Conjugated hepcidin mimetic

Abstract

To provide a method for treating and / or preventing polycythemia vera (PV).SOLUTION: A method includes administering a hepcidin analog made of the following formula (I) or a pharmaceutically acceptable salt or solvate thereof to a subject. In the formula (I) R1-X-Y-R2, each of X and Y represents a specific peptide sequence, a peptide of the formula (I) is optionally pegylated in R1, X or Y, a side chain of amino acid of the peptide is optionally conjugated with a lipophilic substituent or a polymer part.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 895,201, filed September 3, 2019, U.S. Provisional Application No. 62 / 983,515, filed February 28, 2020, U.S. Provisional Application No. 63 / 020,945, filed May 6, 2020, and U.S. Provisional Application No. 63 / 059,747, filed July 31, 2020, all of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on September 1, 2020, is named PRTH_037_05WO_ST25.txt and is 25KB in size.

[0003] The present invention relates in particular to certain hepcidin peptide analogs, including both peptide monomers and peptide dimers, as well as conjugates and derivatives thereof, and the use of the peptide analogs in the treatment and / or prevention of polycythemia vera (PV). [Background technology]

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

[0005] Abnormal hepcidin activity is associated with iron overload disorders, including hereditary hemochromatosis (HH) and iron-loading anemia. Hereditary hemochromatosis is a genetic iron overload disorder primarily caused by hepcidin deficiency or, in some cases, hepcidin resistance. This can lead to excessive dietary iron absorption and the development of iron overload. Clinical manifestations of HH can include liver disease (e.g., cirrhosis and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is regular phlebotomy, which places a significant burden on patients. Iron-loading anemia is a hereditary anemia associated with ineffective red blood cell formation, such as β-thalassemia, which is associated with severe iron overload. Complications due to iron overload are a major cause of morbidity and mortality in these patients. Hepcidin deficiency is the primary cause of iron overload in non-transfused patients and contributes to iron overload in transfused patients. The current treatment for iron overload in these patients is iron chelation, which is very burdensome, sometimes ineffective, and associated with frequent side effects. Hepcidin has many limitations that limit its use as a drug, including a difficult synthetic process due in part to protein aggregation and deposition upon folding, which in turn results in a high cost of goods.

[0006] US Patents US 9,822,157 and US 10,030,061 describe novel hepcidin analogues and their use for treating iron overload diseases, including hereditary hemochromatosis and iron-loading anemia.

[0007] PCT Application Publication No. WO15200916 describes additional novel hepcidin analogs and their use for treating iron overload diseases.

[0008] PCT Application Publication No. WO17117411 describes additional novel hepcidin analogs with improved in vivo half-lives and their use for treating iron overload diseases.

[0009] PCT Application Publication No. WO18048944 describes additional novel hepcidin analogs and their use for treating the prevention of iron overload in a subject and / or reducing serum iron levels in a subject.

[0010] PCT Application Publication No. WO18128828 describes additional novel hepcidin analogs and their uses for treating hepcidin-associated disorders, including the prevention and treatment of iron overload diseases such as hemochromatosis, iron-loading anemias such as thalassemia, and diseases associated with ineffective or enhanced red blood cell formation.

[0011] PCT Application Publication WO17068089 describes additional novel hepcidin analogs (ferroportin inhibitors) and their use for treating thalassemia and hemochromatosis.

[0012] US Patent US9315545 describes additional novel hepcidin analogues and their use for treating diseases of iron metabolism, beta thalassemia, hemochromatosis, iron-loading anemia, alcoholic liver disease or chronic hepatitis C.

[0013] Polycythemia vera (PV) is a chronic, progressive trilineage clonal disorder manifested by increased bone marrow, erythroid, and megakaryocyte proliferation / accumulation and characterized by the World Health Organization (WHO) as a myeloproliferative neoplasm (Arber et al., 2016, 127(20):2391-405). Diagnosis is defined by two criteria: the first is increased erythroid mass, bone marrow biopsy demonstrating trilineage hypercellularity, and the presence of a JAK2V617F or JAK2 exon 12 mutation; the second incorporates polycythemia, bone marrow biopsy confirmation, and subnormal serum erythropoietin levels (Arber et al., 2016, 127(20):2391-405).

[0014] An estimated 148,000 people in the United States are living with PV, with a median age at diagnosis of 61 years (Stein et al., J Clin Oncol. 2015 Nov 20;33(33):3953-60). Symptoms of polycythemia associated with blood hyperviscosity include fatigue, bone pain, headache, dizziness, visual disturbances, atypical chest pain, pruritus, erythromelalgia, and paresthesias (Tefferi et al., Blood Cancer J. 2018,8(1):3). Clinical features include splenomegaly, thrombotic and hemorrhagic complications, and a risk of leukemic transformation.

[0015] Because PV is a disease characterized by increased erythropoiesis, animal models have shown that high-dose hepcidin mimetics can ameliorate this disease by reducing erythropoiesis (Casu et al., Blood. 2016;128(2):265-276). In PV mice expressing the orthologous JAK2 mutation that causes human PV, administration of minihepcidin significantly reduced splenomegaly and normalized hematocrit. These studies indicate that drug-like minihepcidin has potential as a future treatment for non-transfusion β-thalassemia and PV (Casu et al., Blood. 2016;128(2):265-276).

[0016] There is a clear need for new therapeutic agents and methods for treating and preventing PV, including PV in high-risk patients or patients in whom phlebotomy is not acceptable. The present invention addresses this need to treat PV. Summary of the Invention

[0017] In a specific aspect, the present invention provides a method of treating polycythemia vera in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of a hepcidin analog.

[0018] In a more specific aspect, the present invention provides a method of treating polycythemia vera in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a hepcidin analog and a pharmaceutically acceptable carrier, diluent, or excipient.

[0019] In one embodiment, the hepcidin analog has formula (I): R1-XY-R2(I) (SEQ ID NO: 1) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C20 alkanoyl, or pGlu; R2 is NH2 or OH; X is a peptide sequence having formula II, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10(II) (Sequence number 2) 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, or D-His; 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 a peptide sequence having formula III, Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15(III) (SEQ ID NO: 3) 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 peptides of Formula I above are optionally PEGylated at R, X, or Y, and the side chains of the amino acids of the peptides are optionally conjugated to lipophilic substituents or polymer moieties. In a related embodiment, Formula II is shown above, but X is D-Lys.

[0020] In one embodiment, R1 is hydrogen, isovaleric acid, isobutyric acid, or acetyl.

[0021] In certain embodiments of any of the hepcidin analogs or dimers of the present invention, the half-life extending moiety is selected from C12 (lauric acid), C14 (mysteric acid), C16 (palmitic acid), C18 (stearic acid), C20, C12 diacids, C14 diacids, C16 diacids, C18 diacids, C20 diacids, biotin, and isovaleric acid. In certain embodiments, the half-life extending moiety is attached to a linker moiety that is attached to the peptide. In certain embodiments, the half-life extending moiety increases the molecular weight of the hepcidin analog by about 50 D to about 2 KD. In various embodiments, the half-life extending moiety increases the serum half-life, improves solubility, and / or improves bioavailability of the hepcidin analog.

[0022] In certain embodiments, a peptide analog or dimer of the invention comprises an isovaleric acid moiety conjugated to an N-terminal Asp residue.

[0023] In certain embodiments, peptide analogs of the invention comprise an amidated C-terminal residue.

[0024] In certain embodiments, the hepcidin analog or dimer of the invention comprises Asp-Thr-His-Phe-Pro-Cys-Ile-Lys-Phe-Glu-Pro-Arg-Ser-Lys-Gly-Cys-Lys (SEQ ID NO: 19), or a sequence having at least 80%, at least 90%, or at least 94% identity thereto.

[0025] In certain embodiments, the hepcidin analog or dimer of the invention comprises Asp-Thr-His-Phe-Pro-Cys-Ile-Lys-Phe-Pro-Arg-Ser-Lys-Gly-Cys-Lys (SEQ ID NO: 19), or a sequence having at least 80%, at least 90%, or at least 94% identity thereto.

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

[0027] In a further related embodiment, the invention includes a vector comprising a polynucleotide of the invention.

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

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

[0030] In another embodiment, the present invention provides a method for treating polycythemia vera.

[0031] In a further embodiment, the present invention includes a method for treating polycythemia vera in a subject in need thereof, comprising providing an effective amount of a hepcidin analog or pharmaceutical composition of the present invention to the subject. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal, or topical administration. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral or subcutaneous administration. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject at most twice or about twice a day, at most once or about once a day, at most once or about once every two days, at most once or about once a week, or at most once or about once a month.

[0032] In certain embodiments, the hepcidin analog is provided to a subject at a dose of about 10 mg to about 100 mg, about 10 mg to about 80 mg, or about 10 mg to about 50 mg. In more specific embodiments, the hepcidin analog is provided to a subject at a dose of about 20 mg to about 40 mg. In certain embodiments, the hepcidin analog is provided to a subject at a dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg. In certain embodiments, the hepcidin analog is provided to a subject about once per week. In another specific embodiment, the hepcidin analog is provided to a subject about twice per week, e.g., subcutaneously.

[0033] In certain embodiments, any of the disclosed methods further includes determining the subject's hematocrit level at one or more time points after administration of the hepcidin analog and maintaining or adjusting the amount of the hepcidin analog or its pharmaceutically acceptable salt administered to the subject, increasing the amount if the subject's determined hematocrit is greater than 44% or greater than 45%, decreasing the amount if the subject's determined hematocrit is either less than 37.5% or less than 40%, and maintaining the amount if the subject's determined hematocrit is between 37.5% and 45%, between 37.5% and 44%, between 40% and 45%, or between 40% and 44%.

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

[0035] In yet another embodiment, the invention includes a kit comprising a pharmaceutical composition of the invention packaged with reagents, devices, or instructions, or a combination thereof.

[0036] In certain embodiments of any of the methods of the present disclosure, the polycythemia vera is phlebotomy-requiring polycythemia vera or phlebotomy-requiring polycythemia vera in a low-risk patient.

[0037] In certain embodiments of any of the disclosed methods, the subject is a low-risk polycythemia vera patient, a high-risk polycythemia vera patient, a symptomatic polycythemia vera patient requiring phlebotomy, a high-risk patient with polycythemia vera requiring phlebotomy, or a low-risk patient with polycythemia vera requiring phlebotomy.

[0038] In certain embodiments of any of the disclosed methods, the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies with a goal of a hematocrit of 45% or greater in the 24 weeks prior to administering the hepcidin analog or peptide to the subject.

[0039] In certain embodiments of any of the disclosed methods, the subject is administered about 5 mg to about 200 mg of a hepcidin analog or peptide, e.g., about 10 mg to about 100 mg, about 20 mg to about 100 mg, about 20 mg, about 40 mg, about 80 mg, about 100 mg, or about 120 mg.

[0040] In certain embodiments of any of the methods of the present disclosure, the pharmaceutical composition is administered by subcutaneous injection.

[0041] In certain embodiments of any of the disclosed methods, the pharmaceutical composition is administered about weekly over a period of time.

[0042] In certain embodiments of any of the disclosed methods, the amount of hepcidin analog or peptide administered is increased over a period of time.

[0043] In certain embodiments of any of the methods of the present disclosure, the subject is a mammal, for example, a human.

