Hepcidin mimetics for the treatment of hereditary hemochromatosis

Abstract

The present disclosure provides methods for the treatment and / or prevention of iron overload disorders, such as hereditary hemochromatosis.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 210,453, filed June 14, 2021, U.S. Provisional Patent Application No. 63 / 252,001, filed October 4, 2021, and U.S. Provisional Patent Application No. 63 / 349,841, filed June 7, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application has been filed electronically via EFS-Web and contains an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled PRTH_070_02WO_ST25.txt, which was created on Jun. 13, 2022 and is 24 kilobytes in size. The sequence listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates, inter alia, to methods for the treatment and / or prevention of iron overload disorders, such as hereditary hemochromatosis. [Background technology]

[0004] Iron plays a vital role in many cellular and biological activities, from cell division to oxygen transport (see, for example, Casu, C.et al., Blood 2018;131(16):1790-1794). However, excess iron promotes the formation of toxic reactive oxygen species (ROS), which can damage DNA, proteins, and lipid membranes, leading to organ dysfunction or failure. Humans and other vertebrates have evolved regulatory systems to optimize iron absorption and organ distribution. Unfortunately, however, some genetic or acquired disorders of iron homeostasis misregulate iron absorption or distribution, causing organ damage and creating severe infectious or inflammatory conditions with associated morbidity and mortality.

[0005] Hepcidin is a 25-amino acid peptide hormone produced primarily in the liver in proportion to plasma iron concentration and iron stores. Hepcidin is a master regulator of iron homeostasis because it binds to and degrades ferroportin-1, the only known iron exporter expressed on the surface of cells involved in iron absorption, recycling, and storage.

[0006] Given the central role of iron homeostasis in various diseases and disorders, agents capable of regulating iron levels (e.g., in red blood cells and organs) have been developed as therapeutic agents. However, there remains a need for methods of using iron regulators to restore iron homeostasis, for example, for the treatment of hereditary hemochromatosis. The present invention addresses this need. Summary of the Invention

[0007] The present disclosure provides methods, clinically effective doses, and dosing regimens of hepcidin mimetics that restore iron homeostasis in humans, for example, humans with hereditary hemochromatosis. In certain embodiments, the methods modulate pharmacodynamic markers associated with efficacy in treating diseases and disorders associated with dysregulation of iron homeostasis, such as iron overload and disorders, including hereditary hemochromatosis (HH). In certain embodiments, the doses, dosing regimens, and methods disclosed herein are used to treat hereditary hemochromatosis, hereditary hemochromatosis arthropathy, or joint pain associated with hereditary hemochromatosis arthropathy.

[0008] In one aspect, the disclosure provides a method of treating iron overload, e.g., HH, in a human subject, comprising providing an effective amount of a hepcidin mimetic, such as compound 25, to the subject. In certain embodiments, the subject has been diagnosed with hereditary hemochromatosis. In certain embodiments, the subject has undergone or requires phlebotomy treatment prior to the treatment disclosed herein. In certain embodiments, the subject is administered a first effective amount for a first period of time and a second effective amount for a second period of time. In certain embodiments, the subject is administered a third effective amount or a subsequent effective amount for a third or subsequent period of time. In certain embodiments, the dosing frequency during the first period of time and the dosing frequency during the second period of time are the same or different. In certain embodiments, the dosing frequency during the third or subsequent period of time are the same or different from the dosing frequency of the first and second periods of time, respectively, and each other. In certain embodiments, each period independently comprises about 1 week, about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 4 months, about 6 months, or about 1 year. In certain embodiments, the dosage and / or frequency of administration is altered after testing the subject's serum iron and / or TSAT saturation levels after a treatment period to achieve the parameters disclosed herein.

[0009] In a related aspect, the disclosure provides a method of treating hereditary hemochromatosis arthropathy or joint pain associated with hereditary hemochromatosis arthropathy in a human subject, the method comprising administering to the subject an effective amount of a hepcidin mimetic. In certain embodiments, the effective amount comprises a dose ranging from about 5 mg to about 40 mg, and optionally, the subject is administered different doses during different time periods over the course of treatment. In some embodiments, the hepcidin mimetic is compound 25. In certain embodiments, the effective dose is a dose that results in a reduction in the TSAT% of the treated patient of 45% or less, or 40% or less.

[0010] In certain embodiments of the methods disclosed herein, the effective amount of the hepcidin mimetic is about 10 mg to about 40 mg during at least a portion of the course of treatment. In certain embodiments, the effective amount of the hepcidin mimetic is administered to the subject about once per week or about twice per week during at least a portion of the course of treatment. In some embodiments, the subject is administered about 10 mg to about 15 mg, about 15 mg to about 20 mg, or about 10 mg to about 20 mg of the hepcidin mimetic about twice per week during at least a portion of the course of treatment, and in other embodiments, the subject is administered about 20 mg to about 30 mg, about 20 mg to about 40 mg, or about 30 mg to about 40 mg of the hepcidin mimetic about once per week during at least a portion of the course of treatment. In certain embodiments, a subject is administered about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, or about 25 mg of a hepcidin mimetic about twice per week for at least a portion of the course of treatment. In certain embodiments, a subject is administered about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, or about 45 mg of a hepcidin mimetic about twice a week for at least a portion of the course of treatment. In certain embodiments, the hepcidin mimetic is administered subcutaneously.

[0011] In some embodiments of any of the methods disclosed herein, the method results in the subject's TSAT level being reduced to less than about 50% TSAT(%), or less than about 45% TSAT(%), or less than about 40% TSAT(%), or less than 35% TSAT, or less than 30% TSAT. In some embodiments, the method results in the subject's serum iron level being reduced to less than about 150ug / dL. In some embodiments, the method results in the subject's liver iron concentration being substantially maintained, e.g., not significantly changed. In some embodiments, the method results in the subject's serum ferritin and serum transferrin levels or concentrations being substantially maintained, e.g., not significantly changed. In some embodiments, the method results in the subject having improved psychological and / or physical outcomes. In certain embodiments, the subject underwent phlebotomy for at least 6 months prior to treatment, optionally with a phlebotomy frequency of 0.25 to 1 phlebotomy per month, and in certain embodiments, during treatment, the subject required substantially less phlebotomy or no phlebotomy, optionally with a phlebotomy frequency of less than 0.1, less than 0.05, or no phlebotomy per month. In certain embodiments of the disclosed methods, the subject requires reduced or no phlebotomy treatment during the course of the treatment methods disclosed herein. For example, a subject undergoing a course of treatment disclosed herein may not require phlebotomy per month, or may require no more than 1 phlebotomy per month, over the course of treatment disclosed herein. In certain embodiments, a subject undergoing a course of treatment disclosed herein may require less phlebotomy than he was undergoing prior to treatment with a hepcidin mimetic according to the present disclosure. In certain embodiments, treatment according to the present disclosure results in at least a 25%, at least a 50%, or at least a 75% reduction in the number of phlebotomies per month. In certain embodiments, the course of treatment disclosed herein may be for at least 12 weeks, at least 24 weeks, at least 6 months, at least 1 year, at least 2 years, at least 5 years, or longer.

[0012] In some embodiments of any of the methods disclosed herein, the hepcidin mimetic comprises a peptide of any one of Formulas I-VIII disclosed herein, in certain embodiments, the peptide comprises or consists of one of the following sequences or structures: Isovaleric acid-DTHFPICIFGPRSKGWVC-NH 2 (Compound 1, SEQ ID NO:1), Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH 2 (Compound 2, SEQ ID NO:2), Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH 2 (Compound 3, SEQ ID NO:3), Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH 2 (Compound 4, SEQ ID NO: 4), Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH 2 (Compound 5, SEQ ID NO:5), Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH 2 (Compound 6, SEQ ID NO:6), Isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH 2 (Compound 7, SEQ ID NO: 7), Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH 2 (Compound 8, SEQ ID NO: 8), Isovaleric acid-DTHFPICIFGPRS(K(PEG8))GWVC-NH 2 (Compound 9, SEQ ID NO: 9), Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH 2 (Compound 10, SEQ ID NO: 10), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH 2 (Compound 11, SEQ ID NO: 11), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG4))-NH 2 (Compound 12, SEQ ID NO: 12), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH 2 (Compound 13, SEQ ID NO: 13), Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH 2 (Compound 14, SEQ ID NO: 14), Isovaleric acid-DTHFPCIKF)K(Palm))PRSKGWVCK-NH 2 (Compound 15, SEQ ID NO: 15), Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH 2 (Compound 16, SEQ ID NO: 16), Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH 2 (Compound 17, SEQ ID NO: 17), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))NH 2 (Compound 18, SEQ ID NO: 18), Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH 2 (Compound 19, SEQ ID NO: 19), Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH 2 (Compound 20, SEQ ID NO: 20), Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH 2 (Compound 21, SEQ ID NO: 21), Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH 2 (Compound 22, SEQ ID NO: 22), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH 2 (Compound 23, SEQ ID NO: 23), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH 2 (Compound 24, SEQ ID NO:24), Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH 2(Compound 25, SEQ ID NO:25), Isovaleric acid-DTHFPCIKF-K(isoGlu-Palm)-PRSKGCK-NH 2 (Compound 26, SEQ ID NO:26), Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH 2 (Compound 27, SEQ ID NO:27), Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGWECK-NH 2 (Compound 28, SEQ ID NO:28) 、 Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH 2 (Compound 29, SEQ ID NO: 29), Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))CK-NH 2 (Compound 30, SEQ ID NO: 30), Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH 2 (Compound 31, SEQ ID NO: 31), Isovaleric acid-DTHFPCI-K(Dapa-Palm)-FEPRSKGCK-NH 2 (Compound 32, SEQ ID NO: 32), Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGCK-NH 2 (Compound 33, SEQ ID NO: 33), Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH 2 (Compound 34, SEQ ID NO: 34), Isovaleric acid-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH 2 (Compound 35, SEQ ID NO: 35), Isovaleric acid-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH 2 (Compound 36, SEQ ID NO: 36), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))K-NH 2(Compound 37, SEQ ID NO: 37), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))-NH 2 (Compound 38, SEQ ID NO: 38), Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH 2 (Compound 39, SEQ ID NO: 39), Isovaleric acid-DTHFPCIKF-NH 2 (Compound 40, SEQ ID NO: 40), Hy-DTHFPCIKF-NH 2 (Compound 41, SEQ ID NO: 41), Isovaleric acid-DTHFPCIIF-NH 2 (Compound 42, SEQ ID NO: 42), Hy-DTHFPCIIKF-NH 2 (Compound 43, SEQ ID NO: 43), Isovaleric acid-DTKFPCIIF-NH 2 (Compound 44, SEQ ID NO: 44), or Hy-DTKFPCIIF-NH 2 (Compound 45, SEQ ID NO:45).

