prior to any dose to ensure that there were no food particles in the stomach prior to PO administration. Mice were administered the dosing solution by oral gavage in a volume of 200 μl per animal weighing 20 g. The group marked vehicle received the formulation only. Blood was collected 4.5 hours after the last dose and serum was prepared for PD measurement. Serum iron concentrations were measured using a colorimetric method on a Roche cobas c system.

[0470] Example 11 Stability in simulated gastric fluid Blank SGF was prepared by adding 2 g sodium chloride, 7 mL hydrochloric acid (37%) in a final water volume of 1 L, and adjusting the pH to 1.2.

[0471] SGF was prepared by dissolving 320 mg of pepsin (Sigma®, P6887, from porcine gastric mucosa) in 100 mL of blank SGF and stirred for 30 min at room temperature. The solution was filtered through a 0.45 μm membrane, aliquoted and stored at −20° C.

[0472] Experimental compounds of interest (at a concentration of 20 μM) were incubated with pre-warmed SGF at 37° C. Aliquots were taken at various time points (e.g., 0, 0.25, 1, 3, 6, and 24 h) up to 24 h and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid containing 1 μM internal standard). Quenched samples were stored at 4° C. until the end of the experiment and centrifuged at 4,000 rpm for 10 min. Supernatants were diluted 1:1 with deionized water and analyzed using LC-MS. The percentage remaining at each time point was calculated based on the peak area ratio (analyte to internal standard) relative to the initial level at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

[0473] Example 12 Stability in simulated intestinal fluid Blank FaSSIF was prepared by dissolving 0.348 g NaOH, 3.954 g sodium phosphate monobasic monohydrate, and 6.186 g NaCl in a final water volume of 1 liter (pH was adjusted to 6.5).

[0474] FaSSIF was prepared by dissolving 1.2 g of porcine pancreas (Chem-supply, PL378) in 100 mL of blank FaSSIF and stirred at room temperature for 30 min. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at −20° C.

[0475] Experimental compounds of interest (20 μM) were incubated with pre-warmed FaSSIF (1% pancreatin in the final incubation mixture) at 37° C. Aliquots were taken at various time points (e.g., 0, 0.25, 1, 3, 6, and 24 h time points) up to 24 h and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid with 1 μM internal standard). Quenched samples were stored at 4° C. until the end of the experiment and centrifuged at 4,000 rpm for 10 min. Supernatants were diluted 1:1 with deionized water and analyzed using LC-MS. The percentage remaining at each time point was calculated based on the peak area ratio (analyte to internal standard) relative to the initial level at time zero. Half-lives were calculated by fitting to a first-order exponential decay equation using GraphPad.

[0476] Example 13 Modification experiments of peptides prone to "non-specific binding" Compounds of interest (at a concentration of 20 μM) were mixed with pre-warmed FaSSIF (1% pancreatin in the final working solution). The solution mixture was aliquoted and incubated at 37°C. The number of aliquots required was equal to the number of time points (e.g., 0, 0.25, 1, 3, 6, and 24 hours). 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 1 μM internal standard). The remaining steps were the same as in a typical experiment.

[0477] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this Application Data Sheet are hereby incorporated by reference in their entireties.

[0478] At least some of the chemical names or sequences of the compounds of the 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 that the depicted chemical name or sequence differs from the depicted structure, the depicted structure shall prevail. In chemical structures where a chiral center exists within the structure but no specific stereochemistry is depicted for the chiral center, both enantiomers associated with the chiral structure are encompassed by the structure. Similarly, for peptides where E / Z isomers exist but are not specifically mentioned, both isomers are specifically disclosed and encompassed.

[0479] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the spirit and scope of the invention.