[0044] In certain embodiments of any of the disclosed methods, the method reduces the subject's hematocrit level by 45% or less, reduces the subject's hematocrit by at least 3%, and / or increases the subject's serum ferritin. In some embodiments, the subject remains phlebotomy-free during the course of treatment, e.g., about once per week over a period of time. [Brief explanation of the drawings]

[0045] [Figure 1]

[0023] Figure 1 describes data obtained from Cobas Iron2 assay experiments for Compound A and Compound B. A is a graph showing serum iron levels and serum concentrations of Compound A at the indicated times after treatment with Compound A. B is a graph showing serum iron levels and serum concentrations of Compound B at the indicated times after treatment with Compound B. [Figure 2] Figure 1 shows time profiles of hematocrit and RBC indices in male cynomolgus monkeys after subcutaneous administration of vehicle (○) or Compound A at doses of 1 (●), 3 (□), and 10 (■) mg / kg / dose once weekly for four doses with a 28-day recovery period. Compound A induced changes in secondary hematological indices (mean corpuscular hemoglobin concentration, MCHC, and mean corpuscular hemoglobin, MCH) indicative of iron-restricted erythropoiesis. Each point represents the mean ± SD of up to six animals (all groups) during the main treatment period and up to two animals (vehicle and 10 mg / kg Compound A) during the recovery period. Arrows indicate when Compound A was administered. [Figure 3] This shows that Compound A induces significant changes in hematocrit (Hct) and secondary hematological indices. Changes in Hct, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were assessed after subcutaneous administration of vehicle (○) or doses of 0.6 (●), 2 (□), and 6 (■) mg / kg of Compound A once weekly for 13 consecutive weeks, followed by a 35-day recovery period. Arrows indicate days of administration, starting with day 1. Each point represents the mean ± SD of up to 6 animals / sex during the main phase and up to 2 animals / sex during the recovery phase. [Figure 4]Figure 1 shows bilirubin profiles consistent with iron-restricted erythropoiesis in iron-replete cynomolgus monkeys. Total bilirubin levels in male (left) and female (right) cynomolgus monkeys after subcutaneous administration of vehicle (○) or doses of 0.6 (●), 2 (□), and 6 (■) mg / kg of Compound A once weekly for 13 consecutive weeks, followed by a 35-day recovery period. Arrows indicate days of administration, starting with day 1. Each point represents the mean ± SD of up to 6 animals / sex during the main phase and up to 2 animals / sex during the recovery phase. [Figure 5] Platelet profiles are shown in male (left) and female (right) cynomolgus monkeys after 13 consecutive weeks of subcutaneous administration of Compound A at doses of vehicle (○) or 0.6 (●), 2 (□), and 6 (■) mg / kg, followed by a 35-day recovery period. Arrows indicate days of administration, starting with day 1. Each point represents the mean ± SD of up to 6 animals / sex during the main phase and up to 2 animals / sex during the recovery phase. [Figure 6] 1 provides an illustration of the Phase II clinical trial design. [Figure 7] 1 is a timeline showing the timing of therapeutic phlebotomy and treatment with Compound A at the indicated doses for 13 human PV patients. [Figure 8] 1 is a graph showing ferritin (ng / mL) levels in PV patients treated with Compound A at the indicated time points. The number of subjects correlates with that shown in Table 9. [Figure 9] 1 is a graph showing TSAT (%) in PV patients treated with Compound A at the indicated time points. Numbers of subjects correlate with those shown in Table 9. [Figure 10] Graph showing MCV (fL) in PV patients treated with Compound A at the indicated time points. Numbers of subjects correlate with those shown in Table 9. [Figure 11] 1 is a graph showing MCH (pg) in PV patients treated with Compound A at the indicated time points. The number of subjects correlates with that shown in Table 9. [Figure 12] 1 is a graph showing hematocrit (%) in PV patients treated with Compound A at the indicated time points. Numbers of subjects correlate with those shown in Table 9. [Figure 13] 1 is a graph showing red blood cells (106 / uL) in PV patients treated with Compound A at the indicated time points. Subject numbers correlate with those shown in Table 9. [Figure 14] 14-16 are graphs showing platelets (103 / uL) in PV patients before and after treatment with the indicated doses of Compound A at the indicated time points. For Figures 14-16, the number of subjects shown correlates with the number of subjects provided in Table 9 as follows: 1501-01=3, 1501-02=5, 1502-1=1, 1502-02=2, 1502-04=4, 1505-01=6, 1505-02=7, and 1509-01=8. [Figure 15] 1 is a graph showing reticulocytes (%) in PV patients before and after treatment with Compound A at the indicated doses at the indicated time points. [Figure 16] 1 is a graph showing white blood cells ( / uL) in PV patients before and after treatment with Compound A at the indicated doses at the indicated time points. [Figure 17] 1 shows the plasma concentrations of Compound A in PV patients at various times after administration of the indicated amounts of Compound A. A shows data for individual time points, and B shows the average over the specified time interval. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present disclosure relates generally to the use of hepcidin analog peptides to treat and prevent polycythemia vera (PV).

[0047] Hepcidin (also called LEAP-1), a peptide hormone produced by the liver, is a regulator of iron homeostasis in humans and other mammals. Hepcidin acts by binding to its receptor, the iron export channel ferroportin, causing its internalization and degradation. Human hepcidin is a 25-amino acid peptide (Hep25). See Krause et al. (2000) FEBS Lett 480:147-150 and Park et al. (2001) J Biol Chem 276:7806-7810. The structure of the biologically active 25-amino acid form of hepcidin is a simple hairpin with eight cysteines that form four disulfide bonds, as described by Jordan et al. J Biol Chem 284:24155-67. The N-terminal region is required for iron regulatory function, and deletion of the five N-terminal amino acid residues results in loss of iron regulatory function. See Nemeth et al. (2006) Blood 107:328-33.

[0048] Abnormal hepcidin activity is associated with iron overload disorders, including hereditary hemochromatosis (HH) and iron-loaded anemia. Hereditary hemochromatosis is a genetic iron overload disorder primarily caused by hepcidin deficiency or, in some cases, hepcidin resistance. This can lead to excessive dietary iron absorption and the development of iron overload. Clinical symptoms of HH can include liver disease (e.g., cirrhosis and hepatocellular carcinoma), diabetes, and heart failure. Iron-loaded anemia is a genetic anemia associated with ineffective erythropoiesis, such as β-thalassemia, accompanied by severe iron overload.

[0049] Hepcidin has many limitations that limit its use as a drug, including a difficult synthesis process due in part to protein aggregation and precipitation upon folding, which in turn leads to a high cost of goods. The present disclosure provides hepcidin analog peptides that have hepcidin activity and also possess other beneficial physical properties, such as improved solubility, stability, and / or efficacy, so that hepcidin-like biologics can be produced affordably and used to treat and prevent polycythemia vera.

[0050] The present invention also generally relates to hepcidin analog peptides and methods for making and using them. In certain embodiments, hepcidin analogs exhibit one or more hepcidin activities. In certain embodiments, the present invention relates to hepcidin peptide analogs comprising one or more peptide subunits that form a cyclized structure via an intramolecular bond, e.g., an intramolecular disulfide bond. In certain embodiments, the cyclized structure has increased potency and selectivity compared to non-cyclized hepcidin peptides and analogs thereof. In certain embodiments, the hepcidin analog peptides of the present invention exhibit an increased half-life, e.g., when delivered orally, compared to hepcidin or previous hepcidin analogs.

[0051] Definitions and Terminology 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 terms 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.

[0052] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0053] It will be understood that throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" means 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).

[0054] The singular forms "a," "an," and "the" include plural forms unless the context clearly indicates otherwise.

[0055] The term "including" is used to mean "including, but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0056] The terms "patient," "subject," and "individual" can be used interchangeably and 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.

[0057] The term "peptide," as used herein, broadly refers to a sequence of two or more amino acids joined by peptide bonds. It should be understood that the term does not connote a specific length of a polymer of amino acids, nor is it intended to mean or distinguish whether the polypeptide is produced using recombinant techniques, chemical synthesis, enzymatic synthesis, or naturally occurring.

[0058] The term "peptide analog," as used herein, broadly refers to peptide monomers and peptide dimers that share one or more structural features and / or functional activities with hepcidin or a functional region thereof. In certain embodiments, peptide analogs include peptides that share substantial amino acid sequence identity with hepcidin, e.g., peptides that contain one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence of wild-type hepcidin, e.g., human hepcidin. In certain embodiments, peptide analogs contain one or more additional modifications, such as, for example, conjugation to another compound. Any peptide monomer or peptide dimer of the present invention is encompassed by the term "peptide analog." In certain cases, "peptide analogs" may also or alternatively be referred to herein as "hepcidin analogs," "hepcidin peptide analogs," or "hepcidin analog peptides."

[0059] As used herein, the terms "sequence identity," "percent identity," "percent homology," or terms including, for example, "a sequence 50% identical to" refer to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0060] Calculation of sequence similarity or sequence identity (the terms are used interchangeably herein) between sequences can be performed as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0061] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0062] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program in the GCG software package, using a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4:11-17) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0063] The peptide sequences described herein can be used as "query sequences" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol. 215:403-10). Using the NBLAST program, BLAST nucleotide searches can be performed with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. Using the XBLAST program, BLAST protein searches can be performed with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST, as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997), can be used. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0064] As used herein, the term "conservative substitution" refers to the replacement of one or more amino acids with biologically similar residues. Examples include the substitution of amino acid residues with similar characteristics, such as small, acidic, polar, basic, hydrophobic, and aromatic amino acids. See, for example, the table below. In some embodiments of the invention, one or more Met residues are substituted with norleucine (Nle), which is a bioisomer of Met but, in contrast to Met, is not readily oxidized. In some embodiments, one or more Trp residues are substituted with Phe, or one or more Phe residues are substituted with Trp, while in some embodiments, one or more Pro residues are substituted with Npc, or one or more Npc residues are substituted with Pro. Another example of a conservative substitution with a residue not normally found in endogenous mammalian peptides and proteins is, for example, the conservative substitution of Arg or Lys with ornithine, canavanine, aminoethylcysteine, or another basic amino acid. In some embodiments, another conservative substitution is substitution of one or more Pro residues with bhPro or Leu or D-Npc (isonipecotic acid). For more information on phenotypically silent substitutions in peptides and proteins, see, e.g., Bowie et al. Science 247, 1306-1310, 1990. In the following scheme, conservative amino acid substitutions are grouped by physicochemical properties: I: neutral, hydrophilic; II: acid and amide; III: basic; IV: hydrophobic; V: bulky aromatic amino acids. [Table 1]

[0065] In the following scheme, conservative substitutions of amino acids are grouped by physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. [Table 2]

[0066] As used herein, the term "amino acid" or "any amino acid" refers to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. This includes both D- 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 a wide variety of proteins. These are primarily L-stereoisomers, although several D-amino acids occur in bacterial envelopes and some antibiotics. The 20 "standard" naturally occurring amino acids are listed in the table above. "Non-standard" naturally occurring amino acids are pyrrolysine (found in methanogens and other eukaryotes), selenocysteine ​​(present in most eukaryotes as well as many non-eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Non-natural" or "unnatural" amino acids are non-proteinogenic amino acids (i.e., amino acids that are not naturally encoded, i.e., not found in the genetic code), either occurring in nature or chemically synthesized. Over 140 naturally occurring amino acids are known, with thousands more combinations possible. Examples of "non-natural" amino acids include β-amino acids (β 3 and β 2 ), homoamino acids, proline and pyruvate derivatives, tri-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methylamino 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 present on the amino acid.

[0067] As will be apparent to those skilled in the art, 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 sequences disclosed herein incorporate a "Hy-" moiety at the amino terminus (N-terminus) of the sequence and either a "-OH" or "-NH2" moiety at the carboxy terminus (C-terminus) of the sequence. In such cases, unless otherwise indicated, the "Hy-" moiety at the N-terminus of the sequence indicates a hydrogen atom corresponding to the presence of a free primary or secondary amino group at the N-terminus, and the "-OH" or "-NH2" moiety at the C-terminus of the sequence indicates a hydroxyl or amino group, respectively, corresponding to the presence of an amide (CONH2) group at the C-terminus. In each of the sequences of the present invention, a C-terminal "-OH" moiety can be substituted for a C-terminal "-NH2" moiety, and vice versa. It is further understood that the amino- or carboxy-terminal moiety can be a bond, e.g., a covalent bond, particularly in situations where the amino- or carboxy-terminal moiety is attached to a linker or another chemical moiety, e.g., a PEG moiety.

[0068] The term "NH2" 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. Furthermore, the term "Ac" as used herein refers to acetyl protection through acylation of the C-terminus or N-terminus of a polypeptide.

[0069] The term "carboxy" as used herein refers to -CO2H.

[0070] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the nomenclature conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature, as presented 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 these suggestions, they will be made clear to the reader. Some abbreviations useful in describing this invention are defined below in Table 1, below. [Table 3-1] [Table 3-2] [Table 3-3]

[0071] Throughout this specification, naturally occurring amino acids are designated by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.), unless they are referred to by their full names (e.g., alanine, arginine, etc.). For less common or non-naturally occurring amino acids, unless they are referred to by their full names (e.g., sarcosine, ornithine, etc.), the frequently used three-letter or four-letter codes are used for those residues, including 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 (pyridinic acid), ... These include β-glutamic acid), GABA (γ-aminobutanoic acid), β-Pro (pyrrolidine-3-carboxylic acid), 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (4-aminobutyric acid), bhPro (β-homo-proline), bhPhe (β-homo-L-phenylalanine), bhAsp (β-homo-aspartic acid), Dpa (β,β-diphenylalanine), Ida (iminodiacetic acid), hCys (homocysteine), and bhDpa (β-homo-β,β-diphenylalanine).