[0013] In certain embodiments of any of the methods disclosed herein, the reduction in TSAT% levels and / or serum iron levels is a maximum reduction following treatment with the agent, while in other embodiments, the reduction in TSAT% levels and / or serum iron levels is a reduction observed at the trough level of the agent following administration to a subject. In some embodiments, the TSAT% is reduced to about 45% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 10% to about 40%, about 5% to about 20%, or about 10% to about 20% at the time of testing. In some embodiments, the serum iron is reduced to about 150 micrograms / dL or less, about 100 micrograms / dL or less, about 75 micrograms / dL or less, or about 600 micrograms / dL or less at the time of testing. In some embodiments, the TSAT% level and / or serum iron level is reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the levels observed in normal healthy volunteers.

[0014] In various embodiments of any of the methods disclosed herein, an agent, such as a hepcidin mimetic peptide, such as compound 25, is provided to a subject as a salt form and / or in a pharmaceutical composition, and in certain embodiments, is provided parenterally, e.g., subcutaneously. [Brief description of the drawings]

[0015] [Figure 1] 1 provides graphs showing serum iron levels, serum ferritin levels, and total iron in various tissues following treatment of HH mice with 5 mg / kg of Compound 25. [Figure 2A-B] 2A-2B are graphs showing non-heme iron levels in the liver of HH mice after treatment with compound 25 following a low iron diet (FIG. 2A) or a normal iron diet (FIG. 2B). [Diagram 3] An overview of the Phase 2 study design for the treatment of hereditary hemochromatosis (HH) with a hepcidin mimetic is presented. [Figure 4A-B]Figure 4 shows the effect of treatment with Compound 25 on phlebotomy frequency. Figure 4A is a chart showing phlebotomy treatment of HH patients before and after treatment with hepcidin mimetics. Figure 4B is a graph showing the reduction in phlebotomy treatment after treatment with Compound 25. [Figure 5A-B] 5A provides graphs showing TSAT levels in HH patients over time after treatment with hepcidin mimetics. FIG. 5A shows TSAT levels over the course of treatment for an individual patient. For FIG. 5B, in the left graph, baseline transferrin saturation (%) is 45.0000 and after compound 25 is 30.3948. In the right graph, baseline transferrin saturation (%) is 45.0000 and after compound 25 is 36.2500. [Figure 6] Graphs are provided showing the mean serum iron levels in HH patients before treatment and after treatment with hepcidin mimetics. In the graph on the right, baseline is 25.5454 and after compound 25 is 17.6591. [Figure 7] 1 provides graphs showing dose- and concentration-dependent reduction in serum iron and TSAT following treatment with hepcidin mimetics. [Figure 8] 1 provides a graph showing serum ferritin and serum transferrin levels in HH patients before and after treatment with hepcidin mimetics.In the top left graph, baseline is 82.3431, and after compound 25 it is 85.0126.In the bottom left graph, baseline is 82.3431, and after compound 25 it is 114.4775.In the top right graph, baseline is 2.2869, and after compound 25 it is 2.4531.In the bottom right graph, baseline is 2.2869, and after compound 25 it is 2.4169. [Figure 9] 1 provides a graph showing hepatic iron content in HH patients before and after treatment with a hepcidin mimetic. [Figure 10]Summary of HH patient-reported outcomes following treatment with a hepcidin mimetic. The innermost scores for Role Daily Functioning (Mental) and Role Daily Functioning (Physical) correspond to screening, and the outermost scores for Role Daily Functioning (Mental) and Role Daily Functioning (Physical) correspond to post-compound 25 treatment. [Figure 11] 1 is a diagram showing the dose of Compound 25 administered to each subject. [Figure 12] 1 is a graph showing % transferrin saturation over a 6-day period following administration of the indicated doses of Compound 25. [Figure 13] Included are graphs showing TST (%) and MCHC (g / dL) at baseline and after 24 weeks of administration of Compound 25 to HH patients, and at baseline and after 4 and 8 weeks in β-thalassemia transfusion-dependent secondary iron overload patients. [Figure 14] 1 includes graphs showing results in HH patients after 6 months of treatment with Compound 25. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present disclosure identifies therapeutically effective dosages and dosing regimens of hepcidin mimetics useful for treating diseases and disorders associated with iron dysregulation, such as hereditary hemochromatosis (HH). In certain embodiments, the methods disclosed herein are practiced using compound 25 to treat hereditary hemochromatosis.

[0017] 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.

[0018] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0019] 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).

[0020] The singular forms "a," "an," and "the" include plurals unless the context clearly dictates otherwise.

[0021] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0022] 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.

[0023] 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 the polypeptide is produced using recombinant technology, chemical synthesis, enzymatic synthesis, or occurs naturally.

[0024] The term "hepcidin mimic" 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 its functional regions. In certain embodiments, hepcidin mimics 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, hepcidin mimics include one or more additional modifications, such as, for example, conjugation to another compound. Any peptide monomer or peptide dimer disclosed herein is encompassed by the term "hepcidin mimic". In some embodiments, hepcidin mimics have one or more functional activities of hepcidin.

[0025] 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 those found in nature, such as the 23 amino acids that combine 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. "Non-standard" natural amino acids are pyrrolysine (found in methanogens and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "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. Over 140 naturally occurring amino acids are known, with thousands more combinations possible. 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.

[0026] 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 an "-OH" or "-NH" moiety at the C-terminus of the sequence refers to a hydrogen atom corresponding to the presence of a free primary or secondary amino group at the N-terminus. 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, particularly in situations where the amino or carboxy terminus is attached to a linker or another chemical moiety, e.g., a PEG moiety.

[0027] "NH 2 The term "" as used herein 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.

[0028] The term "carboxy" as used herein means -CO 2 Point to H.

[0029] 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 as set forth in "Nomenclature of α-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues 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 below in Table 1, below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0030] 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 abbreviations 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.), they are represented by their conventional three-letter abbreviations or single-letter abbreviations (e.g., 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).

[0031] Additionally, R1 may be substituted with isovaleric acid or equivalent in all sequences. In some embodiments where the peptides of the present invention are conjugated to acidic compounds, such as isovaleric acid, isobutyric acid, valeric acid, etc., the presence of such conjugation is referred to in acid form. Thus, for example, but in no way limiting, instead of indicating the conjugation of isovaleric acid to a peptide by referring to isovaleroyl, in some embodiments, the present application may refer to such conjugation as isovaleric acid.

[0032] The term "L-amino acid" as used herein 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 any L-amino acid residue can be substituted for the "D" isomeric form of the residue, so long as the desired functional group is retained by the peptide.

[0033] 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 designated 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).

[0034] The term "dimer" as used herein broadly refers to a peptide comprising two or more monomeric subunits. A particular dimer comprises 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).

[0035] As used herein, in the context of a particular peptide sequence disclosed herein, parentheses, e.g., (__), represent a side chain conjugation, and square brackets, e.g., [__], represent a non-natural amino acid substitution or an amino acid and a conjugated side chain. In general, when a linker is shown at the N-terminus of a peptide sequence, it indicates that the peptide is dimerized with another peptide and the linker is attached to the N-terminus of the two peptides. In general, when a linker is shown at the C-terminus of a peptide sequence or structure, it indicates that the peptide is dimerized with another peptide and the linker is attached to the C-terminus of the two peptides.

[0036] The term "cyclization," as used herein, refers to a reaction in which a portion of a polypeptide molecule becomes linked to another portion of the polypeptide molecule to form a closed ring, e.g., by forming a disulfide bridge or other similar bond.

[0037] The term "subunit," as used herein, refers to one of a pair of polypeptide monomers that combine to form a dimeric peptide composition.

[0038] 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.

[0039] 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.

[0040] The term "pharmaceutically acceptable salts" as used herein refers to salts or zwitterionic forms of the peptides or compounds of the invention that are water or oil soluble or dispersible, 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 chlorides, bromides, and iodides; dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl bromide 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)+ (wherein 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 possible relevant C2-6 alkenyl groups include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharma- ceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J.Pharm.Sci. 66:2 (1977). Also, for a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Other suitable base salts are formed from bases 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 hemisulfate and hemicalcium salts.

[0041] The term "alkyl" includes saturated aliphatic hydrocarbons, straight or branched, non-cyclic or cyclic, containing 1 to 24 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, and saturated branched alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, and unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

[0042] 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 achieves a desired benefit / risk ratio applicable to any medical treatment.

[0043] Methods of Use of Hepcidin Mimetics Hepcidin targets the major iron transporter ferroportin, causing its internalization and subsequent degradation. Hepcidin regulation is important for providing sufficient iron for cellular function as well as preventing iron toxicity. In hereditary hemochromatosis (HH), excessive absorption of iron from the diet leads to primary iron overload. Under these iron overload conditions where transferrin is saturated (e.g., Transferrin Saturation (TSAT)%>80%), excess iron deposition can lead to organ damage. Furthermore, the presence of labile iron increases overall systemic iron toxicity. The present disclosure identifies methods of administering and using therapeutic hepcidin mimetics for the treatment of diseases and disorders associated with iron overload, such as hereditary hemochromatosis.

[0044] In some embodiments, the methods disclosed herein are applied to the prevention, inhibition, or treatment of a disease or disorder associated with dysregulated iron levels (e.g., a disease or disorder of iron metabolism, a disease or disorder associated with iron overload, and a disease or disorder associated with abnormal hepcidin activity or expression). In certain embodiments, the disease or disorder is a disease of iron metabolism, such as iron overload or another disorder of iron metabolism.

[0045] In certain embodiments, the disease of iron metabolism is hemochromatosis, such as hereditary hemochromatosis, HFE mutant hemochromatosis, ferroportin mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojuvelin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, or neonatal hemochromatosis. In certain embodiments, the disease is hereditary hemochromatosis (HH). In certain embodiments, the disease is HH requiring phlebotomy, such as HH in the maintenance stage.

[0046] In certain embodiments, the disease or disorder is HH associated arthropathy, or HH-associated arthropathy. Chronic arthropathy occurs in 37-80% of HH patients. In these cases, joint pain may be an early sign of the disease and is often the cause of the initial diagnosis of HH. This may be done by X-ray and / or MRI imaging, often combined with the results of a validated joint pain and / or function scoring instrument. In certain embodiments, arthropathy may be associated with age, elevated ferritin, and TSAT levels. Iron accumulation in HH may be associated with increased oxidative stress, disturbed matrix metabolism, and cartilage degeneration, which may contribute to the development of arthropathy similar to osteoarthritis. Persistent arthropathy may reduce quality of life and result in high healthcare utilization and associated costs, especially since up to 16% of HH patients undergo joint replacement surgery.For example, see Whalen, N. “Association of Transferrin Saturation with the Arthropathy of Hereditary Hemochromatosis” 2017; Nguyen, C. “Bone and joint complications in patients with hereditary hemochromatosis: a cross-sectional study of 93 patients” 2020; Carroll, GJ. “Hereditary Hemochromatosis is characterized by a clinically definable arthropathy that correlate with iron load” 2011; Karim, A. “The role of disrupted iron homeostasis in the development and progression of arthropathy” 2022; and Burton, LH. “Systemic administration of a pharmacologic iron chelator reduces cartilage lesion development in the Dunkin-Hartley model of primary osteoarthritis” 2022.