Claims

1. 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 is hydrogen, C １ -C ６ Alkyl, C ６ -C １２ Aryl, C ６ -C １２ Aryl-C １ -C ６ Alkyl, C １ -C ２０ Alkanoyl, or C １ -C ２０ is cycloalkanoyl; R ２ But NH ２ , substituted amino, OH, or substituted hydroxy; X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1, or Tet2, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X2 is Ala, t-BuAla, Thr, substituted Thr, Gly, N-substituted Gly, or Ser; X3 is Ala, t-BuAla, Gly, N-substituted Gly, His, or substituted His; X4 is Ala, t-BuAla, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal; X5 is Ala, t-BuAla, Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X6 is absent or is any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen; X7 is absent or is Ala, t-BuAla, Gly, N-substituted Gly, Ile, Val, Leu, NLeu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X8 is absent or is Ala, t-BuAla, (D)Ala, a-MeAla, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, or 123 triazole; X9 is absent or is Ala, Ile, Gly, N-substituted Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X10 is absent or is Ala, Gly, N-substituted Gly, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X11 is absent or is Ala, Pro, bhPhe, Lys, substituted Lys, or (D)Lys; X12 to X14 are each absent or each independently any amino acid; 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 or may be N-substituted; and iii) at least two of X1 to X14 are independently Ala or aMeAla, and the side chain methyl C of each Ala is C ２ -C １２ Alkanyl or C ２ -C １２ provided that it is cyclized via an alkenyl linker to form a macrocycle; Alkanyl is an alkyl chain, and alkenyl is embedded with at least one double bond. is an alkyl chain, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or b,b-diphenylalanine, bhPhe is b-homophenylalanine, Bip is biphenylalanine, bhPro is b-homoproline, Tic is L-1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotic acid, bhTrp is b-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is ornithine, and Nleu is nor. leucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and the substituted Phe is phenylalanine, where the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted bhPhe is b-homophenylalanine, where the phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine; the substituted Trp is N-methyl-L-tryptophan, a-methyltryptophan, or tryptophan substituted with F, Cl, OH, or t-Bu; the substituted bhTrp is N-methyl-L-b-homotryptophan, a-methyl-b-homotryptophan, or b-homotryptophan substituted with F, Cl, OH, or t-Bu; Tet1 is (S)-(2-amino)-3-(2H-tetrazol-5-yl)propanoic acid and Tet2 is (S)-(2-amino)-4-(1H-tetrazol-5-yl)butanoic acid; 123 Triazole 【Chemistry 30】 and Dab 【Chemistry 31】 or a pharma- ceutically acceptable salt or solvate thereof.

2. X1 and X6, X1 and X7, or X1 and X8 are each Ala, and the side chain methyl C of each Ala is C ２ -C １２ Alkanyl or C ２ -C １２ 2. The hepcidin analog of claim 1, or a pharma- ceutically acceptable salt or solvate thereof, which is cyclized via an alkenyl linker to form a macrocycle.

3. X1 is Glu, Dab, Dap, Orn, Lys, or Tet1; X2 is Thr; X3 is His or 1MeHis; X4 is Dpa, X5 is Ala or Pro; X6 is absent, Ala, Glu, or substituted Lys; X7 is absent or is Ala, Ile, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; X8 is absent or is Ala, Ile, Glu, Asp, 123 triazole, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys; X9 is absent or is bhPhe; X10 is absent or is Ala, Ile, Phe, bhPhe, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; 3. The hepcidin analog of any one of claims 1 to 2, or a pharma- ceutically acceptable salt or solvate thereof, wherein X11 is absent or is Pro, bhPhe, Lys, substituted Lys, or (D)Lys.