[0072] Furthermore, R 1 can be substituted with isovaleric acid or an equivalent in all sequences. In some embodiments, the peptides of the present invention are conjugated to an acidic compound, such as, for example, isovaleric acid, isobutyric acid, valeric acid, etc., and the presence of such conjugates is referred to in their 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.

[0073] The term "L-amino acid," as used herein, refers to a peptide in its "L" isomeric form; conversely, the term "D-amino acid" refers to a peptide in its "D" isomeric form. In certain embodiments, the amino acid residues described herein are in their "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 function is retained by the peptide.

[0074] Unless otherwise indicated, reference is made to the L-isomeric forms of the relevant natural and unnatural amino acids having a chiral center. Where appropriate, the D-isomeric 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).

[0075] As used herein, a "lower homologue of Lys" refers to an amino acid that has the structure of lysine but has one or more fewer carbons in its side chain compared to lysine.

[0076] As used herein, a "higher homologue of Lys" refers to an amino acid that has the structure of lysine but has one or more additional carbon atoms in its side chain compared to lysine.

[0077] The term "DRP" as used herein refers to a disulfide-rich peptide.

[0078] 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. Each monomeric subunit of a dimer may be linked via the same site, or each may be linked via a different site (e.g., C-terminus, N-terminus, or internal site).

[0079] As used herein, in the context of specific peptide sequences disclosed herein, parentheses, e.g., (__), represent a side chain conjugation, and square brackets, e.g., [__], represent a non-natural amino acid substitution or amino acid and a conjugated side chain. Generally, 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. Generally, 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.

[0080] The terms "isostere replacement" or "isostere substitution" are used interchangeably herein and refer to any amino acid or other analog moiety that has similar chemical and / or structural properties as a particular amino acid. In certain embodiments, an isostere substitution is a conservative substitution with a natural or unnatural amino acid.

[0081] The term "cyclization," as used herein, refers to a reaction in which one portion of a polypeptide molecule is linked to another portion of a polypeptide molecule to form a closed ring, such as by a disulfide bridge or other similar bond.

[0082] The term "subunit," as used herein, refers to one of a pair of polypeptide monomers that combine to form a dimeric peptide composition.

[0083] The term "linker moiety," as used herein, broadly refers to a chemical structure that can link or connect two peptide monomer subunits to form a dimer.

[0084] 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 pharmaceutically acceptable salt thereof) and a solvent. The solvent in this context may be, for example, water, ethanol, or another pharmaceutically acceptable species, typically a small 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.

[0085] The term "pharmaceutically acceptable salt," as used herein, refers to a salt or zwitterionic form of a compound of the present invention that is water- or oil-soluble or dispersible, suitable for the treatment of disease without undue toxicity, irritation, or allergic reaction, consistent with a reasonable benefit / risk ratio, and effective for its intended use. Salts can be prepared during the final isolation and purification of the compound or separately by reacting an amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, mesitylene, and the like. The salts of the amino groups in the compounds of the present invention include sulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Additionally, amino groups in the compounds 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 and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. The pharmaceutically acceptable salt may suitably be, 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 N(R1)(R2)(R3)(R4)+ type ions (where R1, R2, R3 and R4 independently typically represent hydrogen, optionally substituted C1-6-alkyl, or optionally substituted C2-6-alkenyl). Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. Examples of possible relevant C2-6-alkenyl groups include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66:2 (1977). For a review of suitable salts, see also 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.

[0086] The term "N(alpha) methylation," as used herein, describes the methylation of the alpha amine of an amino acid, also commonly referred to as N-methylation.

[0087] The term "symmetric methylation" or "Arg-Me-sym" as used herein describes the symmetric methylation of the two nitrogens of the guanidine group of arginine. Additionally, the term "asymmetric methylation" or "Arg-Me-asym" describes the methylation of a single nitrogen of the guanidine group of arginine.

[0088] The term "acylating organic compound," as used herein, refers to various compounds having a carboxylic acid functional group that are used to acylate the N-terminus of an amino acid subunit prior to forming a C-terminal dimer. Non-limiting examples of acylating organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3,3,3-trifluoropropionic acid, 3-fluoromethylbutyric acid, and tetrahedro-2H-pyran-4-carboxylic acid.

[0089] The term "alkyl" includes straight-chain or branched, acyclic or cyclic saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; saturated branched 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.

[0090] 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 disorders). In certain embodiments, a therapeutically effective amount achieves a desired benefit / risk ratio applicable to any medical treatment.

[0091] Hematocrit is the ratio of red blood cell volume to whole blood volume. The normal range of hematocrit differs between the sexes, approximately 45% to 52% for men and approximately 37% to 48% for women. The clinical goal of PV treatment is to achieve a hematocrit of less than 45%. Hematocrit is sometimes referred to herein as the hematocrit level, and a numerical value for the hematocrit level, e.g., 45, is understood to mean a hematocrit of 45%.

[0092] Peptide analogs of hepcidin The present invention provides peptide analogs of hepcidin (collectively "hepcidin analogs"), which can be monomeric or dimeric.

[0093] In some embodiments, the hepcidin analogs of the present invention bind to ferroportin, e.g., human ferroportin. In certain embodiments, the hepcidin analogs of the present invention specifically bind to human ferroportin. As used herein, "specifically bind" refers to the preferential interaction of a specific binding agent with a given ligand over other agents in a sample. For example, a specific binding agent that specifically binds to a given ligand binds to the given ligand in an observable amount or degree that exceeds the amount or degree of any non-specific interactions with other components in the sample under suitable conditions. Suitable conditions are conditions that allow the interaction between a given specific binding agent and a given ligand. These conditions include pH, temperature, concentration, solvent, incubation time, etc., and may vary between a given specific binding agent and ligand pair but can be easily determined by one of skill in the art. In some embodiments, the hepcidin analogs of the present invention bind to ferroportin with greater specificity than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the present invention exhibit ferroportin specificity that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1000%, or 10,000% greater than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein). In some embodiments, the hepcidin analogs of the present invention exhibit ferroportin specificity that is at least about 5-fold, or at least about 10-fold, 20-fold, 50-fold, or 100-fold greater than a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0094] In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity. In some embodiments, the activity is in vitro or in vivo activity, for example, the in vivo or in vitro activity described herein. In some embodiments, the hepcidin analogs of the present invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more than 99% of the activity exhibited by a hepcidin reference compound (e.g., any one of the hepcidin reference compounds provided herein).

[0095] In some embodiments, the hepcidin analogs of the present invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99% of the ferroportin binding capacity exhibited by a reference hepcidin. In some embodiments, the hepcidin analogs of the present invention exhibit a lower IC for binding to ferroportin (e.g., human ferroportin) compared to the reference hepcidin. 50 In some embodiments, the hepcidin analogs of the present invention have an IC that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than a reference hepcidin in a ferroportin competitive binding assay. 50 It has.

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

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

[0098] In some embodiments, the peptides or peptide dimers of the invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or greater than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or greater than 1000% in vivo activity for inducing a reduction of free plasma iron in an individual as compared to a reference hepcidin, wherein activity is measured according to the methods described herein.

[0099] In some embodiments, the activity is an in vitro or in vivo activity, eg, an in vivo or in vitro activity described herein. In certain embodiments, hepcidin analogs of the invention exhibit 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200-fold, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% greater activity than a reference hepcidin, wherein the activity is in vitro activity for inducing degradation of ferroportin, e.g., as measured according to the Examples herein, or the activity is in vivo activity for reducing free plasma iron, e.g., as measured according to the Examples herein.

[0100] In some embodiments, the hepcidin analog of the present invention mimics the hepcidin activity of Hep25, the biologically active human 25-amino acid form, and is herein referred to as "minihepcidin." As used herein, in certain embodiments, a compound (e.g., a hepcidin analog) with "hepcidin activity" means that when administered to a subject (e.g., a mouse or human) (e.g., parenterally injected or orally administered), the compound has the ability to reduce the plasma iron concentration in the subject in a dose-dependent and time-dependent manner. See, for example, Rivera et al. (2005), Blood 106:2196-9. In some embodiments, the peptides of the invention reduce plasma iron concentrations in a subject by at least about 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

[0101] In some embodiments, the hepcidin analogs of the present invention have in vitro activity as assayed by their ability to cause internalization and degradation of ferroportin in ferroportin-expressing cell lines as taught in Nemeth et al. (2006) Blood 107:328-33. In some embodiments, in vitro activity is measured by the dose-dependent loss of fluorescence in cells engineered to display ferroportin fused to green fluorescent protein as in Nemeth et al. (2006) Blood 107:328-33. Aliquots of cells are incubated for 24 hours with gradient concentrations of a reference preparation of Hep25 or minihepcidin. As provided herein, EC 50 Values ​​are provided as the concentration of a given compound (e.g., a hepcidin analog peptide or peptide dimer of the invention) that causes 50% of the maximal loss of fluorescence produced by the reference compound. EC 50 In certain embodiments, preferred hepcidin analogs of the present invention have an EC of about 1,000 nM or less in an in vitro activity assay. 50 In certain embodiments, hepcidin analogs of the invention have an EC value of less than any one of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM in an in vitro activity assay (e.g., as described in Nemeth et al. (2006) Blood 107:328-33 or in the Examples herein). 50 In some embodiments, the hepcidin analog or biotherapeutic composition (e.g., any one of the pharmaceutical compositions described herein) has an EC 50 It has a value.

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

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

[0104] In certain embodiments, the hepcidin analogs of the present invention exhibit a longer half-life than a reference hepcidin. In certain embodiments, the hepcidin analogs of the present invention exhibit a half-life of at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 4 days, at least about 7 days, at least about 10 days, under a given set of conditions (e.g., temperature, pH). The hepcidin analogs have a half-life of at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months or more, or any intermediate half-life or intermediate range, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 4 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months or more, or any intermediate half-life or intermediate range. In some embodiments, the half-life of the hepcidin analogs of the present invention is extended by conjugation to one or more lipophilic substituents or half-life extending moieties, such as any of the lipophilic substituents or half-life extending moieties disclosed herein. In some embodiments, the half-life of the hepcidin analogs of the present invention is extended by conjugation to one or more polymer moieties, such as any of the polymer moieties or half-life extending moieties disclosed herein. In certain embodiments, the hepcidin analogs of the present invention have the above-mentioned half-lives under a given set of conditions, where the temperature is about 25°C, about 4°C, or about 37°C, and the pH is physiological pH or about pH 7.4.

[0105] In certain embodiments, hepcidin analogs of the invention comprising a conjugated half-life extending moiety have an increased serum half-life after oral, intravenous, or subcutaneous administration compared to the same analog but lacking the conjugated half-life extending moiety. In certain embodiments, the serum half-life of a hepcidin analog of the invention after oral, intravenous, or subcutaneous administration is at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 72 hours, or at least 168 hours. In certain embodiments, this is 12-168 hours, 24-168 hours, 36-168 hours, or 48-168 hours.

[0106] In certain embodiments, hepcidin analogs of the present invention containing a conjugated half-life extending moiety result in a reduction in serum iron concentrations after oral, intravenous, or subcutaneous administration to a subject. In certain embodiments, the subject's serum iron concentration is reduced by less than 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the serum iron concentration in the absence of administration of the hepcidin analog to the subject. In certain embodiments, the reduced serum iron concentration is maintained for at least 1 hour, at least 4 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours after administration to the subject. In certain embodiments, this is maintained for 12-168 hours, 24-168 hours, 36-168 hours, or 48-168 hours. In one embodiment, the subject's serum iron concentration is reduced by less than 20% about 4 hours or about 10 hours after administration, e.g., intravenously, orally, or subcutaneously, to the subject. In one embodiment, the subject's serum iron concentration is reduced by less than 50% or less than 60% about 24 to about 30 hours after administration, e.g., intravenously, orally, or subcutaneously.

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

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

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

[0110] In certain embodiments, the invention provides hepcidin analogs described herein, wherein the hepcidin analog exhibits at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times greater, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater solubility than a reference hepcidin in a particular solution or buffer, e.g., water or a buffer known in the art or disclosed herein.

[0111] In certain embodiments, the invention provides hepcidin analogs described herein, wherein the hepcidin analogs exhibit reduced aggregation, such that aggregation of the peptide in solution is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times less, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% less than a reference hepcidin in a particular solution or buffer, e.g., water, or a buffer known in the art or disclosed herein.