[0047] In one aspect, the present disclosure provides a method of treating iron overload, e.g., HH, HH associated with arthropathy, or HH associated arthropathy, in a human subject, the method comprising providing to the subject an effective amount of a hepcidin mimetic, including but not limited to those disclosed herein, such as compound 25. In certain embodiments, the hepcidin mimetic is provided subcutaneously. In certain embodiments, the subject has been diagnosed with hereditary hemochromatosis. In certain embodiments, the subject has undergone or requires phlebotomy treatment prior to the treatment disclosed herein. In certain embodiments, prior to treatment with a hepcidin mimetic according to the disclosed methods, the subject has undergone at least 3, at least 4, at least 5, or at least 6 phlebotomy treatments per year. In certain embodiments, the subject has undergone about 5 to about 6 phlebotomies per year. In certain embodiments, the subject's HH is in a maintenance phase, and the subject is treated with phlebotomy less than once per week, e.g., about once per month, or about once every 2 to 4 months. In certain embodiments, the subject has confirmed HH and has undergone stable phlebotomy for at least 3 months or at least 6 months prior to treatment according to the present disclosure, with a frequency of about 0.25 to 1 phlebotomy per month. In certain embodiments, the subject has no clinically significant laboratory abnormalities and / or has not undergone iron chelation therapy or erythrocytapheresis. In certain embodiments, the patient has difficulty with phlebotomy due to venous access problems, for example, due to needle sticks accumulated over time from multiple phlebotomies. In certain embodiments, the subject has iron deficiency anemia, and in certain embodiments, the subject has needle phobia.

[0048] In certain embodiments, the effective amount of the hepcidin mimetic, e.g., compound 25, is about 1 mg to about 100 mg, or about 10 mg to about 80 mg. In certain embodiments, the effective amount of the hepcidin mimetic, e.g., compound 25, is about 5 mg to about 80 mg, or about 10 mg to about 80 mg. In certain embodiments, the effective amount of the hepcidin mimetic, e.g., compound 25, is about 10 mg to about 40 mg, or about 5 mg to about 50 mg. In certain embodiments, the effective amount of the hepcidin mimetic, e.g., compound 25, is about 10 mg to about 40 mg, or about 5 mg to about 50 mg. In certain embodiments, the effective amount of the hepcidin mimetic is administered to the subject about once per week or about twice per week. In some embodiments, the subject is administered about 10 mg to about 20 mg, about 10 mg to about 15 mg, or about 15 mg to about 20 mg of the hepcidin mimetic about twice per week, while in other embodiments, the subject is administered about 20 mg to about 30 mg, about 20 mg to about 40 mg, or about 30 mg to about 40 mg of the hepcidin mimetic about once per week. In certain embodiments, the subject is administered about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, or about 25 mg of the hepcidin mimetic about twice per week. In certain embodiments, a subject is administered about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, or about 45 mg of a hepcidin mimetic about twice a week. In certain embodiments, the hepcidin mimetic is administered subcutaneously. In certain embodiments, the hepcidin mimetic is compound 25. In certain embodiments, the amount administered to a subject may vary over the course of treatment.

[0049] In embodiments, the method reduces the transferrin saturation (TSAT) level and / or serum iron level and / or mean corpuscular hemoglobin concentration (MCHC) level in the subject by at least 30%, at least 40%, or at least 60%. In some embodiments, the effective amount reduces the transferrin saturation (TSAT) level and / or serum iron in the subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the TSAT% and / or serum iron level and / or MCHC level is reduced by about 40% to about 90%, about 50% to about 90%, or 50% to 75%. In some embodiments, the TSAT% level and / or serum iron level and / or MCHC level is reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the level observed in normal healthy volunteers of the same mammalian type. In certain embodiments, the reduction in the TSAT% level and / or serum iron level and / or MCHC level is the maximum reduction observed in the subject after treatment with the drug, and in other embodiments, the reduction in the TSAT% level and / or serum iron level is the reduction observed at the trough level of the drug after administration to the subject. In some embodiments, the reduction in the TSAT% level and / or serum iron level and / or MCHC level is the average reduction observed, for example, in multiple subjects.

[0050] In some embodiments, the subject's TSAT level is reduced to 60% or less TSAT, 50% or less TSAT, 45% or less TSAT, 40% or less TSAT, 30% or less TSAT, 20% or less TSAT, or 10% or less TSAT. In certain embodiments, the subject's TSAT level is reduced to 45% or less TSAT, or 40% or less TSAT. In certain embodiments, the subject's TSAT level is reduced to 40% or less TSAT, or 35% or less TSAT. In certain embodiments, the TSAT level is reduced to about 20% TSAT to about 50% TSAT, for example, about 25% TSAT to about 40% TSAT. In certain embodiments, the TSAT% and / or serum iron level is reduced by at least 10%, at least 20%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 90%. In certain embodiments, the TSAT% value is reduced to about 0% to about 60%, about 0% to about 40%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 60%, about 20% to about 40%, or about 20% to about 30%. In some embodiments, the TSAT% is reduced to 50% or less TSAT, 45% or less TSAT, 40% or less TSAT, 35% or less TSAT, 30% or less TSAT, 20% or less TSAT, 15% or less TSAT, 10% or less TSAT, about 10% TSAT to about 45% TSAT, about 5% TSAT to about 20% TSAT, or about 10% TSAT to about 20% TSAT at the time of testing. In some embodiments, the TSAT% level and / or serum iron level is reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the level observed in normal, healthy volunteers of the same mammal type.

[0051] In certain embodiments, the serum iron level is reduced to about 0 uM to about 30 uM, about 0 uM to about 5 uM, about 0 uM to about 20 uM, about 0 uM to about 15 uM, about 0 uM to about 10 uM, or about 0 uM to about 5 uM. In certain embodiments, the subject's serum iron level is reduced to 20 umol / L or less, 18 umol / L or less, 16 umol / L or less, 14 umol / L or less, 12 umol / L or less, 10 umol / L or less, 8 umol / L or less, 6 umol / L or less, or 4 umol / L or less. In some embodiments, the subject's serum iron level is reduced to about 2 umol / L to about 100 umol / L, for example, 2 to about 10 umol / L, or about 10 umol / L to about 50 umol / L, or about 10 umol / L to about 30 umol / L. In some embodiments, the subject's serum iron level is reduced to about 2 umol / L to about 100 umol / L, e.g., 2 to about 10 umol / L, or about 10 umol / L to about 50 umol / L, or about 10 umol / L to about 30 umol / L. In some embodiments, the subject's serum iron level is reduced to about 50 mcg / dL to about 200 mcg / dL, or about 60 mcg / dL to about 170 mcg / dL. In certain embodiments, the serum iron level is reduced to less than about 150 ug / dL, less than about 100 ug / dL, or less than about 75 ug / dL.

[0052] In some embodiments, the subject's MCHC level is reduced by at least 5%, at least 10%, at least 20%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 90%. In certain embodiments, the MCHC value (g / dL) is reduced to less than about 35, less than about 34, less than about 33, less than about 32, less than about 31, less than about 30, or less than about 29.

[0053] In embodiments, the method reduces the transferrin saturation (TSAT) level and / or serum iron and / or MCHC level in the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 60%. In some embodiments, the effective amount reduces the transferrin saturation (TSAT) level and / or serum iron and / or MCHC level in the subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the TSAT% level and / or serum iron level and / or MCHC level is reduced by about 40% to about 90%, about 50% to about 90%, or 50% to 75%. In some embodiments, the TSAT% level and / or serum iron level and / or MCHC level is reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the level observed in normal healthy volunteers of the same mammal type. In certain embodiments, the reduction in TSAT% levels and / or serum iron levels and / or MCHC levels is the maximum reduction observed in a subject following treatment with the agent, while in other embodiments, the reduction in TSAT% levels and / or serum iron levels and / or MCHC levels is the reduction observed in trough levels of the agent following administration to a subject. In some embodiments, the reduction in TSAT% levels and / or serum iron levels and / or MCHC levels is the average reduction observed, e.g., in multiple subjects.

[0054] In some embodiments, the TSAT% level and / or serum iron level and / or MCHC level are reduced to levels less than 200%, less than 150%, less than 100T, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of those observed in normal, healthy volunteers of the same mammal type.

[0055] In certain embodiments, after treatment with a hepcidin mimetic, such as compound 25, HH patients exhibit TSAT levels of 45% or less or 40% or less, serum iron levels of less than 150 ug / dL, less than 100 ug / dL, or less than 75 ug / dL, and / or MCHC levels of less than 35 g / dL or less than 30 g / dL. In certain embodiments, these levels occur at trough concentrations of the hepcidin mimetic, for example, about 7 days after the last dose.

[0056] In certain embodiments, the reduction in the subject's transferrin saturation (TSAT) level and / or serum iron level and / or MCHC level occurs for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week after administration of the hepcidin mimetic. In certain embodiments, the subject is provided with the hepcidin mimetic, for example, about twice per week or about once per week, as a single dose or over a period of time. In certain embodiments, the reduction in the subject's transferrin saturation (TSAT) level and / or serum iron level and / or MCHC level is at least 50%, or at least 60%, or at least 80% for at least 1 day, at least 2 days, at least 3 days, or at least 4 days after administration. In certain embodiments, the reduction in TSAT and / or serum iron levels and / or MCHC is maintained over the course of treatment, which may be, for example, once per week or twice per week for at least 2 months, at least 4 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years or more. In certain embodiments, the dosing regimen for the subject may vary throughout the course of treatment, but in certain embodiments, administration of the hepcidin mimetic (e.g., compound 25) occurs about once per week or about twice per week over the course of treatment, with dosages ranging from about 5 mg to about 40 mg, or from about 10 mg to about 40 mg per dose. In certain embodiments, the subject is administered different dosages (at the same frequency or different frequencies), or the same dosage at different frequencies, during different periods of the course of treatment, but in certain embodiments, the frequency throughout the course of treatment is about once or twice per week, with each dosage administered being within the range of about 5 mg to about 40 mg. In some embodiments, the agent is a hepcidin mimetic peptide disclosed herein, such as a peptide of any one of formulas I-VIII or any of compounds 1-34 (e.g., compound 25). In certain embodiments, the reduction in the subject's transferrin saturation (TSAT) level and / or serum iron level is at least 60% for at least one day.

[0057] In certain embodiments, a subject requires fewer phlebotomies following treatment with a hepcidin mimetic according to the disclosed methods, such as compound 25. In some embodiments, a subject requires less than 0.1 phlebotomies per month, or less than 0.05 phlebotomies per month during treatment according to the present disclosure.