4. 4. The hepcidin analog of any one of claims 1 to 3, or a pharma- ceutically acceptable salt or solvate thereof, wherein X1 is GIu, X2 is Thr, X3 is His, X4 is DPA, and X5 is Pro. (a) X7 is Ala, Ile, or a substituted Lys; and / or (b) X8 is Ala, Lys, or (D) Lys; and / or (c) X9 is absent or is bhF, and / or 5. The hepcidin analog of any one of claims 1 to 4, or a pharma- ceutically acceptable salt or solvate thereof, wherein (d) X10 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. (a) the peptide is according to formula II: R １ -Ala’-Thr-His-[Dpa]-Pro-X6-X7-Ala’-X9-X10-X11-X12-X13-X14-R ２ (II) In the formula, R １ , R ２ , X6 to X7, and X9 to X14 are as described in claim 1, Ala' is alanine, and the side chain methyl C of each of Ala' is C ２ -C １２ Alkanyl or C ２ -C １２ cyclized via an alkenyl linker to form a macrocycle; or (b) the peptide is according to formula VI: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala'-X7-Ala'-X9-X10-X11-X12-X13-X14-R 2 (VI) 6. The hepcidin analog of any one of claims 1 to 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein R1, R2, and X8 to X14 are as described in claim 1, Ala' is alanine, and the side chain methyl C of each of Ala' is cyclized via a C2-C12 alkanyl or C2-C12 alkenyl linker to form a macrocycle. (a) X10 is absent, Lys, substituted Lys, (D)Lys, or substituted (D)Lys; or (b) X10 is Lys(Ahx_Palm), or (c) X10 is Lys(L1Z) or (D) Lys(L1Z), where L1 is a linker and Z is a half-life extending moiety; Optionally (i) L1 is Ahx, or (ii) L1 is PEG-Ahx; or (iii) The hepcidin analog of any one of claims 1 to 6, or a pharma- ceutically acceptable salt or solvate thereof, wherein L1 is iso-Glu-PEG-Ahx.

8. 8. The hepcidin analog of claim 7, or a pharma- ceutically acceptable salt or solvate thereof, wherein 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, and n is an integer from 1 to 100, or 10K, 20K, or 30K. (a) Z is Palm; (b) -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 IsoGlu_Palm, -1PEG2_1PEG2_Dap_C18_diacid, -1PEG2_1PEG2_IsoGlu_C10_Diaic Acid, -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, Where: PEG11_OMe is -[C(O)-CH 2 -CH 2 -(OCH 2 CH 2 ) 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)-CH 2 -CH 2 -(OCH 2 CH 2 ) 12 -NH-; Ado is -[C(O)-(CH 2 ) 11 -NH]-; Cn acid is -C(O)(CH 2 ) n-2 -CH 3 and C18 acid is -C(O)-(CH 2 ) 16 -Me; Palm is -C(O)-(CH 2 ) 14 -Me; isoGlu is isoglutamic acid; isoGlu_Palm 【Chemistry 32】 and Ahx is -[C(O)-(CH 2 ) 5 -NH]-; Cn_diacid is -C(O)-(CH 2 ) n-2 -COOH, where n is 10, 12, 14, 16, 18, or 22; or (c) X6 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n _diacid), Lys(1PEG2_1PEG2_IsoGlu_C n _diacid) is 【Chemical 33】 8. The hepcidin analog of claim 7, or a pharma- ceutically acceptable salt or solvate thereof, wherein:

10. (a) R １ is IVA or isovaleric acid, or (b) The hepcidin analog of any one of claims 1 to 9, or a pharma- ceutically acceptable salt or solvate thereof, wherein the peptide is a linear peptide.

11. 1. A hepcidin analog comprising or consisting of a peptide, or a pharma- ceutically acceptable salt or solvate thereof, said peptide comprising: (a) any one of the peptides listed in Table 6A; or (b) any one of the peptides listed in Table 6B or Table 6C; or (c) A hepcidin analog, which is any of the peptides listed in Table 7, or a pharma- ceutically acceptable salt or solvate thereof.