[0112] In some embodiments, the present invention provides hepcidin analogs described herein, wherein the hepcidin analogs exhibit less degradation (i.e., greater degradation stability) than reference hepcidin, for example, by more than or less than about 10%, more than or less than about 20%, more than or less than about 30%, more than or less than about 40%, or more than or less than about 50%. In some embodiments, degradation stability is determined by any suitable method known in the art. In some embodiments, suitable methods known in the art for determining degradation stability include the method described in Hawe et al. J Pharm Sci, VOL.101, NO.3, 2012, p 895-913, which is incorporated herein in its entirety. In some embodiments, such methods are used to select potent sequences with extended shelf life.

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

[0114] The various hepcidin analog monomer and dimer peptides of the present invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may contain non-natural or unnatural amino acids, including, but not limited to, modified amino acids. In certain embodiments, modified amino acids include natural amino acids that have been chemically modified to include a group, groups, or chemical moiety not naturally occurring on the amino acid. The hepcidin analogs of the present invention may further include D-amino acids. Still further, the hepcidin analog peptide monomers and dimers of the present invention may include amino acid analogs. In certain embodiments, the peptide analogs of the present invention include any of those described herein in which one or more natural amino acid residues of the peptide analog are replaced with a non-natural or unnatural amino acid, or a D-amino acid.

[0115] In certain embodiments, hepcidin analogs of the invention comprise one or more modified or unnatural amino acids. For example, in certain embodiments, hepcidin analogs include one or more of Daba, Dapa, Pen, Sar, Cit, Cav, HLeu, 2-Nal, 1-Nal, d-1-Nal, d-2-Nal, Bip, Phe(4-OMe), Tyr(4-OMe), βhTrp, βhPhe, Phe(4-CF3), 2-2-indan, 1-1-indan, cyclobutyl, βhPhe, hLeu, Gla, Phe(4-NH2), hPhe, 1-Nal, Nle, 3-3-diPhe, cyclobutyl-Ala, Cha, Bip, β-Glu, Phe(4-Guan), homoamino acids, D-amino acids, and various N-methylated amino acids. Those skilled in the art will understand that other modified or unnatural amino acids, and various other substitutions of natural amino acids with modified or unnatural amino acids, can be made to achieve similar desired results, and that such substitutions are within the teachings and spirit of the present invention.

[0116] The present invention includes any of the hepcidin analogs described herein, eg, in free or salt form.

[0117] The compounds described herein include isotopically labeled compounds that are identical to the compounds described in the various formulas and structures presented herein, except for the fact that one or more atoms are replaced with an atom having an atomic mass or mass number different from that usually found in nature. Examples of isotopes that can be incorporated into the compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, for example, 3 H and 14 Incorporation of radioactive isotopes such as 1C is useful in drug and / or substrate tissue distribution assays. 2 Substitution with isotopes such as H may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0118] The hepcidin analogs of the present invention comprise any of the peptide monomers or dimers described herein linked to a linker moiety, including certain linker moieties described herein.

[0119] Hepcidin analogs of the present invention include peptides, e.g., monomers or dimers, comprising peptide monomer subunits having at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to a hepcidin analog peptide sequence described herein (e.g., any one of the peptides disclosed herein), including, but not limited to, any of the amino acid sequences shown in Tables 2 and 3.

[0120] In certain embodiments, a peptide analog of the invention or a monomeric subunit of a dimeric peptide analog of the invention comprises or consists of 7-35 amino acid residues, 8-35 amino acid residues, 9-35 amino acid residues, 10-35 amino acid residues, 7-25 amino acid residues, 8-25 amino acid residues, 9-25 amino acid residues, 10-25 amino acid residues, 7-18 amino acid residues, 8-18 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues, and optionally one or more additional non-amino acid moieties, e.g., conjugated chemical moieties, e.g., half-life extending moieties, PEG, or linker moieties. In certain embodiments, a monomeric subunit of a hepcidin analog comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues. In certain embodiments, a monomeric subunit of a hepcidin analog of the invention comprises or consists of 10-18 amino acid residues and, optionally, one or more additional non-amino acid moieties, e.g., conjugated chemical moieties, e.g., PEG or linker moieties. In various embodiments, a monomeric subunit comprises or consists of 7-35 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues. In certain embodiments of any of the various formulas described herein, X comprises or consists of 7 to 35 amino acid residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues, 10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25 amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues.

[0121] In certain embodiments, hepcidin analogs according to the present invention include any and all hepcidin analogs disclosed in PCT Patent Application Publication Nos. WO2014 / 145561, WO2015 / 200916, or WO2017 / 117411, the entireties of which are incorporated herein by reference.

[0122] Peptide Monomeric Hepcidin Analogues In a specific aspect, the present invention provides a method of treating polycythemia vera in a subject in need thereof, comprising administering to the subject a hepcidin analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof.

[0123] In a more specific aspect, the present invention provides a method of treating polycythemia vera in a subject in need thereof, comprising administering to the subject an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a hepcidin analog or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, or excipient.

[0124] In one embodiment, the hepcidin analog has formula (I): R1-XY-R2(I) (SEQ ID NO: 1) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C20 alkanoyl, or pGlu; R2 is NH2 or OH; X is a peptide sequence having formula II, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10(II) (Sequence number 2) 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, or D-His; 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 a peptide sequence having formula III, Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15(III) (SEQ ID NO: 3) 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 peptides of formula I above are optionally PEGylated at R1, X or Y, and the side chains of the amino acids of the peptides are optionally conjugated to lipophilic substituents or polymer moieties.

[0125] In a related embodiment, Formula II is shown above for X1, X2, X4, X5, X6, X7, X8, X9, and X1, but X3 is His, Lys, D-His, or D-Lys.

[0126] In one embodiment, R1 is hydrogen, isovaleric acid, isobutyric acid, or acetyl. In another embodiment, R1 is isovaleric acid or isobutyric acid. In a particular embodiment, R1 is isovaleric acid.

[0127] In one embodiment, X is a peptide sequence having formula IV: X1-Thr-His-X4-X5-X6-X7-X8-Phe-X10(IV) (SEQ ID NO: 4) 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.

[0128] In another embodiment, X is a peptide sequence having the formula V: X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10(V) (SEQ ID NO: 5) 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.

[0129] In certain embodiments, the peptide is according to Formula VI: R 1 -XYR 2 (VI) (SEQ ID NO: 6) or a pharmaceutically acceptable salt thereof, wherein: R 1 is hydrogen, isovaleric acid, isobutyric acid or acetyl, R 2 is -NH2 or -OH, X is a peptide sequence having formula VII, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10(VII) (SEQ ID NO: 7) 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 a peptide sequence having formula VIII, Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10(VIII) (SEQ ID NO: 8) 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 of formula I above may optionally comprise R 1 PEGylated at X, Y, or 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.

[0130] In a further particular embodiment, the peptide has the following sequence: DTHFPICIFGPRSKGWVC (SEQ ID NO: 9), DTHFPCIIFGPRSKGWVCK (SEQ ID NO: 10), DTHFPCIIFEPRSKGWVCK (SEQ ID NO: 11), DTHFPCIIFGPRSKGWACK (SEQ ID NO: 12), DTHFPCIIFGPRSKGWVCKK (SEQ ID NO: 13), DTHFPCIIFVCHRPKGCYRRVCR (SEQ ID NO: 14), DTHFPCIKFGPRSKGWVCK (SEQ ID NO: 15), DTHFPCIKFKPRSKGWVCK (SEQ ID NO: 16), DTHFPCIIFGPRSRGWVCK (SEQ ID NO: 17), DTHFPCIKFGPKSKGWVCK (SEQ ID NO: 18), DTHFPCIKFEPRSKGCK (SEQ ID NO: 19), DTHFPCIKFEPKSKGWECK (SEQ ID NO: 20), DTHFPCIKFEPRSKKCK (SEQ ID NO: 21), DTHFPCIKFEPRSKGCKK (SEQ ID NO: 22), DTHFPCIKFKPRSKGCK (SEQ ID NO: 23), DTHFPCIKFEPKSKGCK (SEQ ID NO: 24), DTHFPCIKF (SEQ ID NO: 25), DTHFPCIIF (SEQ ID NO: 26) or DTKFPCIIF (SEQ ID NO: 27), The peptide is optionally PEGylated at R1, X, or Y, and the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer moieties.

[0131] In further specific embodiments, the hepcidin analog or peptide has the following sequence: Isovaleric acid-DTHFPICIFGPRSKGWVC-NH2 (SEQ ID NO: 9), Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH2 (SEQ ID NO: 10), Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH2 (SEQ ID NO: 11), Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH2 (SEQ ID NO: 12), Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH2 (SEQ ID NO: 13), Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH2 (SEQ ID NO: 14), Isovaleric acid-DTHFPCI(K(PEG))FGPRSKGWVCK-NH (SEQ ID NO: 28), Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH2 (SEQ ID NO: 16), Isovaleric acid-DTHFPICIFGPRS(K(PEG))GWVC-NH (SEQ ID NO: 29), Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH2 (SEQ ID NO: 30), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG))-NH2 (SEQ ID NO: 31), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG))-NH (SEQ ID NO: 32), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH2 (SEQ ID NO: 33), Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 34), Isovaleric acid-DTHFPCIKF)K(Palm))PRSKGWVCK-NH2 (SEQ ID NO: 35), Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH2 (SEQ ID NO: 36), Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH2 (SEQ ID NO: 37), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))NH2 (SEQ ID NO: 38), Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 39), Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40), Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH2 (SEQ ID NO: 41), Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH2 (SEQ ID NO: 42), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH2 (SEQ ID NO: 43), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG))-NH2 (SEQ ID NO: 44), Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45), Isovaleric acid-DTHFPCIKF-K(isoGlu-Palm)-PRSKGCK-NH2 (SEQ ID NO: 46), Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47), Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGWECK-NH2 (SEQ ID NO: 20), Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48), Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))CK-NH2 (SEQ ID NO: 21), Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH2 (SEQ ID NO: 49), Isovaleric acid-DTHFPCI-K(Dapa-Palm)-FEPRSKGCK-NH2 (SEQ ID NO: 50), Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGCK-NH2 (SEQ ID NO: 23), Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH2 (SEQ ID NO: 24), Isovaleric acid-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH2 (SEQ ID NO: 51), Isovaleric acid-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH2 (SEQ ID NO: 52), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))K-NH2 (SEQ ID NO: 53), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))-NH2 (SEQ ID NO: 54), Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH2 (SEQ ID NO: 55), Isovaleric acid-DTHFPCIKF-NH2 (SEQ ID NO: 25), Hy-DTHFPCIKF-NH2 (SEQ ID NO: 25), Isovaleric acid-DTHFPCIIF-NH2 (SEQ ID NO: 26), Hy-DTHFPCIIKF-NH2 (SEQ ID NO: 26), Isovaleric acid-DTKFPCIIF-NH2 (SEQ ID NO: 27) or Hy-DTKFPCIIF-NH2 (SEQ ID NO: 27).

[0132] In a more particular embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH2 (SEQ ID NO: 10).

[0133] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH2 (SEQ ID NO: 11).

[0134] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH2 (SEQ ID NO: 28).

[0135] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH2 (SEQ ID NO: 16).

[0136] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH2 (SEQ ID NO: 31).

[0137] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 34).

[0138] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKF(K(Palm))PRSKGWVCK-NH2 (SEQ ID NO: 35).

[0139] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH2 (SEQ ID NO: 36).

[0140] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))-NH2 (SEQ ID NO: 38).

[0141] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 39).

[0142] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40).

[0143] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH2 (SEQ ID NO: 41).

[0144] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH2 (SEQ ID NO: 42).

[0145] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH2 (SEQ ID NO: 43).

[0146] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH2 (SEQ ID NO: 44).

[0147] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45).

[0148] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46).

[0149] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47).

[0150] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48).

[0151] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCI(K(Dapa-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 50).

[0152] In a more specific embodiment, the hepcidin analog or peptide is isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH2 (SEQ ID NO: 24).

[0153] In more particular embodiments, the hepcidin analog or peptide is selected from the group consisting of: [ka] Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40), [ka] Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45), [ka] Isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46), [ka] Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) and [ka] Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48), as well as pharmaceutically acceptable salts of any of the foregoing, wherein the amino acid is an L-amino acid.