[0058] In one embodiment, the method includes treating a subject with HH, where prior to treatment, the subject is at a stable phlebotomy frequency of 0.25 to 1.0 times per month for at least 6 months by administering to the subject an effective amount of a hepcidin mimetic, such as compound 25. In certain embodiments, the subject is administered about 5 to about 20 mg (e.g., about 5 mg, about 10 mg, about 15 mg, or about 20 mg) of a hepcidin mimetic twice per week and / or about 10 to about 40 mg (e.g., about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, or about 40 mg) of a hepcidin mimetic once per week. In certain embodiments, the subject is administered different dosages at different frequencies for different periods of time. In certain embodiments, the subject is administered the hepcidin mimetic for at least 1 month, at least 2 months, at least 4 months, at least 6 months, or at least 1 year. In certain embodiments, the subject's phlebotomy frequency is reduced to less than 0.1 phlebotomy per month, or less than 0.05 phlebotomy per month over the course of treatment.

[0059] In certain embodiments, the subject has improved arthropathy following treatment, as determined, for example, by methods available in the art, such as, for example, by x-ray, MRI, joint pain, and / or the use of function scoring instruments.

[0060] In certain embodiments, the subject has reduced oxidative stress, reduced disturbed matrix metabolism, and / or reduced cartilage degeneration following treatment.

[0061] In certain embodiments, the methods disclosed herein result in a subject having reduced circulating transferrin saturation (TSAT) and / or reduced toxic non-transferrin bound iron (NTBI), and / or reduced iron accumulation in organs such as the liver, pancreas, heart and bone.

[0062] If untreated, iron overload can lead to hepatomegaly, diabetes, skin hyperpigmentation, cardiomyopathy, diastolic dysfunction, heart failure, cirrhosis, etc. In certain embodiments, the methods of treatment disclosed herein reduce, alleviate, or ameliorate any of these symptoms or conditions associated with iron overload, such as HH.

[0063] In certain embodiments, the method further comprises determining the TSAT level and / or serum iron level and / or MCHC level in the subject before and / or after treatment with a hepcidin mimetic. In certain embodiments, the subject has a reduction in TSAT and / or serum iron and / or MCHC of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 80% and is provided with the agent. In certain embodiments, the method comprises measuring the TSAT% level in the subject before and / or after providing the agent to the subject, and in some embodiments, the method comprises providing an additional agent to the subject to achieve or maintain a reduced TSAT% level, e.g., 45% TSAT or less or 40% TSAT or less. In certain embodiments, the agent is any of those disclosed herein, e.g., a hepcidin mimetic peptide, such as a peptide of any one of formulas I-VIII or any of compounds 1-34 (e.g., compound 25).

[0064] In some embodiments, the method includes determining the TSAT% and / or serum iron level and / or MCHC level in a subject before and after administering a hepcidin mimetic to the subject, and then determining whether the subject's TSAT% and / or serum iron level and / or MCHC level is reduced to a desired level after administration of the hepcidin mimetic. In some embodiments, the subject is given increasing amounts of the hepcidin mimetic until the TSAT% and / or serum iron level and / or MCHC level is reduced to a desired level. In some embodiments, the subject is given multiple different doses of the hepcidin mimetic over the course of treatment (and potentially at different frequencies, e.g., once a week and / or twice a week), and the subject's TSAT% and / or serum iron level and / or MCHC level are monitored at various times during the course of treatment to identify the minimum or appropriate dose and / or concentration required to reduce the TSAT% and / or serum iron level to a desired level, and / or to identify how frequently the subject should be treated to maintain the subject's TSAT% and / or serum iron level and / or MCHC level at a desired level. In certain embodiments, the method includes measuring the TSAT% level in the subject before and / or after administering the hepcidin mimetic to the subject, and in some embodiments, the method includes providing additional hepcidin mimetic to the subject to achieve or maintain a reduced TSAT% level, e.g., a TSAT of 45% or less. In certain embodiments, the hepcidin mimetic is a peptide of any one of formulas I-VIII or any of compounds 1-34 (e.g., compound 25).

[0065] In certain embodiments, the present disclosure provides a method of treating HH, HH associated with arthropathy, or HH associated arthropathy, the method comprising: a) administering subcutaneously to a subject diagnosed with HH, e.g., phlebotomy-dependent HH, HH associated with arthropathy, or HH-associated arthropathy, about 5 to about 25 mg (optionally 10 mg or 20 mg) of a hepcidin mimetic disclosed herein, e.g., Compound 25; b) optionally determining the TSAT% of the subject after step a), e.g., about 7 days after step a), at a trough drug level; c) If the subject's TSAT% is greater than about 40% or greater than about 45%, (i) administering to a subject an increasing amount of a hepcidin mimetic, e.g., about 20 mg to about 80 mg, optionally about 20 mg or about 40 mg, subcutaneously on a schedule of about once a week or about twice a week; or (ii) administering to the subject the same or an increased amount of a hepcidin mimetic, e.g., about 10 mg or about 20 mg or about 40 mg, subcutaneously on an increased dosing schedule, e.g., about twice a week. In certain embodiments, the method includes monitoring the subject's TSAT% level, for example about 7 days after the first weekly dose, and adjusting dosing by increasing the dose or increasing the frequency of dosing if the TSAT% is greater than 40% or greater than 45%. The method may further include determining and / or monitoring the subject's serum iron and / or MCHC levels and adjusting the amount or frequency of dosing based on either or both.

[0066] In various embodiments of any of the methods disclosed herein, the TSAT% level and / or serum iron level and / or MCHC level is reduced by a percentage or is reduced below a particular TSAT% level or serum iron level and / or MCHC level, e.g., to be associated with a therapeutically effective agent or dosing regimen. In certain embodiments, the reduction in the TSAT% level and / or serum iron level and / or MCHC level is maintained for a period of time, e.g., 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. As can be appreciated, the percentage reduction in any of these levels can be a percentage reduction in a particular patient, for example, where TSAT% levels and / or serum iron levels and / or MCHC levels are monitored to determine dosing or dosing regimens for the patient, or the percentage reduction in TSAT% levels and / or serum iron levels and / or MCHC levels can be a reduction compared to a predetermined value (e.g., an average or mean TSAT% level, or serum iron level, or MCHC level associated with a particular patient population). In certain embodiments, the reduction in TSAT% is a reduction compared to normal healthy volunteers.

[0067] Serum iron can be measured by a variety of methods, including colorimetric assays. TSAT represents the percentage of transferrin iron-binding capacity that is actually accounted for by iron in serum. It is calculated by multiplying serum iron by 100 and dividing by total iron-binding capacity (Coyne. Kidney International 69:54-58).

[0068] Hepcidin Mimetics The methods disclosed herein can be implemented with a variety of hepcidin mimetics, including but not limited to those described herein, such as compound 25. In certain embodiments, the agent regulates serum iron levels, for example, by temporarily sequestering or redistributing iron to various tissues and / or preventing further absorption of iron from food. In some embodiments, the iron sequestering compound prevents excretion of iron. In various embodiments, the agent affects serum iron levels and / or iron loading or distribution in various tissues / organs. It is understood that the present disclosure further relates to pharma- ceutically acceptable salts and solvates of any of the agents disclosed herein.

[0069] In certain embodiments, the hepcidin mimetic is described in any of the following: U.S. Patents US9,822,157 and US10,030,061, which describe hepcidin analogs and their use for treating iron overload (including hereditary hemochromatosis and iron loading anemia), PCT Publication WO15200916, which describes additional hepcidin analogs and their use for treating iron overload, PCT Publication WO17117411, which describes additional hepcidin analogs with improved in vivo half-lives and their use for treating iron overload, PCT Publication WO18048944, which describes additional hepcidin analogs and their use for treating and / or preventing iron overload in a subject and / or reducing serum iron levels in a subject, PCT Publication WO18048944, which describes additional hepcidin analogs and their use for treating and / or preventing iron overload in a subject, and PCT Publication WO18048944, which describes additional hepcidin analogs and their use for treating and / or preventing iron overload in a subject. PCT published application WO18128828 describes additional hepcidin analogs and their use to treat hepcidin-related disorders, including the prevention and treatment of iron overload diseases such as hemochromatosis, iron-loaded anemias such as thalassemia, and diseases associated with ineffective or enhanced erythropoiesis; PCT published application WO17068089 describes additional hepcidin analogs (ferroportin inhibitors) and their use to treat thalassemia and hemochromatosis; and U.S. patent US9315545 describes additional novel analogs and their use to treat diseases of iron metabolism, beta thalassemia, hemochromatosis, iron-loaded anemia, alcoholic liver disease, or chronic hepatitis C.

[0070] In certain embodiments, the hepcidin mimetic comprises or consists of a peptide of Formula I: R1-XY-R2 (I) or a pharma- ceutically acceptable salt or solvate thereof; During the ceremony, R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C20 alkanoyl, or pGlu; R2 is NH 2 or OH, X is an amino acid sequence of formula II, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (II) During the ceremony, X1 is Asp, Ala, Ida, pGlu, bhAsp, Leu, D-Asp, or absent; X2 is Thr, Ala, or D-Thr; X3 is His, Lys, D-His, or Lys; X4 is Phe, Ala, Dpa, or D-Phe; X5 is Pro, Gly, Arg, Lys, Ala, D-Pro, or bhPro; X6 is Ile, Cys, Arg, Lys, D-Ile, or D-Cys; X7 is Cys, Ile, Leu, Val, Phe, D-Ile, or D-Cys; X8 is Ile, Arg, Phe, Gln, Lys, Glu, Val, Leu, or D-Ile; X9 is Phe or bhPhe; X10 is Lys, Phe, or absent; When Y is absent, X7 is Ile; Y is an amino acid sequence of formula III, Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15 (III) During the ceremony, Y1 is Gly, Cys, Ala, Phe, Pro, Glu, Lys, D-Pro, Val, Ser, or absent; Y2 is Pro, Ala, Cys, Gly, or absent; Y3 is Arg, Lys, Pro, Gly, His, Ala, Trp, or absent; Y4 is Ser, Arg, Gly, Trp, Ala, His, Tyr, or absent; Y5 is Lys, Met, Arg, Ala, or absent; Y6 is Gly, Ser, Lys, Ile, Ala, Pro, Val, or absent; Y7 is Trp, Lys, Gly, Ala, Ile, Val, or absent; Y8 is Val, Thr, Gly, Cys, Met, Tyr, Ala, Glu, Lys, Asp, Arg, or absent; Y9 is Cys, Tyr, or absent; Y10 is Met, Lys, Arg, Tyr, or absent; Y11 is Arg, Met, Cys, Lys, or absent; Y12 is Arg, Lys, Ala, or absent; Y13 is Arg, Cys, Lys, Val, or absent; Y14 is Arg, Lys, Pro, Cys, Thr, or absent; Y15 is Thr, Arg, or absent; The peptide comprising formula I is optionally PEGylated on R1, X, or Y, the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer moieties; Ida is iminodiacetic acid, pGlu is pyroglutamic acid, bhAsp is β-homoaspartic acid, and bhPro is β-homoproline.