12. A method for treating a pulmonary circulation comprising the steps of: 【Chemistry 53】 R 1 is C 1 -C 20 alkanoyl; R 2 is OH, NH 2 , or phenyl-C 1-8 alkylene-amino; R 5 is H or C 1-6 alkyl; L x is -CH 2 CH=CHCH 2 -, -CH 2 CH=CHCH 2 -, -(CH 2 ) 2 CH=CH(CH 2 ) 2 -, -(CH 2 ) 2 CH=CH(CH 2 ) 2 -, -(CH 2 ) 2 C(=CH 2 )C(=CH 2 )(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 4 -, or -CH 2 C(=CH 2 )C(=CH 2 )CH 2 -, X2 is Thr, (NMe)Thr, or Thr_psi; X3 is His or His_psi; X4 is DIP or DIP_psi; X5 is Pro, X6 is Ala, Sar, Lys (Ahx_Palm), Lys_Ahx_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_Dap_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1P EG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_Dap_C18_diacid, -NHCH 2 CH 2 N + (CH 3 ) 2 -CH 2 C(O)-, or Lys_1PEG2_1PEG2_Ahx_Palm, X7 is Arg, Tba, Tle, Ile, Ala, or Lys (calcine); X8 is Ala, (a-Me)Ala, bhPhe, Lys, or (D)Lys; X9 is Dip, bhF, or NMe_Lys_Ahx_Palm; X10 is Arg, (D)Arg, Lys_Ahx_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1PEG2_1PEG2_Ahx_C18_diacid, dK_betaine, or (D)Lys; X11 is Arg, (D)Arg, or (D)Lys; Optionally (a) X10 is (D)Arg, Lys_Ahx_Palm, Lys_1PEG2_1PEG2_Ahx_C18_diacid, Lys_1PEG2_1PEG2_DMG_N_2ae_C18_diacid, Lys_1PEG2_1PEG2_IsoGlu_Palm, Lys_1PEG2_1PEG2_IsoGlu_C18_diacid, Lys_1PEG2_1PEG2_Ahx_C18_diacid, dK_betaine, or (D)Lys; and / or 2. The hepcidin analog of claim 1, or a pharma- ceutically acceptable salt or solvate thereof, wherein (b) X11 is (D)Arg or (D)Lys.

13. 13. A composition for use in a method of binding to or inducing ferroportin internalization and degradation, said method comprising contacting said ferroportin with at least one said hepcidin analog or a pharma- ceutically acceptable salt or solvate thereof, said composition comprising a hepcidin analog or a pharma- ceutically acceptable salt or solvate thereof according to any one of claims 1 to 12. (a) a method for treating a disorder of iron metabolism in a subject in need thereof, optionally wherein the disorder of iron metabolism is an iron overload disorder; or (b) A method for treating a disease or disorder associated with dysregulated hepcidin signaling in a subject in need thereof, optionally wherein said disease or disorder is hemochromatosis, thalassemia, or polycythemia vera. A composition for use in A composition comprising the hepcidin analog of any one of claims 1 to 12, or a pharma- ceutically acceptable salt or solvate thereof.

15. The composition comprising: (a) provided to said subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, aerosol, sublingual, buccal, parenteral, rectal, vaginal, or topical administration routes; and / or (b) provided to the subject at most twice a day, at most once a day, at most once every two days, at most once a week, or at most once a month; and / or (c) provided to the subject in a dosage of about 1 mg to about 100 mg; The composition of claim 14.

16. 13. A device for delivering the hepcidin analog or a pharma- ceutically acceptable salt or solvate thereof, optionally orally or subcutaneously, to a subject, the device comprising a pharmaceutical composition comprising the hepcidin analog or a pharma- ceutically acceptable salt or solvate thereof according to any one of claims 1 to 12 and a pharma- ceutically acceptable carrier, excipient, or vehicle.

17. 13. A kit comprising a pharmaceutical composition comprising the hepcidin analog of any one of claims 1 to 12, or a pharma- ceutically acceptable salt or solvate thereof, and a pharma- ceutically acceptable carrier, excipient, or vehicle, packaged with a reagent, device, or instructions, or a combination thereof.

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