[0154] In some embodiments, the hepcidin analogs of the present invention are active in dimeric conformation, especially when free cysteine ​​residues are present in the peptide.In certain embodiments, this occurs as a synthesized dimer, or when dimerized under oxidizing conditions, especially when free cysteine ​​monomer peptides are present.In some embodiments, the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

[0155] In certain embodiments, a hepcidin analog dimer of the invention is a peptide dimer comprising two hepcidin analog peptide monomers of the invention.

[0156] In certain embodiments, the invention includes polypeptides comprising an amino acid sequence set forth herein, or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity to any of these amino acid sequences. In related embodiments, the invention includes dimers comprising two polypeptides, each comprising an amino acid sequence set forth herein, or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity to any of these amino acid sequences.

[0157] In certain embodiments, the monomeric subunits may be dimerized by a disulfide bridge between two cysteine ​​residues, one in each peptide monomeric subunit, or by another suitable linker moiety, including those described herein. Some of the monomeric subunits have been shown to have C- and / or N-termini that both contain free amines. Thus, to generate peptide dimeric inhibitors, the monomeric subunits may be modified to eliminate the free amine at either the C- or N-terminus, thereby allowing dimerization at the remaining free amine. For example, in some instances, the termini of one or more monomeric subunits are acylated with an acylating organic compound selected from the group consisting of 2-me-trifluorobutyl, trifluoropentyl, acetyl, octonyl, butyl, pentyl, hexyl, palmityl, trifluoromethylbutyric acid, cyclopentanecarboxylic acid, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, tetrahydro-2H-pyran-4carboxylic acid, succinic acid, and glutaric acid. In some instances, the monomeric subunits contain both a free carboxyl terminus and a free amino terminus, allowing the user to selectively modify the subunits to achieve dimerization at the desired terminus. Thus, those skilled in the art will understand that the monomeric subunits of the present invention can be selectively modified to achieve a single, specific amine for the desired dimerization.

[0158] It is further understood that the C-terminal residue of the monomer subunits disclosed herein may be an amide unless otherwise indicated. Furthermore, it is understood that in certain embodiments, dimerization at the C-terminus is facilitated by using a suitable amino acid having a side chain with an amine functionality, as is generally understood in the art. With respect to the N-terminal residue, it is generally understood that dimerization can be achieved via the free amine of the terminal residue, or by using a suitable amino acid side chain with a free amine, as is generally understood in the art.

[0159] It is further understood that the side chains of one or more internal residues contained in the hepcidin analog peptide monomers of the present invention can be utilized for dimerization purposes. In such embodiments, the side chains are, in some embodiments, suitable natural amino acids (e.g., Lys) or non-natural amino acids, as defined herein, that include a side chain suitable for conjugation to, for example, a suitable linker moiety.

[0160] The linker moiety connecting the monomeric subunits can have any structure, length, and / or size compatible with the teachings herein. In at least one embodiment, the linker moiety is selected from the non-limiting group consisting of cysteine, lysine, DIG, PEG4, PEG4-biotin, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, IDA-Palm, ADA, Boc-IDA, glutaric acid, isophthalic acid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, triazine, Boc-triazine, IDA-biotin, PEG4-biotin, AADA, suitable aliphatic compounds, aromatic compounds, heteroaromatic compounds, and polyethylene glycol-based linkers having a molecular weight of approximately 400 Da to approximately 40,000 Da. Non-limiting examples of suitable linker moieties are provided in Table 2. In certain embodiments, any of these linker moieties may alternatively link a half-life extending moiety to a hepcidin analog. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0161] Those skilled in the art will appreciate that the C-terminal and N-terminal and internal linker moieties disclosed herein are non-limiting examples of suitable linker moieties, and that the present invention can include any suitable linker moiety.

[0162] In certain embodiments of any of the hepcidin analog peptide dimers, the N-terminus of each peptide monomer subunit is connected by a linker moiety.

[0163] In certain embodiments of any of the hepcidin analog peptide dimers, the C-terminus of each peptide monomer subunit is connected by a linker moiety.

[0164] In certain embodiments, the side chains of one or more internal amino acid residues (eg, Lys residues) contained in each peptide monomer subunit of a hepcidin analog peptide dimer are connected by a linker moiety.

[0165] In certain embodiments of the hepcidin analog peptide dimer, the C-terminal, N-terminal, or internal amino acid (e.g., lysine side chain) of each peptide monomer subunit is connected by a linker moiety, and at least two cysteine ​​or Pen residues of the hepcidin analog peptide dimer are linked by a disulfide bridge. In some embodiments, the peptide dimer has the general structure shown below. A non-limiting schematic example of such a hepcidin analog is shown in the following diagram: [ka]

[0166] Peptide Analog Conjugates In certain embodiments, the hepcidin analogs of the present invention, including both monomers and dimers, contain one or more conjugated chemical substituents, such as lipophilic substituents and polymer moieties, collectively referred to herein as half-life extending moieties. Without wishing to be bound by any particular theory, it is believed that the lipophilic substituents bind to albumin in the bloodstream, thereby preventing the hepcidin analog from enzymatic degradation and thus increasing its half-life. Furthermore, it is believed that the polymer moiety increases half-life, reduces clearance in the bloodstream, and in some cases, can enhance permeability through epithelia and retention in the lamina propria. Furthermore, it is believed that in some cases, these substituents can enhance permeability through epithelia and retention in the lamina propria. Those skilled in the art will be familiar with suitable techniques for preparing compounds used in the context of the present invention. For non-limiting examples of suitable chemistries, see, for example, WO98 / 08871, WO00 / 55184, WO00 / 55119, Madsen et al (J. Med. Chem. 2007, 50, 6126-32), and Knudsen et al. 2000 (J. Med. Chem. 43, 1664-1669).

[0167] In one embodiment, the side chain of one or more amino acid residues (e.g., Lys residues) in the hepcidin analogs of the present invention is further conjugated (e.g., covalently bonded) to a lipophilic substituent or other half-life extending moiety. The lipophilic substituent may be covalently bonded to an atom in the amino acid side chain or may be conjugated to the amino acid side chain via one or more spacer or linker moieties. The spacer or linker moiety, if present, may provide spacing between the hepcidin analog and the lipophilic substituent. In certain embodiments, the half-life extending moiety is conjugated to the hepcidin analog via a linker moiety, which in certain embodiments is a linker moiety shown in Table 2 or a linker moiety disclosed or shown in any of Tables 2-7.

[0168] In certain embodiments, the lipophilic substituent or half-life extending moiety comprises a hydrocarbon chain having 4 to 30 C atoms, e.g., at least 8 or 12 C atoms, preferably 24 or fewer C atoms, or 20 or fewer C atoms. The hydrocarbon chain can be linear or branched, saturated or unsaturated. In certain embodiments, the hydrocarbon chain is substituted with a moiety that forms part of the bond to an amino acid side chain or spacer, such as an acyl group, a sulfonyl group, an N atom, an O atom, or an S atom. In some embodiments, the hydrocarbon chain is substituted with an acyl group, and thus the hydrocarbon chain can form part of an alkanoyl group, e.g., palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0169] Lipophilic substituents can be conjugated to any amino acid side chain in the hepcidin analog of the present invention.In certain embodiments, the amino acid side chain comprises a carboxyl, hydroxyl, thiol, amide or amine group to form ester, sulfonyl ester, thioester, amide or sulfonamide with a spacer or lipophilic substituent.For example, lipophilic substituents can be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys, or Dbu, Dpr or Orn.In certain embodiments, lipophilic substituents are conjugated to Lys.The amino acid shown as Lys in any of the formulas provided herein can be replaced by, for example, Dbu, Dpr or Orn to which a lipophilic substituent is added.

[0170] In further embodiments of the invention, alternatively or additionally, the side chains of one or more amino acid residues in the hepcidin analogs of the invention may be conjugated to polymeric or other half-life extending moieties, e.g., to increase solubility and / or half-life and / or bioavailability in vivo (e.g., in plasma). Such modifications are also known to reduce clearance (e.g., renal clearance) of therapeutic proteins and peptides.

[0171] 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 the molecular weight. As used herein, PEO, PEE, or POG refer to oligomers or polymers of ethylene oxide. While the three names are chemically equivalent, PEG tends to refer to oligomers and polymers with molecular weights less than 20,000 g / mol, PEO to polymers with molecular weights greater than 20,000 g / mol, and POE to polymers of any molecular weight. PEG and PEO are liquids or low-melting solids, depending on their molecular weights. Throughout this disclosure, the three names are used interchangeably. PEG is prepared by polymerization of ethylene oxide and is commercially available in a wide range of molecular weights, from 300 g / mol to 10,000,000 g / mol. PEG and PEO with different molecular weights find use in different applications and have different physical properties (e.g., viscosity) due to the chain length effect, but their chemical properties are nearly identical. The polymer moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymer moieties include polyethylene glycol (PEG), PEG homopolymers or copolymers, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See Int. J. Hematology 68:1 (1998), Bioconjugate Chem. 6:150 (1995), and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992). PEGs prepared for the purpose of half-life extension, such as mono-activated alkoxy-terminated polyalkylene oxides (POA), such as monomethoxy-terminated polyethylene glycol (mPEG), are also contemplated, as are bis-activated polyethylene oxides (glycols) or other PEG derivatives. Suitable polymers vary substantially in weight, ranging from about 200 to about 40,000 daltons, and are typically selected for the purposes of the present invention. In particular embodiments, PEGs having molecular weights of 200-2,000 daltons or 200-500 daltons are used.Different forms of PEG can also be used depending on the initiator used in the polymerization process; for example, a common initiator is monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated as mPEG. Other suitable initiators are known in the art and are suitable for use in the present invention.

[0172] Low molecular weight PEGs are also available as pure oligomers, referred to as monodisperse, uniform, or discrete, which are used in certain embodiments of the present invention.

[0173] 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 typically have multiple PEG chains grafted to the polymer backbone. PEGs can also be linear. A number often included in the name of PEGs indicates their average molecular weight (e.g., a PEG with n=9 has an average molecular weight of approximately 400 daltons and is classified as PEG400).

[0174] As used herein, "PEGylation" refers to the act of attaching (e.g., covalently) a PEG structure to a hepcidin analog of the invention, and in certain embodiments, is referred to as a "PEGylated hepcidin analog." In certain embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG portion of the conjugated half-life extending moiety is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, it is PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8. In some embodiments, the spacer is PEGylated. In certain embodiments, the PEG of the PEGylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the PEGylated spacer is PEG3 or PEG8.

[0175] In some embodiments, the present invention includes hepcidin analog peptides (or dimers thereof) conjugated with PEG, covalently attached, for example, via an amide, a thiol, by click chemistry, or by any other suitable means known in the art. In certain embodiments, PEG is attached via an amide bond; therefore, the particular PEG derivative used is appropriately functionalized. For example, in certain embodiments, PEG11, O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol, has both an amine and a carboxylic acid for attachment to the peptides of the present invention. In certain embodiments, PEG25 contains a diacid and 25 glycol moieties.

[0176] Other suitable polymer moieties are poly-amino acids, such as poly-lysine, poly-aspartic acid, and poly-glutamic acid (see, e.g., Gombotz, et al. (1995), Bioconjugate Chem., vol. 6:332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57, and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73, :721-729). The polymer moiety can be linear or branched. In some embodiments, it has a molecular weight of 500 to 40,000 Da, e.g., 500 to 10,000 Da, 1000 to 5000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da.

[0177] In some embodiments, a hepcidin analog of the present invention may include two or more such polymer moieties, in which case the total molecular weight of all such moieties will generally be within the ranges provided above.

[0178] In some embodiments, the polymer moiety may be attached (covalently) to the amino, carboxyl, or thiol groups of the amino acid side chains. Particular examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, although the carboxyl groups of Asp and Glu residues may also be used.

[0179] Those skilled in the art will be familiar with suitable techniques that can be used to carry out the conjugation reaction. For example, a PEG moiety bearing a methoxy group can be conjugated to a Cys thiol group via a maleimide bond using a commercially available reagent from Nektar Therapeutics AL. For details of suitable chemistries, see also WO2008 / 101017 and the references listed above. Maleimide-functionalized PEG can also be conjugated to the side chain sulfhydryl group of a Cys residue.