[0071] In certain embodiments, any of the peptides disclosed herein comprises a disulfide bond between two Cys amino acid residues present in the peptide, e.g., the thiol groups of two cysteine ​​residues in the peptide form a disulfide bond.

[0072] In certain embodiments, R1 is hydrogen, isovaleric acid, isobutyric acid, or acetyl.

[0073] In certain embodiments, X is an amino acid sequence of formula IV: X1-Thr-His-X4-X5-X6-X7-X8-Phe-X10(IV) During the ceremony, X1 is Asp, Ida, pGlu, bhAsp, or absent; X4 is Phe or Dpa; X5 is Pro or bhPro, X6 is Ile, Cys, or Arg; X7 is Cys, Ile, Leu, or Val; X8 is Ile, Lys, Glu, Phe, Gln, or Arg; X10 is Lys or absent.

[0074] In certain embodiments, X is an amino acid sequence of formula V: X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10(V) During the ceremony, X1 is Asp, Ida, pGlu, bhAsp, or absent; X4 is Phe or Dpa; X5 is Pro or bhPro, X8 is Ile, Lys, Glu, Phe, Gln or Arg; X10 is Lys or absent.

[0075] In certain embodiments, the peptide has formula VI: R 1 -XYR 2 (VI) or a pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is hydrogen, isovaleric acid, isobutyric acid, or acetyl; R 2 NH 2 or OH, X is an amino acid sequence of formula VII, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10(VII) During the ceremony, X1 is Asp, Ida, pGlu, bhAsp, or absent; X4 is Phe or Dpa; X5 is Pro or bhPro, X8 is Ile, Lys, Glu, Phe, Gln, or Arg; X10 is Lys or absent; Y is an amino acid sequence of formula VIII, Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10(VIII) During the ceremony, Y1 is Gly, Glu, Val, or Lys; Y3 is Arg or Lys; Y5 is Arg or Lys; Y6 is Gly, Ser, Lys, Ile, or Arg; Y7 is Trp or absent, Y8 is Val, Thr, Asp, Glu, or absent; Y10 is Lys or absent; The peptide contains a disulfide bond between two Cys. The peptide may optionally be 1 PEGylated on X or Y; the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer moieties; Ida is iminodiacetic acid, pGlu is pyroglutamic acid, bhAsp is β-homoaspartic acid, and bhPro is β-homoproline.

[0076] In certain embodiments, the peptide comprises or consists of one of the following sequences: DTHFPICIFGPRSKGWVC (SEQ ID NO: 46), DTHFPCIIFGPRSKGWVCK (SEQ ID NO:47), DTHFPCIIFEPRSKGWVCK (SEQ ID NO: 48), DTHFPCIIFGPRSKGWACK (SEQ ID NO:49), DTHFPCIIFGPRSKGWVCKK (SEQ ID NO:50), DTHFPCIIFVCHRPKGCYRRVCR (SEQ ID NO:51), DTHFPCIKFGPRSKGWVCK (SEQ ID NO:52), DTHFPCIKFKPRSKGWVCK (SEQ ID NO:53), DTHFPCIIFGPRSRGWVCK (SEQ ID NO:54), DTHFPCIKFGPKSKGWVCK (SEQ ID NO:55), DTHFPCIKFEPRSKGCK (SEQ ID NO:56), DTHFPCIKFEPKSKGWECK (SEQ ID NO:57), DTHFPCIKFEPRSKKCK (SEQ ID NO:58), DTHFPCIKFEPRSKGCKK (SEQ ID NO:59), DTHFPCIKFKPRSKGCK (SEQ ID NO: 60), DTHFPCIKFEPKSKGCK (SEQ ID NO:61), DTHFPCIKF (SEQ ID NO:62), DTHFPCIIF (SEQ ID NO: 63), or DTKFPCIIF (SEQ ID NO: 64), the peptide is optionally PEGylated on R1, X, or Y; the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer moieties; The peptide optionally comprises a disulfide bond between two Cys amino acid residues of the peptide.

[0077] In certain embodiments, the peptide comprises or consists of one of the following sequences: Isovaleric acid-DTHFPICIFGPRSKGWVC-NH 2 (SEQ ID NO: 1), Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH 2 (SEQ ID NO:2) 、 Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH 2 (SEQ ID NO:3) 、 Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH 2 (SEQ ID NO:4) 、 Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH 2 (SEQ ID NO:5) 、 Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH 2 (SEQ ID NO:6) 、 Isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH 2 (SEQ ID NO:7) 、 Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH 2 (SEQ ID NO:8) 、 Isovaleric acid-DTHFPICIFGPRS(K(PEG8))GWVC-NH 2 (SEQ ID NO:9) 、 Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH 2 (SEQ ID NO:10) 、 Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH 2 (SEQ ID NO:11) 、 Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG4))-NH 2 (SEQ ID NO:12) 、 Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH 2 (SEQ ID NO:13) 、 Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH 2 (SEQ ID NO:14) 、 Isovaleric acid-DTHFPCIKF)K(Palm))PRSKGWVCK-NH 2 (SEQ ID NO:15) 、 Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH 2 (SEQ ID NO:16) 、 Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH 2 (SEQ ID NO:17) 、 Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))NH 2 (SEQ ID NO:18) 、 Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH 2 (SEQ ID NO:19) 、 Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH 2 (SEQ ID NO:20) 、 Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH 2 (SEQ ID NO:21) 、 Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH 2 (SEQ ID NO:22) 、 Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH 2 (SEQ ID NO:23) 、 Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH 2(SEQ ID NO:24) 、 Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH 2 (SEQ ID NO:25) 、 Isovaleric acid-DTHFPCIKF-K(isoGlu-Palm)-PRSKGCK-NH 2 (SEQ ID NO:26) 、 Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH 2 (SEQ ID NO:27) 、 Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGWECK-NH 2 (SEQ ID NO:28) 、 Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH 2 (SEQ ID NO:29) 、 Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))CK-NH 2 (SEQ ID NO: 30), Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH 2 (SEQ ID NO:31) 、 Isovaleric acid-DTHFPCI-K(Dapa-Palm)-FEPRSKGCK-NH 2 (SEQ ID NO:32) 、 Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGCK-NH 2 (SEQ ID NO: 33), Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH 2 (SEQ ID NO:34), Isovaleric acid-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH 2 (SEQ ID NO:35), Isovaleric acid-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH 2(SEQ ID NO:36), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))K-NH 2 (SEQ ID NO:37), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))-NH 2 (SEQ ID NO:38), Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH 2 (SEQ ID NO:39), Isovaleric acid-DTHFPCIKF-NH 2 (SEQ ID NO: 40), Hy-DTHFPCIKF-NH 2 (SEQ ID NO:41), Isovaleric acid-DTHFPCIIF-NH 2 (SEQ ID NO: 42), Hy-DTHFPCIIKF-NH 2 (SEQ ID NO:43), Isovaleric acid-DTKFPCIIF-NH 2 (SEQ ID NO: 44), or Hy-DTKFPCIIF-NH 2 (SEQ ID NO:45), Optionally, the peptide comprises a disulfide bond between two Cys amino acid residues of the peptide. 。

[0078] In certain embodiments, the peptide is [ka] Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH 2 (SEQ ID NO: 20), [ka] Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH 2 (SEQ ID NO: 25), [ka] Isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH 2 (SEQ ID NO:26), [ka] Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH 2 (SEQ ID NO: 27), and [ka] Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH 2 (SEQ ID NO: 29), The amino acids are L-amino acids.

[0079] The peptides disclosed herein, including hepcidin mimetics, may be produced using methods known in the art, including chemical synthesis, biosynthesis or in vitro synthesis using recombinant DNA methods, and solid phase synthesis. See, for example, PCT Application Publication Nos. WO2014 / 145561 and WO2015 / 200916, 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 peptides disclosed herein can be purified using protein purification techniques known in the art, such as reverse-phase high performance liquid chromatography (HPLC), ion exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem. 12(16):3121-3126, and Scopes (1982) Protein Purification, Springer-Verlag, NY, which are incorporated herein by reference. Alternatively, the peptides may be produced by recombinant DNA techniques known in the art.

[0080] In certain embodiments, the peptides disclosed herein may be PEGylated. As used herein, "polyethylene glycol" or "PEG" is a polyether compound of the general formula H-(O-CH2-CH2)n-OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on their molecular weight. PEG, PEO, or POE, 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 Da, PEO to polymers with molecular weights greater than 20,000 Da, and POE to polymers of any molecular weight. PEG and PEO are liquids or low melting solids depending on their molecular weight. 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 Da to 10,000,000 Da. 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. PEG 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). PEG prepared for the purpose of half-life extension, for example, 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 PEGs vary substantially in weight, for example, from about 200 Da to about 40,000 Da, or from about 200 Da to about 60,000 Da, any of which may be used for purposes of the present disclosure. In certain embodiments, PEGs having molecular weights of 200 Da to 2,000 Da or 200 Da to 500 Da are used. Different forms of PEG may also be used, depending on the initiator used in the polymerization process, with common initiators being monofunctional methyl ether PEG, or methoxypoly(ethylene glycol) (abbreviated mPEG). Low molecular weight PEGs are also available as pure oligomers, referred to as monodisperse, uniform, or discrete. These are used in certain embodiments of the present disclosure.

[0081] 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 the 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.

[0082] As used herein, "PEGylation" refers to the act of covalently attaching a PEG structure to a peptide inhibitor of the invention, which is referred to as a "PEGylated peptide inhibitor." In certain embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 Da to about 40,000 Da.

[0083] In various embodiments, the agent is present in a pharmaceutical composition that includes one or more pharma- ceutically acceptable diluents, carriers, or excipients. A pharma- ceutically acceptable carrier, diluent, or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. The term "pharma- ceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, sterile saline and phosphate-buffered saline at weakly acidic or physiological pH can be used. Suitable pH buffering agents may be, for example, phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetate (e.g., as sodium acetate), or mixtures thereof. The term further encompasses any carrier agent listed in the United States Pharmacopoeia for use in animals, including humans. EXAMPLES

[0084] The following examples show certain specific embodiments of the present invention.Unless otherwise specified in detail, the following examples are carried out using standard techniques that are well known and routine 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)

[0085] K(): In the peptide sequences provided herein, a compound or chemical group is presented in parentheses immediately following a lysine residue, and it is understood that the compound or chemical group within the parentheses is the side chain conjugated to the lysine residue. Thus, for example, but in no way limiting, K-[(PEG8)]- indicates that a PEG8 moiety is conjugated to the side chain of this lysine.

[0086] Palm: indicates the conjugation of palmitic acid (palmitoyl).