[0180] As used herein, disulfide bond oxidation can be performed in a single step or is a two-step process. For a single oxidation step, trityl protecting groups are often used during assembly, allowing for deprotection during cleavage followed by solution oxidation. If a second disulfide bond is required, there are options for native or selective oxidation. For selective oxidation, which requires orthogonal protecting groups, Acm and trityl are used as protecting groups for cysteines. Cleavage results in the removal of one protecting pair of cysteines, allowing oxidation of this pair. A second oxidative deprotection step of the Acm groups protected by cysteines is then performed. For native oxidation, trityl protecting groups are used on all cysteines, allowing for native folding of the peptide.

[0181] Those skilled in the art will be familiar with suitable techniques that can be used to carry out the oxidation step.

[0182] In certain embodiments, the hepcidin analogs of the present invention comprise a half-life extending moiety, which may be selected from, but is not limited to, Ahx-Palm, PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-lauric acid, isoGlu-myristic acid, and isoGlu-isovaleric acid.

[0183] In certain embodiments, the hepcidin analog comprises a half-life extending moiety having the structure shown below, where n=0-24 or n=14-24. [ka]

[0184] In certain embodiments, the hepcidin analogs of the present invention comprise a conjugated half-life extending moiety as shown in Table 3. [Table 5-1] [Table 5-2]

[0185] In certain embodiments, the half-life extending moiety is conjugated directly to the hepcidin analog, while in other embodiments, the half-life extending moiety is conjugated to the hepcidin analog peptide via a linker moiety, e.g., any of those shown in Tables 2 or 4. [Table 6-1] [Table 6-2] [Table 6-3]

[0186] With respect to the linker structures shown in Table 4, reference to n=1-24 or n=1-25, etc. (e.g., L4, L8, or L13) indicates that n can be any integer within the range described. For example, for L4 shown in Table 4, n can be 1, 2, 3, etc., and when n=5, L4 has the structure shown in L3 (Ahx).

[0187] In certain embodiments, the hepcidin analogs of the invention comprise any of the linker moieties shown in Table 4 and any of the half-life extending moieties shown in Table 3, including any of the following combinations shown in Table 5: [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0188] In certain embodiments, the hepcidin analog comprises two or more linkers, hi certain embodiments, the two or more linkers are concatamerized, i.e., linked to one another. In related embodiments, the invention includes polynucleotides that encode a polypeptide having a peptide sequence present in any of the hepcidin analogs described herein.

[0189] Furthermore, the present invention includes vectors, eg, expression vectors, comprising the polynucleotides of the present invention.

[0190] Treatment method Polycythemia vera (PV) is a chronic, progressive trilineage clonal disorder manifested by increased bone marrow, erythroid, and megakaryocyte proliferation / accumulation and characterized by the World Health Organization (WHO) as a myeloproliferative neoplasm (Arber et al., Blood, 2016, 127(20):2391-405). Diagnosis is defined by two criteria: the first is increased erythroid mass, bone marrow biopsy demonstrating trilineage hypercellularity, and the presence of JAK2V617F or JAK2 exon 12 mutations; the second is polycythemia, bone marrow biopsy confirmation, and subnormal serum erythropoietin levels (Arber et al., Blood, 2016, 127(20):2391-405).

[0191] During erythropoiesis in normocytic individuals, erythropoiesis is regulated by erythropoietin, which is JAK2-dependent; however, when JAK2 is constitutively activated, erythropoietin-independent erythropoiesis leads to polycythemia. Approximately 95% of PV patients have the JAK2V617F mutation (Rampal et al., Blood. 2014, 123(22):e123-33). PV can progress to myelofibrosis or undergo leukemic transformation. Polycythemia in PV men is characterized by an Hgb > 16.5 g / dL or Hct > 49%, and in PV women by an Hgb > 16.0 g / dL or Hct > 48%. A hematocrit above 44% in patients with PV is associated with a sharp increase in the number of thromboembolic complications (Pearson et al., Lancet. 1978;2:1219-1221). A bone marrow biopsy is required to confirm PV originating from essential thrombocythemia and should demonstrate hypercellularity for age with trilineage proliferation.

[0192] An estimated 148,000 people in the United States are living with PV, with a median age at diagnosis of 61 years (Stein et al., J Clin Oncol. 2015 Nov 20;33(33):3953-60). Symptoms of polycythemia associated with blood hyperviscosity include fatigue, bone pain, headache, dizziness, visual disturbances, atypical chest pain, pruritus, erythromelalgia, and paresthesias (Tefferi et al., Blood Cancer J. 2018,8(1):3). Clinical features include splenomegaly, thrombotic and hemorrhagic complications, and a risk of leukemic transformation.

[0193] PV is classified into two risk categories that define subsequent treatment regimens: high-risk (age ≥ 60 years and a history of thrombosis) and low-risk (age < 60 years and no history of thrombosis) (Tefferi & Barbui, Am J Hematol. 2017 Jan;92(1):94-108). In the United States, alternative classifications of high-risk (age ≥ 60 years) and low-risk (age < 60 years) may be used. All PV patients undergo therapeutic phlebotomy to reduce hematocrit and once-daily aspirin (81 mg) to prevent thrombohemorrhagic complications. Before the introduction of therapeutic phlebotomy, the median survival time for untreated PV was less than 2 years, with most deaths attributed to thrombotic complications (Tefferi et al., 2018). With current treatment, the median survival from the time of diagnosis is 24 years for patients under 60 years of age compared with 14 years for patients over 60 years of age. The hematocrit targets for therapeutic phlebotomy are <45% in men, <42% in women, and <36% during pregnancy (Streiff et al., Blood. 2002, 99(4):1144-9), corresponding to Hgb levels of 15, 14, and 12 g / dL, respectively. The goal of therapeutic phlebotomy is to create a chronic state of iron deficiency, thereby reducing erythropoiesis. Furthermore, iron-deficient red blood cells, due to their smaller size, are less viscous than normocytic blood. Fortunately, evaluations have confirmed that after exercise, chronically iron-deficient PV patients do not experience the aerobic deficit associated with iron deficiency that is expected in their normocytic counterparts (Rector et al., Medicine (Baltimore). 1982 Nov;61(6):382-9).

[0194] Cytoreductive therapy is recommended for high-risk patients, as well as those for whom phlebotomy is not acceptable, who demonstrate progressive splenomegaly, or who have a disease progression exceeding 1500x10 9It is used in low-risk patients with platelet counts greater than 1 / L or progressive leukocytosis. Cytoreductive agents include, but are not limited to, hydroxyurea, interferon alpha, ruxolitinib (Jakafi®), and busulfan. In the United States, hydroxyurea is the first-line treatment for PV patients over 40 years of age because it effectively reverses bone marrow suppression and reduces the risk of thrombosis compared with phlebotomy alone. However, concerns about the long-term risk of secondary leukemia with hydroxyurea are justified. After a median follow-up of more than 8 years, the Polycythemia Vera Study Group reported that 5.4% of evaluated patients with PV developed leukemia after receiving hydroxyurea, compared with 1.5% of patients treated with phlebotomy alone (Fruchtman et al., Semin Hematol. 1997, 34:17-23). Patients who are intolerant or resistant to hydroxyurea can be managed with pegylated IFN-α or busulfan, with IFN-α preferred in patients under 65 years of age and busulfan preferred in the elderly.

[0195] Approximately one in four patients with PV is considered uncontrolled due to an inadequate response or intolerance to hydroxyurea. Jakafi® (ruxolitinib) is a JAK1 / JAK2 inhibitor approved by the U.S. Food and Drug Administration for PV patients with resistance or intolerance to hydroxyurea. Approval was based on a Phase 3 study called the Response: Randomized, Open-Label, Multicenter Phase 3 Study of Efficacy and Safety in Subjects with Polycythemia Vera Who Are Resistant or Intolerant to Hydroxyurea: Comparison of the JAK Inhibitor INC424 Tablets Versus Best Available Therapy (BAT). The trial evaluated patients with phlebotomy-dependent PV and splenomegaly who were intolerant or resistant to hydroxyurea and randomized to ruxolitinib (n=110) or best available therapy (n=112). Coprimary endpoints included hematocrit control (<45%) and splenomegaly reduction (≥35%) at 32 weeks. After week 32, patients randomized to best available therapy could cross over to ruxolitinib. At week 32, 77% of patients randomized to ruxolitinib met at least one component of the primary endpoint, whereas only 1% of patients receiving BAT achieved the primary endpoint. The majority of ruxolitinib-treated patients (91%) who achieved the primary endpoint had a confirmed response at week 48, and the probability of maintaining the primary response through 1 year was 94%. The incidence of thromboembolic events was low in the ruxolitinib group, with only one event (portal vein thrombosis) reported by week 32 compared with six events in patients receiving BAT. Investigators in the Response trial concluded that in patients with PV who had an inadequate response to or were intolerant to hydroxyurea, ruxolitinib was superior to BAT in controlling hematocrit without phlebotomy, normalizing blood counts, reducing spleen volume, and improving PV-related symptoms, including itching, fatigue, and night sweats.

[0196] Patients in the United States undergo approximately eight phlebotomies per year, a procedure that causes pain, discomfort, and inconvenience (Boccia et al., Blood, 2017, 130(Suppl 1), 5271). A significant amount of time is spent undergoing phlebotomy, involving approximately half a day of work per procedure. Furthermore, many redox / metabolic cycles occur (more are needed around this time); therefore, if the ultimate goal of phlebotomy therapy in polycythemia vera is to achieve a state of chronic iron deficiency that limits red blood cell formation, this could theoretically be achieved with hepcidin / hepcidin mimetics.

[0197] Hepcidin, a 25-amino acid peptide, regulates whole-body iron homeostasis and is produced by the liver in response to plasma iron concentration and iron stores. Hepcidin inhibits the cellular iron exporter ferroportin (FPN-1), which is expressed on the surface of cells involved in iron absorption, recycling, and storage. Hepcidin mediates systemic iron restriction and exogenous administration of exogenous hepcidin mimetics, as well as reductions in Hgb levels and splenomegaly in a mouse PV model (Casu et al., Blood. 2016;128(2):265-276).

[0198] Constitutive activation of JAK2 leads to red blood cell-independent erythropoietin production, resulting in polycythemia. An approach to prevent the effects of mutant Jak2 is to induce iron restriction with hepcidin or hepcidin-mimetic peptides. Low iron levels inhibit erythropoietin signaling downstream of Jak2, thus providing an override signal. When hepcidin-mimetic peptides were administered to transgenic mice expressing the human Jak2 gene with a polycythemia-causing mutation, increased erythropoiesis and hematocrit, characteristic of polycythemia, were reversed to the normal range. (Casu et al. Blood. 2016;128(2):265-276)

[0199] In some embodiments, the present invention provides a method for treating a subject suffering from a disease or disorder associated with polycythemia vera, comprising administering to the subject a hepcidin analog of the present invention. In some embodiments, the hepcidin analog administered to the subject is present in a composition (e.g., a pharmaceutical composition). It is understood that throughout, references to hepcidin analogs include pharmaceutically acceptable salts of such hepcidin analogs.

[0200] In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by increased ferroportin activity or expression, comprising administering to the individual a hepcidin analog or composition of the present invention in an amount sufficient to bind (partially or fully) to and agonize ferroportin in the subject. In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by dysregulated iron metabolism, comprising administering to the subject a hepcidin analog or composition of the present invention.

[0201] In some embodiments, the methods of the present invention include providing a hepcidin analog or composition of the present invention to a subject in need thereof. In certain embodiments, the subject in need thereof has been diagnosed with or determined to be at risk for developing a disease or disorder characterized by dysregulated iron levels (e.g., a disease or disorder of iron metabolism, a disease or disorder associated with iron overload, and a disease or disorder associated with abnormal hepcidin activity or expression). In certain embodiments, the subject is a mammal (e.g., a human).

[0202] In certain embodiments, the disease or disorder is polycythemia vera. In certain embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy. In some embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy in a low-risk patient. In some embodiments, the subject is a high-risk polycythemia vera patient. In some embodiments, the subject is a low-risk polycythemia vera patient. In some embodiments, the subject is a symptomatic polycythemia vera patient requiring phlebotomy. In some embodiments, the subject is a low-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject is a high-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject is a high-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies to target a hematocrit of 45% or less in the 24 weeks prior to administering the pharmaceutical composition to the subject. In some embodiments, the subject is a mammal, e.g., a human.