[0087] As used herein, "C()" refers to the cysteine ​​residue involved in a particular disulfide bridge. For example, in hepcidin, there are four disulfide bridges, the first between two C(1) residues, the second between two C(2) residues, the third between two C(3) residues, and the fourth between two C(4) residues. Thus, in some embodiments, the sequence of hepcidin is as follows: Hy-DTHFPIC(1)IFC(2)C(3)GC(2)C(4)HRSKC(3)GMC(4)C(1)KT-OH (SEQ ID NO:65), and the sequences of other peptides may optionally be written in the same manner.

[0088] Example 1 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.

[0089] Procedure for solid phase peptide synthesis 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 a solution containing Fmoc amino acid, HBTU, and DIPEA (1:1:1.1) in DMF was added to the swollen resin [HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIPEA: diisopropylethylamine, DMF: dimethylformamide]. HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate) was used instead of HBTU to improve coupling efficiency in difficult regions. Removal of the Fmoc protecting group was achieved by treatment with a DMF, piperidine (2:1) solution.

[0090] Procedure for cleaving peptide from 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 TFA removal, 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).

[0091] 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 A (buffer A: 0.05% TFA in water, buffer B: 0.043% TFA, 90% acetonitrile in water).

[0092] Peptide oxidation procedure. Method A (single disulfide oxidation). Oxidation of unprotected peptides of the invention was carried out by dissolving iodine (1 mg per mL) in MeOH in a solution (ACN:H 2 This was achieved by dropwise addition of the peptide in 50 mL of HO, 7:3, 0.5% TFA. After stirring for 2 min, ascorbic acid was added in small portions until the solution was clear and the sample was immediately loaded onto the HPLC for purification.

[0093] Method B (selective oxidation of two disulfides). When more than two disulfides are present, selective oxidation is often performed. Oxidation of free cysteines was performed using NH 4 CO 3 This was achieved in solution. After stirring for 24 h, the solution was acidified to pH 3 with TFA and subsequently lyophilized prior to purification. The resulting single oxidized peptide (containing the ACM-protected cysteine) was then oxidized / selectively deprotected using iodine solution. The peptide (1 mg per 2 mL) was dissolved in MeOH / H 2 80:20 Iodine dissolved in 0 and dissolved in reaction solvent was added to the reaction (final concentration: 5 mg / mL) at room temperature. The solution was stirred for 7 minutes, after which ascorbic acid was added in small portions until the solution was clear. The solution was then loaded directly onto the HPLC.

[0094] Method C (Natural Oxidation). In cases where more than two disulfides were present and selective oxidation was not performed, natural oxidation was performed. Natural oxidation was achieved using 100 mM NH4CO3 (pH 7.4) solution in the presence of oxidized and reduced glutathione (peptide / GSH / GSSG, 1:100:10 molar ratio) (peptide:GSSG:GSH, 1:10, 100). After stirring for 24 h, the solution was acidified to pH 3 with TFA and subsequently lyophilized prior to RP-HPLC purification.

[0095] Procedure for cysteine ​​oxidation to generate dimers. Oxidation of unprotected peptides of the present invention was achieved by dropwise addition of iodine (1 mg per mL) in MeOH to the peptide in solution (ACN:H2O, 7:3, 0.5% TFA). After stirring for 2 min, ascorbic acid was added in small portions until the solution was clear, and the sample was immediately loaded onto HPLC for purification.

[0096] Dimerization procedure. Glyoxylic acid (DIG), IDA, or Fmoc-β-Ala-IDA were preactivated as N-hydroxysuccinimide esters by treating 1 equivalent (abbreviated as "eq") of acid with both N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) in 2.2 equivalents of NMP (N-methylpyrrolidone) at a final concentration of 0.1 M. For the PEG13 and PEG25 linkers, these chemicals were purchased and preformed as the activated succinimide esters. Approximately 0.4 equivalents of the activated ester were slowly added in small portions to the peptide (1 mg / mL) in NMP. The solution was left stirring for 10 min, after which 2-3 additional aliquots of approximately 0.05 equivalents of linker were slowly added. The solution was left stirring for an additional 3 h, after which the solvent was removed under vacuum and the residue was purified by reverse-phase HPLC. An additional step of stirring the peptide in 20% piperidine in DMF (2×10 min) was performed before further reverse phase HPLC purification.

[0097] Those skilled in the art will appreciate that standard methods of peptide synthesis can be used to produce the compounds of the invention.

[0098] Linker Activation and Dimerization The peptide monomer subunits were linked to form hepcidin analog peptide dimers, as described below.

[0099] Small scale DIG linker activation procedure: 5 mL of NMP was added to a glass vial containing IDA diacid (304.2 mg, 1 mmol), N-hydroxysuccinimide (NHS, 253.2 mg, 2.2 equiv, 2.2 mmol) and a stir bar. The mixture was stirred at room temperature to completely dissolve the solid starting material. N,N'-dicyclohexylcarbodiimide (DCC, 453.9 mg, 2.2 equiv, 2.2 mmol) was then added to the mixture. A precipitate appeared within 10 min and the reaction mixture was further stirred overnight at room temperature. The reaction mixture was then filtered to remove precipitated dicyclohexylurea (DCU). The activated linker was kept in a sealed vial before being used for dimerization. The nominal concentration of the activated linker was approximately 0.20 M.

[0100] For dimerization using PEG linkers, no pre-activation step was involved: commercially available pre-activated bifunctional PEG linkers were used.

[0101] Dimerization procedure: 2 mL of anhydrous DMF was added to a vial containing peptide monomer (0.1 mmol). The pH of the peptide was adjusted to 8-9 with DIEA. Then, activated linker (IDA or PEG13, PEG25) (0.48 eq, 0.048 mmol relative to monomer) was added to the monomer solution. The reaction mixture was stirred at room temperature for 1 h. Completion of the dimerization reaction was monitored using analytical HPLC. The time taken for completion of the dimerization reaction varied depending on the linker. After completion of the reaction, the peptide was precipitated in cold ether and centrifuged. The supernatant ether layer was discarded. The precipitation step was repeated twice. The crude dimer was then purified using reverse phase HPLC (Luna C18 support, 10u, 100A, mobile phase A: water with 0.1% TFA, mobile phase B: acetonitrile (ACN) with 0.1% TFA, gradient of 15% B and change to 45% B over 60 min, flow rate 15ml / min). Fractions containing the pure product were then lyophilized on a lyophilizer.

[0102] Conjugation of half-life extending moieties Peptide conjugation was performed on-resin. Lys(ivDde) ​​was used as the major amino acid. After assembly of the peptide on-resin, selective deprotection of the ivDde group was performed using 2% hydrazine in DMF for 3×5 min. Linker activation and acylation using HBTU for 3 h, 1-2 equivalents of DIEA, and Fmoc removal followed by a second acylation with a lipid acid afforded the conjugated peptide.

[0103] Example 2 Pharmacodynamics of hepcidin mimetic peptides in hereditary hemochromatosis mice The ability of hepcidin mimetics to modulate iron and other markers was determined in a mouse model of hereditary hemochromatosis (HH). HH mice (129S-Hjv tm1NcaMice (14-18 years old, ...

[0104] In HH mice, hepatic iron accumulation was prevented in the group treated with Compound 25 for 2 weeks under less severe iron overload conditions (Figure 2A and Figure 2B). HH mice were iron-loaded to different levels by maintaining them on either a low-iron or normal iron diet for 2 weeks to achieve different levels of iron overload at the start of therapy. Mice were then treated with Compound 25 (2.5 mg / kg, Q2D) under a normal iron diet. Organ iron concentrations were assessed by ICP-MS method (total iron) or colorimetric assay (non-heme iron). Statistical analysis: one-way ANOVA with Dunnett's multiple comparisons or t-test with Welch's correction.

[0105] As shown in Figure 2A, "non-diseased" mice maintained on a low iron diet to prevent iron accumulation until the start of therapy showed a reduction in liver iron, demonstrating that compound 25 prevents excess absorption of iron from the diet, thus preventing elevated TSAT% and iron accumulation in the liver. As shown in Figure 2B, mice maintained on a normal diet for 2 weeks prior to treatment to allow partial iron overload also showed a reduction in iron liver. In this case, compound 25 not only prevented further iron accumulation in the liver (compared to vehicle), but also redistributed iron away from the liver.

[0106] These studies demonstrate that starting from a high iron overload state (TSAT%>90%, severe disease), a decrease in TSAT% as a result of iron sequestration in splenic macrophages (capable of storing large amounts of iron) is associated with reduced iron accumulation in organs (e.g., pancreas, kidney). They also show that trough drug levels, especially TSAT% below normal levels, are serum biomarkers of clinical efficacy (e.g., organ iron overload).

[0107] These studies indicate that treatment with hepcidin mimetic peptides, such as compound 25, could potentially benefit hemochromatosis patients with primary and secondary iron overload by preventing and reversing iron loading, for example by lowering serum TSAT% and labile iron through iron sequestration in splenic macrophages.

[0108] Example 3 Efficacy of hepcidin mimetic peptides in patients with hereditary hemochromatosis A clinical efficacy study was conducted in HH patients. Subjects received Compound 25 subcutaneously for up to 24 weeks. Subjects were started at an initial dose of 10mg subcutaneously per week. Dose was increased to 20mg per week, then 40mg and 80mg per week as needed, based on tolerability and the pharmacodynamic marker TSAT. In addition, twice weekly subcutaneous dosing schedules of 10mg, 20mg, 30mg, and 40mg were tested (days 1 and either 4 or 5). The majority of patients received a dose of 20mg or less per week. Subject safety and blood iron parameters (serum iron, serum ferritin, transferrin, and TSAT) were collected to monitor the pharmacodynamic effects of Compound 25. Phlebotomy need and effect on QoL data (36-Item Short Form Health Survey [SF-36] and Patient's Global Impression of Change [PGI-C]) were also collected and tabulated.

[0109] The dose and schedule of each individual subject was determined based on the pharmacodynamic (PD) marker TSAT measured at two time points after administration (one at peak PD effect one day after administration and one at trough PD effect). The intention of the dose and schedule adjustment was to reduce TSAT and serum iron levels. If necessary, the dose of Compound 25 was increased sequentially from 10 mg to 20 mg, and if necessary, to 30 mg, 40 mg, and 80 mg every week until TSAT was less than about 40% at peak PD effect one day after administration and at trough PD effect before the next administration of Compound 25. Administration was once a week or twice a week. The trough PD effect was the TSAT value measured 7 days after administration of Compound 25 (and before the next administration) for once-weekly administration, or 7 days after the first administration in a week for twice-weekly administration. For twice-weekly regimens, doses were given at least 3 days apart (e.g., on the 1st and 4th or 5th days of each week). Compound 25 doses were increased up to a maximum of 40 mg twice weekly. To facilitate dose escalation and identification of therapeutic doses, researchers assessed TSAT values ​​on the day subjects visited the clinic for any dose adjustments.