[0203]

[0013] Thus, in one embodiment, a method is provided for treating a subject suffering from or diagnosed with polycythemia vera, comprising administering to the subject an amount of a hepcidin analog or composition disclosed herein effective to treat polycythemia vera. In certain embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy. In some embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy in a low-risk patient. In some embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy in a high-risk patient. In some embodiments, the subject is a low-risk polycythemia vera patient. In some embodiments, the subject is a high-risk polycythemia vera patient. In some embodiments, the subject is a symptomatic polycythemia vera patient requiring phlebotomy. In some embodiments, the subject is a low-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies to target a hematocrit of 45% or less in the 24 weeks prior to administering the pharmaceutical composition to the subject. In some embodiments, the subject is a mammal, e.g., a human.

[0204] In certain embodiments, the present disclosure provides a method of treating polycythemia vera in a human subject in need thereof, comprising administering to the subject an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof, peptide, or composition disclosed herein, e.g., a peptide having a structure of SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. In certain embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy. In some embodiments, the polycythemia vera is polycythemia vera requiring phlebotomy in a low-risk patient or a high-risk patient. In some embodiments, the subject is a low-risk polycythemia vera patient. In some embodiments, the subject is a high-risk polycythemia vera patient. In some embodiments, the subject is a symptomatic polycythemia vera patient requiring phlebotomy. In some embodiments, the subject is a low-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject is a high-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies to target a hematocrit of 45% or less in the 24 weeks prior to administering the pharmaceutical composition to the subject.

[0205] In certain embodiments of any of the methods disclosed herein, the effective amount is about 5 mg to about 200 mg, or about 10 mg to about 100 mg, e.g., about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 100 mg, or about 120 mg.

[0206] In one embodiment, the disclosure provides a method of treating a human subject with polycythemia vera requiring phlebotomy, comprising subcutaneously administering to the subject an effective amount of a hepcidin analog disclosed herein, e.g., a peptide having the structure of SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. In certain embodiments, the effective amount is about 10 mg to about 100 mg, e.g., about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, or about 100 mg, and the hepcidin analog is administered to the subject about twice a week, about once a week, about once every other week, or about once a month. In certain embodiments, a subject is administered about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg of a hepcidin analog or a pharmaceutically acceptable salt thereof about once per week. In certain embodiments, when a hepcidin analog is administered to a woman, a reduced amount, for example, about 10 mg to about 60 mg, for example, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg of a hepcidin analog, may be administered. In some embodiments, a hepcidin analog or a pharmaceutically acceptable salt thereof is administered about once every two weeks or about once per month. In some embodiments, the hepcidin analog or a pharmaceutically acceptable salt thereof is administered multiple times over a period of time, for example, at least 6 months, at least or about 1 year, at least or about 2 years, at least or about 5 years, or over the life of the subject.

[0207] In certain embodiments of the methods disclosed herein, the hepcidin analog or a pharmaceutically acceptable salt or peptide thereof is administered in a composition (e.g., a pharmaceutical composition), and in some embodiments, the hepcidin analog or a pharmaceutically acceptable salt or peptide thereof (or composition) is administered by subcutaneous injection. In some embodiments, the hepcidin analog or a pharmaceutically acceptable salt or peptide thereof (or composition) is administered approximately weekly for a period of time, for example, as long as necessary. In some embodiments, the hepcidin analog or peptide (or composition) is administered approximately every 3 days, approximately twice a week, approximately once every 4 days, approximately once every 5 days, approximately every week, approximately once every 2 weeks, approximately once a month, approximately once every 6 weeks, approximately once every 8 weeks, approximately once every 2 months, or approximately once every 3 months. In certain embodiments, it is administered approximately once a week or approximately once every 2 weeks. In certain embodiments, it is administered approximately once a week. In some embodiments, it is administered about once every two weeks, about once a month, or about once every two months.

[0208] In various embodiments of the methods disclosed herein, an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof is an amount sufficient to achieve a plasma or serum concentration of the hepcidin analog or a pharmaceutically acceptable salt thereof in a subject of about 5 ng / mL to about 3500 ng / mL, about 100 ng / mL to about 3000 ng / mL, about 5 ng / mL to about 900 ng / mL, or about 5 ng / mL to about 250 ng / mL, or about 20 ng / mL to 150 ng / mL. The effective amount optimally maintains the patient in a desired hematocrit range, as defined herein.

[0209] In certain embodiments, the disclosure provides a method of treating PV in a subject, comprising providing to the subject an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof, wherein the amount of the hepcidin analog or a pharmaceutically acceptable salt thereof provided to the subject achieves a plasma or serum concentration of the hepcidin analog or a pharmaceutically acceptable salt thereof of about 2 ng / mL to about 3500 ng / mL, about 5 ng / mL to about 3500 ng / mL, about 100 ng / mL to about 3000 ng / mL, about 5 ng / mL to about 900 ng / mL, about 5 ng / mL to about 250 ng / mL, or about 20 ng / mL to 150 ng / mL. In certain embodiments, the plasma or serum concentration achieved is at least about 25 ng / mL, at least about 50 ng / mL, at least about 100 ng / mL, at least about 200 ng / mL, at least about 500 ng / mL, at least about 1000 ng / mL, at least about 1500 ng / mL, at least about 2000 ng / mL, at least about 2500 ng / mL, or at least about 3000 ng / mL. In certain embodiments, the plasma or serum concentration achieved is, for example, about 200 ng / mL to about 3200 ng / mL, or about 1000 ng / mL to about 3200 ng / mL, or about 1000 ng / mL to about 2000 ng / mL, or about 2000 ng / mL to about 3000 ng / mL for Compound A. In certain embodiments, the plasma or serum concentration achieved is at least 4 ng / mL, at least 5 ng / mL, at least 8 ng / mL, at least 10 ng / mL, at least 12 ng / mL, at least 15 ng / mL, at least 17 ng / mL, or at least 20 ng / mL, e.g., for Compound A. In certain embodiments, the plasma or serum concentration achieved is at least about 4 ng / mL or at least about 17 ng / mL, e.g., for Compound A. In certain embodiments, this plasma or serum level is achieved after a single administration and maintained until the next administration of the hepcidin analog, e.g., Compound A or Compound B. In certain embodiments, this plasma or serum level is achieved and maintained for at least 4 days, at least 5 days, at least 6 days, or at least 1 week after administration of the hepcidin analog, e.g., Compound A or Compound B.In certain embodiments, the plasma or serum concentration achieved is, for example, for Compound A, at least about 20 ng / mL, at least about 30 ng / mL, at least about 50 ng / mL, at least about 100 ng / mL, at least about 150 ng / mL, at least about 200 ng / mL, at least about 500 ng / mL, at least about 1000 ng / mL, at least about 1500 ng / mL, at least about 2000 ng / mL, at least about 2500 ng / mL, or at least about 3000 ng / mL. In certain embodiments, the plasma or serum concentration achieved is, for example, for Compound A, at least about 50 ng / mL within 20-48 hours after administration, at least about 100 ng / mL within 20-48 hours after administration, at least about 250 ng / mL within 20-48 hours after administration, at least about 400 ng / mL within 20-48 hours after administration, at least about 500 ng / mL within 20-48 hours after administration, at least about 800 ng / mL within 20-48 hours after administration, or at least about 1000 ng / mL within 20-48 hours after administration. In certain embodiments, the plasma or serum concentration achieved is, for example, about 25 ng / mL to about 1000 ng / mL within 20 to 48 hours after administration, about 100 ng / mL to about 1000 ng / mL within 20 to 48 hours after administration, about 200 ng / mL to about 1000 ng / mL within 20 to 48 hours after administration, or about 400 ng / mL to about 850 ng / mL within 20 to 48 hours after administration, for example, Compound A. In certain embodiments, the plasma or serum concentration achieved is, for example, about 25 ng / mL to about 125 ng / mL, or about 50 ng / mL to about 125 ng / mL, for example, Compound B. In certain embodiments, the plasma or serum concentration achieved is at least about 25 ng / mL, at least about 50 ng / mL, or at least about 100 ng / mL, for example, Compound B. In certain embodiments, the plasma or serum concentration is the maximum plasma or serum concentration following administration of the hepcidin analog or a pharmaceutically acceptable salt thereof.In certain embodiments, this plasma or serum concentration is maintained for a period of time after administration of the hepcidin analog or pharmaceutically acceptable salt thereof, e.g., at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 9 days, at least 12 days, or at least 2 weeks. In certain embodiments, the plasma or serum concentration achieved is, for example, for Compound A, from about 200 ng / mL to about 3200 ng / mL, or from about 1000 ng / mL to about 3200 ng / mL, or from about 1000 ng / mL to about 2000 ng / mL, or from about 2000 ng / mL to about 3000 ng / mL, e.g., for about 4 hours, about 8 hours, about 12 hours, about 24 hours, or about 48 hours. In certain embodiments, the plasma or serum concentration achieved is at least about 200 ng / mL, at least about 500 ng / mL, at least about 1000 ng / mL, at least about 1500 ng / mL, at least about 2000 ng / mL, at least about 2500 ng / mL, or at least about 3000 ng / mL, for example, for Compound A, over at least about 4 hours, about 8 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours. In certain embodiments, the plasma or serum concentration achieved is from about 25 ng / mL to about 125 ng / mL, or from about 50 ng / mL to about 125 ng / mL, for example, for Compound B, over at least about 4 hours, about 8 hours, about 12 hours, about 24 hours, or about 48 hours. In certain embodiments, the plasma or serum concentration achieved is, for example, for Compound B, at least about 25 ng / mL, at least about 50 ng / mL, or at least about 100 ng / mL, for example, for about 4 hours, about 8 hours, about 12 hours, about 24 hours, or about 48 hours. In certain embodiments. In certain embodiments, the hepcidin analog or a pharmaceutically acceptable salt thereof is administered subcutaneously. In certain embodiments, the subject has polycythemia vera requiring phlebotomy.In certain embodiments, a subject is administered about 10 mg to about 100 mg, e.g., about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, or about 100 mg, of a hepcidin analog or a pharmaceutically acceptable salt thereof, and the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject about twice a week, about once a week, about once every other week, or about once a month. In certain embodiments, a subject is administered about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg of a hepcidin analog or a pharmaceutically acceptable salt thereof about once a week or about once every two weeks. In certain embodiments, a subject is administered about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg of a hepcidin analog or a pharmaceutically acceptable salt thereof about once per week.

[0210] In certain embodiments, a subject is treated with a hepcidin analog disclosed herein or a pharmaceutically acceptable salt thereof, e.g., a hepcidin analog having the structure of SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, or Compound A or Compound B, in combination with a cytoreductive therapy, e.g., hydroxyurea, interferon, or ruxolitinib. In certain embodiments, when a hepcidin analog is used in combination with a cytoreductive therapy, a reduced amount of the hepcidin analog may be administered, e.g., about 10 mg to about 60 mg, e.g., about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg.

[0211] Any of the methods disclosed herein may further include determining the subject's hematocrit after administration of a hepcidin analog. As shown in FIG. 7, hematocrit levels can be maintained at a desired level by adjusting the amount of hepcidin analog administered to the patient. Thus, the methods may be used to identify and administer a concentration / dose of hepcidin analog that is optimal for maintaining hematocrit below a target level. In some embodiments, the target level for humans is 45% or less, e.g., as measured herein. In certain embodiments, the target level for men is 45% or less (e.g., about 37% to about 45%), and in some cases, the target threshold level for women (e.g., non-pregnant) is 43% or less, or 42% or less (e.g., about 35% to about 42% or 43%), and in some cases, the target level for pregnant women is 36% or less (e.g., about 30% to about 36%). In some embodiments, the methods are implemented to achieve a hematocrit of less than 45% (or a defined level, e.g., 42%) for the patient based on an understanding of the relationship between hepcidin analog dose and hematocrit response (i.e., hematocrit reduction). Thus, the present disclosure provides dosing regimens that can maximize the time a patient's hematocrit is below a desired target level and minimize fluctuations above this target level.

[0212] In certain embodiments, the subject's hematocrit is determined between about 1 day and about 7 days after administration of the hepcidin analog. In certain embodiments, if the subject's hematocrit is determined to be greater than 45%, a higher dose of the hepcidin analog is administered to the subject at the next scheduled treatment compared to the dose administered immediately before the hematocrit was determined. In certain embodiments, if the subject's hematocrit is determined to exceed a desired target level for the subject's gender and pregnancy status, a higher dose of the hepcidin analog is administered to the subject at the next scheduled treatment compared to the dose administered immediately before the hematocrit was determined. In certain embodiments, if the subject's hematocrit is determined to be below a threshold level, e.g., less than 42%, less than 40%, less than 37.5%, less than 36%, or less than 35%, a lower dose of the hepcidin analog is administered to the subject at the next scheduled treatment compared to the dose administered immediately before the hematocrit was determined. In some embodiments, if a subject's hematocrit is determined to be within an acceptable range, e.g., 35%-42%, 35%-45%, 37.5%-45%, 40%-45%, or 40%-44%, the subject is administered the same dose of a hepcidin analog at the next scheduled treatment compared to the dose administered immediately prior to the hematocrit determination. The acceptable range may vary depending on the subject's gender and pregnancy status. In certain embodiments, the acceptable range for men is about 37%-45%, the acceptable range for non-pregnant women is about 35%-42% or 43%, and the acceptable range for pregnant women is about 30%-36%.