[0110] Patients had a previous phlebotomy frequency of at least 0.25 per month (e.g., at least 3 phlebotomies in the past 12 months or at least 4 phlebotomies in the past 15 months) and a phlebotomy frequency of less than 1 per month, hemoglobin greater than 11.5 g / dL, and serum ferritin less than 300 ng / mL at the time of screening (prior to phlebotomy screening). Methods: This single-arm, open-label, dose-ranging Phase 2 study investigated a subcutaneous hepcidin mimetic (compound 25) in patients (pts) with confirmed HH who had undergone documented stable phlebotomy (phl) for at least 6 months (mos) prior to treatment with a phlebotomy frequency of 0.25 to 1 per month. Patients with clinically significant laboratory abnormalities and those undergoing iron chelation therapy or erythrocyte apheresis were excluded. As outlined in Figure 3, patients were given individually titrated compound 25 doses once or twice weekly to maintain transferrin saturation (TSAT) below 45% and followed for 6 months. Individual doses are shown in Figure 11. Endpoints included TSAT, serum iron, serum transferrin and serum ferritin, liver iron content (LIC) measured by Ferriscan MRI, adverse events, and patient-reported outcomes on the Patient Global Impression of Change (PGI-C) and Medical Outcomes Research Questionnaire Short Form Health Survey (SF-36). Compound 25 was formulated in a buffered aqueous solution. Results: Sixteen patients (10 males / 6 females) were enrolled. Mean age and weight were 62.5 years and 88.1 kg, respectively. LIC values ​​were maintained at pre-study levels with minimal use of phlebotomy during the study period. In the 6 months prior to the study, the mean phlebotomy (phl) rate was 0.27 phl / month compared to 0.03 phl / month during the study (p<0.0001). Study endpoints included safety, reduction in phlebotomy, serum iron, TSAT, transferrin, ferritin, liver iron content by MRI, and adverse events. Compound 25 was able to eliminate phlebotomy in the majority of subjects during the treatment period (Figures 4A and 4B). Treatment with Compound 25 also showed a statistically significant reduction in mean TSAT levels (Figures 5A and 5B). Mean baseline TSAT was 45% compared to 30.4% during the study (p=0.0025). For certain patients with a TSAT above 45% at baseline, treatment with Compound 25 reduced TSAT to less than or close to 45% if phlebotomy was not possible. Treatment with Compound 25 also resulted in a reduction in mean serum iron (Figure 6). Serum iron was reduced from 24.5 μmol / L before the study to a mean of 17.7 μmol / mL during the study (p=0.0059), or from 137 ug / dL at baseline to 98.6 ug / dL after treatment with Compound 25. There was a dose- and concentration-dependent reduction in serum iron and TSAT (Figure 7). Serum ferritin and transferrin levels remained relatively constant from baseline to after treatment with Compound 25 (Figure 8). Treatment with Compound 25 maintained liver iron content without statistically significant differences at baseline or after treatment with Compound 25 (Figure 9). There were no significant changes in blood parameters such as hematocrit, red blood cells, white blood cells, or platelets. Patient-reported outcomes were determined and improvements were noted in the Role Physical and Role Mental subcomponents of the SF-36 following treatment with Compound 25 (Figure 10). Compound 25 was generally well tolerated. All treatment-related adverse events were characterized as CTCAE grade 1 or 2.

[0111] A summary of the results from a 6-month open-label study in 16 HH patients in the maintenance phase of iron reduction with stable pre-study phlebotomies for ≥6 months (requiring ≥3 phlebotomies per 12 months or ≥4 phlebotomies per 15 months) is shown in FIG. Conclusions: Compound 25 demonstrated pharmacological efficacy in reducing serum iron and TSAT levels. These pharmacodynamic effects corresponded to reduced need for phlebotomy, control of LIC, and clinically meaningful changes in patient-reported outcomes. These data indicate that Compound 25 controls LIC in the absence of phlebotomy. Compound 25 was well tolerated in patients with HH. These data support Compound 25 and other hepcidin mimetics as treatments for HH.

[0112] Example 4 Pharmacodynamic control of hepcidin mimetic peptides Iron is an important component of normal cell function, and metabolic dysregulation contributes to the formation and / or progression of disease. Compound 25 controls body iron stores by blocking dietary iron adsorption and rapidly redistributing serum iron to splenic macrophages. Restoration of iron homeostasis by Compound 25 has been demonstrated in subjects with healthy iron stores, iron deficiency, and tissue iron overload.

[0113] In this example, the pharmacodynamic control of a subcutaneously administered compound was investigated in healthy volunteers and HH patients.

[0114] In the first study, healthy human volunteers were administered a single dose of placebo or Compound 25 (1 mg, 3 mg, 10 mg, 20 mg, 40 mg, or 80 mg) subcutaneously. Subjects' serum TSAT levels were determined as a surrogate for serum iron stores over a period of 6 days following dosing, and subjects' mean corpuscular hemoglobin concentration (MCHC) levels were determined as a surrogate for red blood cell integrity over a period of 6 days following dosing. As shown in Figure 12, subjects showed a dose-responsive reduction in TSAT levels in less than one day, which gradually increased over the next 5-6 days.

[0115] In the second study, maintenance phase HH patients and transfusion-dependent β-thalassemia patients were subcutaneously administered Compound 25, and their TSAT and MCHC levels were determined before and 24 weeks after administration of Compound 25. As shown in Figure 13, the subjects' TSAT and MCHC levels were reduced after treatment.

[0116] These studies demonstrate dose-related and consistent pharmacodynamic control of Compound 25 in humans, as well as improvements in both serum iron stores and RBC integrity following treatment of HH patients with Compound 25, supporting the use of Compound 25 and other hepcidin mimetics to treat HH, including but not limited to HH and arthropathy due to excess iron.

[0117] 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 herein and / or listed in this application data sheet are hereby incorporated by reference in their entireties.

[0118] 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. A composition for use in a method of treating hereditary hemochromatosis, hereditary hemochromatosis arthropathy, or joint pain associated with hereditary hemochromatosis arthropathy in a human subject, said composition comprising an effective amount of a hepcidin mimetic, said method comprising administering said composition to said subject, said effective amount comprising a dosage in the range of about 5 mg to about 40 mg, and optionally, said subject being administered different dosages during different periods over the course of treatment.

2. The composition according to claim 1, wherein said subject is administered said effective amount of said hepcidin mimetic about once a week or about twice a week during at least a portion of the course of treatment.

3. Said subject is (a) administered said hepcidin mimetic in an amount of about 5 mg to about 20 mg about twice a week during at least a portion of the course of treatment, or (b) administered said hepcidin mimetic in an amount of about 10 mg to about 40 mg about once a week during at least a portion of the course of treatment, the composition according to claim 2.

4. The composition according to claim 1, wherein said effective amount causes a reduction in the transferrin saturation (TSAT) level and / or serum iron level of said subject.

5. (a) The TSAT level of said subject is reduced to less than 45%, or (b) The TSAT level of said subject is reduced to less than 40%, the composition according to claim 4.

6. The composition according to claim 5, wherein the TSAT level of said subject is maintained at less than 45% over the course of treatment with said hepcidin mimetic, and optionally, said course of treatment comprises at least 24 weeks.

7. The hepcidin mimetic is a peptide having the formula I: R1-X-Y-R2 (I) or a pharmaceutically acceptable salt or solvate thereof, wherein, R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C20 alkanoyl, or pGlu, R2 is NH ２ or OH, X is the amino acid sequence of Formula II, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (II) wherein, X1 is Asp, Ala, Ida, pGlu, bhAsp, Leu, D-Asp, or absent, X2 is Thr, Ala, or D-Thr, X3 is His, Lys, D-His, or Lys, X4 is Phe, Ala, Dpa, or D-Phe, X5 is Pro, Gly, Arg, Lys, Ala, D-Pro, or bhPro, X6 is Ile, Cys, Arg, Lys, D-Ile, or D-Cys, X7 is Cys, Ile, Leu, Val, Phe, D-Ile, or D-Cys, X8 is Ile, Arg, Phe, Gln, Lys, Glu, Val, Leu, or D-Ile, X9 is Phe or bhPhe, and X10 is Lys, Phe, or absent, when Y is absent, X7 is Ile, and Y is the amino acid sequence of Formula III, Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15 (III) wherein, Y1 is Gly, Cys, Ala, Phe, Pro, Glu, Lys, D-Pro, Val, Ser, or absent, Y2 is Pro, Ala, Cys, Gly, or absent, Y3 is Arg, Lys, Pro, Gly, His, Ala, Trp, or absent, Y4 is Ser, Arg, Gly, Trp, Ala, His, Tyr, or absent, Y5 is Lys, Met, Arg, Ala, or absent, Y6 is Gly, Ser, Lys, Ile, Ala, Pro, Val, or absent, Y7 is Trp, Lys, Gly, Ala, Ile, Val, or absent, Y8 is Val, Thr, Gly, Cys, Met, Tyr, Ala, Glu, Lys, Asp, Arg, or absent, Y9 is Cys, Tyr, or absent, Y10 is Met, Lys, Arg, Tyr, or absent, Y11 is Arg, Met, Cys, Lys, or absent, Y12 is Arg, Lys, Ala, or absent, Y13 is Arg, Cys, Lys, Val, or absent, Y14 is Arg, Lys, Pro, Cys, Thr, or absent, and Y15 is Thr, Arg, or absent, The peptide of formula I is optionally PEGylated on R1, X, or Y, The side chains of the amino acids of the peptide are optionally conjugated to a lipophilic substituent or a polymer moiety, The peptide of formula I optionally has a disulfide bond formed between the thiol groups of two cysteine residues, and The composition according to claim 1, wherein Ida is iminodiacetic acid, pGlu is pyroglutamic acid, bhAsp is β-homoaspartic acid, and bhPro is β-homoproline.

8. The composition according to claim 7, wherein R1 is hydrogen, isovaleric acid, isobutyric acid or acetyl.

9. X is the amino acid sequence of Formula IV, X1-Thr-His-X4-X5-X6-X7-X8-Phe-X10 (IV) wherein, X1 is Asp, Ida, pGlu, bhAsp, or absent, X4 is Phe or Dpa, X5 is Pro or bhPro, X6 is Ile, Cys, or Arg, X7 is Cys, Ile, Leu, or Val, and X8 is Ile, Lys, Glu, Phe, Gln, or Arg, X10 is Lys or absent, the composition according to claim 7 or 8.

10. X is the amino acid sequence of Formula V, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10 (V) wherein, X1 is Asp, Ida, pGlu, bhAsp, or absent, X4 is Phe or Dpa, X5 is Pro or bhPro, X8 is Ile, Lys, Glu, Phe, Gln or Arg, and X10 is Lys or absent, the composition according to claim 7.

11. The peptide is of Formula VI: R １ -X-Y-R ２ (VI) or a pharmaceutically acceptable salt thereof, wherein, R １ is hydrogen, isovaleric acid, isobutyric acid, or acetyl, R ２ is NH ２ or OH, X is the amino acid sequence of Formula VII, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10 (VII) wherein, X1 is Asp, Ida, pGlu, bhAsp, or absent, X4 is Phe or Dpa, X5 is Pro or bhPro, X8 is Ile, Lys, Glu, Phe, Gln, or Arg, and X10 is Lys or absent, Y is the amino acid sequence of formula VIII, Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10 (VIII) wherein, Y1 is Gly, Glu, Val, or Lys, Y3 is Arg or Lys, Y5 is Arg or Lys, Y6 is Gly, Ser, Lys, Ile, or Arg, Y7 is Trp or absent, Y8 is Val, Thr, Asp, Glu, or absent, Y10 is Lys or absent, the peptide has a disulfide bond formed between the thiol groups of two cysteine residues, the peptide is optionally PEGylated on R １ , X, or Y, the side chains of the amino acids of the peptide are optionally conjugated to a lipophilic substituent or a polymer moiety, Ida is iminodiacetic acid, pGlu is pyroglutamic acid, bhAsp is β-homoaspartic acid, bhPro is β-homoproline, the composition according to claim 7.

12. the peptide has one of the following sequences, DTHFPICIFGPRSKGWV C (SEQ ID NO: 46), DTHFPCIIFGPRSKGWVCK (SEQ ID NO: 47), DTHFPCIIFEPRSKGWVCK (SEQ ID NO: 48), DTHFPCIIFGPRSKGWACK (SEQ ID NO: 49), DTHFPCIIFGPRSKGWVCKK (SEQ ID NO: 50), DTHFPCIIFVCHRPKGCYRRVCR (SEQ ID NO: 51), DTHFPCIKFGPRSKGWVCK (SEQ ID NO: 52), DTHFPCIKFKPRSKGWVCK (SEQ ID NO: 53), DTHFPCIIFGPRSRGWVCK (SEQ ID NO: 54), DTHFPCIKFGPKSKGWVCK (SEQ ID NO: 55), DTHFPCIKFEPRSKGCK (SEQ ID NO: 56), DTHFPCIKFEPKSKGWECK (SEQ ID NO: 57), DTHFPCIKFEPRSKKCK (SEQ ID NO: 58), DTHFPCIKFEPRSKGCKK (SEQ ID NO: 59), DTHFPCIKFKPRSKGCK (SEQ ID NO: 60), DTHFPCIKFEPKSKGCK (SEQ ID NO: 61), DTHFPCIKF (SEQ ID NO: 62), DTHFPCIIF (SEQ ID NO: 63), or DTHFPCIIF (SEQ ID NO: 64), the peptide is optionally PEGylated on R1, X, or Y, the side chain of the amino acid of the peptide is optionally conjugated to a lipophilic substituent or a polymer moiety, the composition according to claim 7.

13. the peptide has one of the following sequences or structures, isovaleric acid - DTHFPICIFGPRSKGWVC - NH ２ (Compound 1, SEQ ID NO: 1), Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH ２ (Compound 2, SEQ ID NO: 2) 、 Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH ２ (Compound 3, SEQ ID NO: 3) 、 Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH ２ (Compound 4, SEQ ID NO: 4) 、 Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH ２ (Compound 5, SEQ ID NO: 5) 、 Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH ２ (Compound 6, SEQ ID NO: 6) 、 Isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH ２ (Compound 7, SEQ ID NO: 7) 、 Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH ２ (Compound 8, SEQ ID NO: 8) 、 Isovaleric acid-DTHFPICIFGPRS(K(PEG8))GWVC-NH ２ (Compound 9, SEQ ID NO: 9) 、 Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH ２ (Compound 10, SEQ ID NO: 10) 、 Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH ２ (Compound 11, SEQ ID NO: 11) 、 Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG4))-NH ２ (Compound 12, SEQ ID NO: 12) 、 Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH ２(Compound 13, SEQ ID NO: 13) 、 Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH ２ (Compound 14, SEQ ID NO: 14) 、 Isovaleric acid-DTHFPCIKF)K(Palm))PRSKSGWVCK-NH ２ (Compound 15, SEQ ID NO: 15) 、 Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH ２ (Compound 16, SEQ ID NO: 16) 、 Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH ２ (Compound 17, SEQ ID NO: 17) 、 Isovaleric acid-DTHFPCIKFGPRSKGWVCK(K(Palm))NH ２ (Compound 18, SEQ ID NO: 18) 、 Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH ２ (Compound 19, SEQ ID NO: 19) 、 Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKSGWVCK-NH ２ (Compound 20, SEQ ID NO: 20) 、 Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH ２ (Compound 21, SEQ ID NO: 21) 、 Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH ２ (Compound 22, SEQ ID NO: 22) 、 Isovaleric acid-DTHFPCIKFGPRSKGWVCK(K(PEG3-Palm))-NH ２ (Compound 23, SEQ ID NO: 23) 、 Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH ２ (Compound 24, SEQ ID NO: 24) 、 Isovaleric acid-DTHFPCIK(isoGlu-Palm)FEPRSKGC-NH ２ (Compound 25, SEQ ID NO: 25) 、 Isovaleric acid-DTHFPCIKF-K(isoGlu-Palm)-PRSKGC-NH ２ (Compound 26, SEQ ID NO: 26) 、 Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGC-NH ２ (Compound 27, SEQ ID NO: 27) 、 Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGWECK-NH ２ (Compound 28, SEQ ID NO: 28) 、 Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GC-NH ２ (Compound 29, SEQ ID NO: 29) 、 Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))C-NH ２ (Compound 30, SEQ ID NO: 30), Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH ２ (Compound 31, SEQ ID NO: 31) 、 Isovaleric acid-DTHFPCIK(Dapa-Palm)-FEPRSKGC-NH ２ (Compound 32, SEQ ID NO: 32) 、 Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGC-NH ２ (Compound 33, SEQ ID NO: 33), Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGC-NH ２ (Compound 34, SEQ ID NO: 34) Isoleucine-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH ２ (Compound 35, SEQ ID NO: 35), Isoleucine-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH ２ (Compound 36, SEQ ID NO: 36), Isoleucine-DTHFPCIKFEPRSKG(K(Dapa-Palm))K-NH ２ (Compound 37, SEQ ID NO: 37), Isoleucine-DTHFPCIKFEPRSKG(K(Dapa-Palm))-NH ２ (Compound 38, SEQ ID NO: 38), Isoleucine-DTHFPCIKF(K(PEG11-Palm))PRSKSarCK-NH ２ (Compound 39, SEQ ID NO: 39), Isoleucine-DTHFPCIKF-NH ２ (Compound 40, SEQ ID NO: 40), Hy-DTHFPCIKF-NH ２ (Compound 41, SEQ ID NO: 41), Isoleucine-DTHFPCIIF-NH ２ (Compound 42, SEQ ID NO: 42), Hy-DTHFPCIIKF-NH ２ (Compound 43, SEQ ID NO: 43), Isoleucine-DTKFPIIF-NH ２ (Compound 44, SEQ ID NO: 44), or Hy-DTKFPIIF-NH ２ (Compound 45, SEQ ID NO: 45), Optionally, the peptide has a disulfide bond formed between the thiol groups of two cysteine residues, the composition according to claim 7.

14. The peptide is Isoleucine-DTHFPCIK(isoGlu-Palm)FEPRSKGCK-NH ２ (Compound 25, SEQ ID NO: 25), and The composition according to claim 7 or 8, wherein optionally, the peptide has a disulfide bond formed between the thiol groups of two cysteine residues.

15. wherein the peptide is 【Chemical Formula 1】 isovaleryl-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH ２ (Compound 20, SEQ ID NO: 20), 【Chemical Formula 2】 isovaleryl-DTHFPCI(K(isoGlu-Palm))FEPRSKGC-K-NH ２ (Compound 25, SEQ ID NO: 25), 【Chemical Formula 3】 isovaleryl-DTHFPCIKF(K(isoGlu-Palm))PRSKGC-K-NH ２ (Compound 26, SEQ ID NO: 26), 【Chemical Formula 4】 isovaleryl-DTHFPCIKFEP(K(isoGlu-Palm))SKGC-K-NH ２ (Compound 27, SEQ ID NO: 27), and 【Chemical Formula 5】 isovaleryl-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-K-NH ２ (Compound 28, SEQ ID NO: 28), and is selected from the group consisting of The composition according to claim 7 or 8, wherein the amino acid is an L-amino acid.

16. The method according to claim 1, wherein the method includes measuring the TSAT and / or serum iron levels in the subject before and after administration of the hepcidin mimetic to the subject, and optionally, the TSAT level is measured at the trough level of the hepcidin mimetic after administration to the subject.

17. The composition according to claim 1, wherein the hepcidin mimetic is administered subcutaneously to the subject.

18. The composition according to claim 1, wherein the subject has received phlebotomy at a phlebotomy frequency of optionally 0.25 to 1 time per month for at least 6 months before treatment.

19. The composition according to claim 18, wherein during the treatment, the subject required substantially less phlebotomy or no phlebotomy at a phlebotomy frequency of optionally less than 0.1 time per month, less than 0.05 time per month, or no phlebotomy.

20. A composition for use in a method of treating hereditary hemochromatosis in a human subject, the composition comprising an effective amount of a compound 25 having the following formula: [Chemical Formula 6] Or a pharmaceutically acceptable salt thereof, the method comprising administering the composition to the subject, the effective amount comprising a dosage in the range of about 5 mg to about 40 mg, and optionally, the subject being administered different dosages during different periods throughout the treatment process.

21. A composition for use in a method of treating hereditary hemochromatosis in a human subject, the composition comprising an effective amount of isovaleryl-DTHFPCI (K(isoGlu-Palm))FEPRSKGCK-NH ２ A peptide having the sequence of (SEQ ID NO: 25), or a pharmaceutically acceptable salt thereof, the method comprising administering the composition to the subject, the thiol groups of two cysteine residues in the peptide optionally forming a disulfide bond, the effective amount comprising a dosage in the range of about 5 mg to about 40 mg, and optionally, the subject being administered different dosages during different periods throughout the treatment process.

22. A composition for use in a method of treating hereditary hemochromatosis arthritis or joint pain associated with hereditary hemochromatosis arthritis in a human subject, the composition comprising an effective amount of a hepcidin mimetic, the method comprising administering the composition to the subject.

23. The composition according to claim 22, wherein the effective amount comprises a dosage in the range of about 5 mg to about 40 mg, and optionally, the subject is administered different dosages during different periods over the course of treatment. **Claim 24** The composition according to claim 22 or 23, wherein the hepcidin mimetic is Compound 25.

Images