[0213] In certain embodiments, the method includes determining the subject's hematocrit level multiple times over the course of treatment to monitor the effectiveness of the dosage and modifying the dosage as needed to maintain the subject's hematocrit within a target range, e.g., 30%-35%, 35%-41%, 35%-45%, 37.5%-45%, 40%-45%, or 40%-43%. In certain embodiments, the subject's hematocrit level is determined about every 2 weeks, about every 3 weeks, about every 4 weeks, or about every 8 weeks over the course of treatment. In certain embodiments, it is determined about every 4 weeks over the course of treatment. In certain embodiments, the target range is within the acceptable range for the subject's gender and pregnancy status. In certain embodiments, any of these methods include maintaining or adjusting the amount of hepcidin analog or a pharmaceutically acceptable salt thereof administered to the subject, including increasing the amount if the subject's determined hematocrit is greater than 45%, decreasing the amount if the subject's determined hematocrit is either less than 37.5% or less than 40%, and maintaining the amount if the subject's determined hematocrit is between 37.5% and 45% or between 40% and 44%.

[0214] In one embodiment, the present disclosure provides a method for treating PV, comprising subcutaneously administering to a patient diagnosed with PV a hepcidin analog, e.g., Compound A, at a dose of about 10 mg to about 80 mg about once per week for a period of at least 7 weeks, wherein the subject does not require or have therapeutic phlebotomy during the 7-week period. In certain embodiments, the dose is about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg. In certain embodiments, the subject has undergone one or more therapeutic phlebotomy prior to treatment with the hepcidin analog. In certain embodiments, the subject has undergone one or more therapeutic phlebotomy within 8 weeks prior to treatment with the hepcidin analog. In certain embodiments, the subject is male, and the hematocrit level is maintained below 45% for at least 7 weeks. In certain embodiments, the subject is a non-pregnant woman and the hematocrit level is maintained below 42% or below 43% for at least 7 weeks. In certain embodiments, the subject is a pregnant woman and the hematocrit level is maintained below 36% for at least 7 weeks. In certain embodiments, the hematocrit level is maintained for at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 6 months, at least 1 year, or at least 2 years after the initial administration of the hepcidin analog and during treatment.

[0215] In one embodiment, the present disclosure provides a method for treating PV, comprising subcutaneously administering to a patient diagnosed with PV a dose of about 10 mg to about 80 mg of a hepcidin analog, e.g., Compound A, about once per week for a period of at least seven weeks, wherein the subject does not require or have therapeutic phlebotomy during the seven weeks. In certain embodiments, the subject underwent one or more therapeutic phlebotomy sessions prior to treatment with the hepcidin analog. In certain embodiments, the subject underwent one or more therapeutic phlebotomy sessions within eight weeks of treatment with the hepcidin analog. In certain embodiments, the subject's hematocrit level is measured one or more times during the seven weeks, and if the subject's hematocrit level exceeds the acceptable range, the dose for the following week is increased, or if the subject's hematocrit level is below the acceptable range, the dose for the following week is decreased. In certain embodiments, the subject is male, and the acceptable range is about 37% to about 45%. In certain embodiments, the increased or decreased dosage is also about 10 mg to about 80 mg. In certain embodiments, the dosage is increased or decreased by about 5 mg, about 10 mg, about 15 mg, or about 20 mg. In certain embodiments, the dosage is about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg. In one embodiment, the subject is non-pregnant, and the tolerance range is about 35% to about 43%. In one embodiment, the subject is pregnant, and the tolerance range is about 30% to about 36%. In certain embodiments, the subject does not require or have phlebotomy for at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 6 months, at least 1 year, or at least 2 years after the initial administration of the hepcidin analog and during treatment. In certain embodiments, treatment continues for at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 6 months, at least 1 year, or at least 2 years.

[0216] In various embodiments of the methods disclosed herein, the methods include administering an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof multiple times over a period of time, e.g., the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to a subject about once per week or about twice per week over a period of time. The effective amount may vary or be the same between administrations. The period of time may be, for example, 1 week to 10 years, 1 month to 10 years, 1 month to 5 years, 1 month to 2 years, or 4 months to 1 year. In certain embodiments, the hepcidin analog is selected from the group consisting of: (a) isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof, (b) isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof, (c) isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof, (d) isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof, and (e) isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof, wherein optionally the hepcidin analog comprises a disulfide bond between two Cys amino acids. In certain embodiments, the hepcidin analog or its pharmaceutically acceptable salt is administered to the subject via subcutaneous administration at a dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg approximately once a week for a certain period of time. The effective amount may vary between administrations or may be the same. In certain embodiments, the dosage is adjusted based on the subject's hematocrit level over a certain period of time. Thus, in certain embodiments, the method further includes determining the subject's hematocrit after one or more of the multiple administrations of the hepcidin analog or its pharmaceutically acceptable salt, and the dosage given to the subject during the next administration is maintained or adjusted based on the hematocrit value. For example, if the subject's determined hematocrit exceeds the acceptable range based on the subject's gender and pregnancy status, the next dose may be increased compared to the previously administered amount; if the subject's determined hematocrit is below the acceptable range, the next dose may be decreased compared to the previously administered amount; or if the subject's determined hematocrit is within the acceptable range, the next dose may be the same as the previously administered amount. In certain embodiments, the subject's hematocrit is measured about 3, 4, 5, 6, or 7 days after administration of the hepcidin analog or a pharmaceutically acceptable salt thereof. The subject's hematocrit level may be determined after each administration of the hepcidin analog or a pharmaceutically acceptable salt thereof, or may be determined only after a specific administration of the hepcidin analog or a pharmaceutically acceptable salt thereof, for example, once every two weeks, once a month, once every two months, once every four months, or once every six months.

[0217] In various embodiments of the methods and treatment regimens disclosed herein, the methods result in a subject's hematocrit level being 45% or less. In certain embodiments, the subject's hematocrit is maintained within a range of about 37.5% to about 45% (or within the tolerance range for the subject's gender and pregnancy status) for a period of time, e.g., at least 1 month, at least 2 months, at least 6 months, or longer. In certain embodiments, the methods or treatment regimens result in a reduction in hematocrit (Hct%) of at least 3%, at least 5%, or at least 10%, and / or a reduction in phlebotomy (e.g., in patients requiring phlebotomy) of at least 10%, at least 20%, at least 40%, or at least 50%. As used herein, a 3% reduction in hematocrit means an absolute reduction, e.g., a 46% to 43% reduction.

[0218] In various embodiments of the methods disclosed herein, the method results in an increase in the subject's serum ferritin level. In certain embodiments, the serum ferritin level increases by at least 20%, at least 30%, at least 50%, at least 100%, or at least 200% during the treatment regimen, or for at least 1 month, at least 2 months, at least 6 months, or longer. In certain embodiments, the subject's serum ferritin level is maintained within a range of about 25 ng / mL to about 150 ng / mL for a period of time, for example, at least 1 month, at least 2 months, at least 6 months, or longer.

[0219] In various embodiments of the methods disclosed herein, the methods result in or cause a decrease in the subject's transferrin saturation (TSAT) level and / or serum iron level by at least 60% or at least 80%. In some embodiments, the subject's TSAT level is reduced by less than 40%. In some embodiments of the methods disclosed herein, the methods result in a small increase or no change in TSAT and / or serum iron levels, and in certain embodiments, TSAT and / or serum iron levels remain below normal levels.

[0220] In various embodiments of the methods disclosed herein, the methods result in an increase in the subject's MCV and / or MCH. In certain embodiments, the MCV and / or MCH increase by at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, or at least 200% during the treatment regimen, or for at least 1 month, at least 2 months, at least 6 months, or more.

[0221] In various embodiments of the methods disclosed herein, the methods result in a decrease in the subject's hematocrit and / or red blood cell count by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 80%. In some embodiments, the subject's TSAT levels are reduced by less than 40%. In some embodiments of the methods disclosed herein, the methods result in little or no increase in TSAT and / or serum iron levels, and in certain embodiments, TSAT and / or serum iron levels remain below normal levels.

[0222] In various embodiments of the methods disclosed herein, the methods result in the subject not needing or having therapeutic phlebotomy for, e.g., at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.

[0223] In various embodiments, the methods disclosed herein can be performed on low-risk or high-risk PV patients. In certain embodiments, low-risk PV patients are PV patients under 60 years of age. In certain embodiments, high-risk PV patients are PV patients 60 years of age or older.

[0224] In certain embodiments, the treatment regimen comprises two or more administrations, three or more administrations, four or more administrations, or continuous administration of a hepcidin analog over a period of time, for example, about once per week or about once every two weeks for at least one month, at least two months, at least six months, or more.

[0225] In various embodiments of the methods disclosed herein, the methods do not result in a substantial change in the subject's platelet count, e.g., an increase or decrease of more than 50%. In certain embodiments, the subject's platelet count does not increase or decrease by more than 10%, 20%, 30%, 40%, or 50%.

[0226] In various embodiments of the methods disclosed herein, the methods do not result in a substantial change in the subject's red blood cell count, e.g., an increase or decrease of more than 50%. In certain embodiments, the subject's red blood cell count does not increase or decrease by more than 10%, 20%, 30%, 40%, or 50%.

[0227] In various embodiments of the methods disclosed herein, the methods do not result in a substantial change in the subject's white blood cell or leukocyte cell count, e.g., an increase or decrease of more than 50%. In certain embodiments, the subject's white blood cell or leukocyte cell count does not increase or decrease by more than 10%, 20%, 30%, 40%, or 50%.

[0228] In certain embodiments of any of the methods of the present disclosure, the method provides a therapeutic benefit to the subject, which may include relief or alleviation of one or more symptoms of PV, including, but not limited to, itching, hair loss, fatigue, headache, visual disturbances, night sweats, and thrombotic events.

[0229] In some embodiments, the methods of the present invention comprise providing a hepcidin analog of the present invention (i.e., a first therapeutic agent) in combination with a second therapeutic agent to a subject in need thereof. In certain embodiments, the second therapeutic agent is provided to the subject before, simultaneously with, and / or after the hepcidin analog is administered to the subject. In certain embodiments, the second therapeutic agent is an iron chelator. In certain embodiments, the second therapeutic agent is selected from the iron chelators deferoxamine and deferasirox (Exjade™). In another embodiment, the method comprises administering a third therapeutic agent to the subject.

[0230] The present invention provides compositions (e.g., pharmaceutical compositions) comprising one or more hepcidin analogs of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent. A pharmaceutically acceptable carrier, excipient, or diluent refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0231] The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical industry and are described, for example, in "Remington's Pharmaceutical Sciences," 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH can be used. Suitable pH buffers can be, for example, phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (Tris), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetate (e.g., sodium acetate), or mixtures thereof. The term also encompasses any carrier agent listed in the United States Pharmacopoeia for use in animals, including humans.

[0232] In certain embodiments, the composition comprises two or more hepcidin analogs disclosed herein. In certain embodiments, the combination is selected from one of the following: (i) any two or more of the hepcidin analog peptide monomers disclosed herein, (ii) any two or more of the hepcidin analog peptide dimers disclosed herein, or (iii) any one or more of the hepcidin analog peptide monomers disclosed herein and any one or more of the hepcidin analog peptide dimers disclosed herein.

[0233] It should be understood that the inclusion of a hepcidin analog of the invention (i.e., one or more hepcidin analog peptide monomers of the invention or one or more hepcidin analog peptide dimers of the invention) in a pharmaceutical composition also encompasses the inclusion of a pharmaceutically acceptable salt or solvate of the hepcidin analog of the invention. In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, excipients, or vehicles.

[0234] In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog or a pharmaceutically acceptable salt or solvate thereof for treating various conditions, diseases, or disorders disclosed herein or elsewhere (see, e.g., methods of treatment herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide monomer or a pharmaceutically acceptable salt or solvate thereof for treating various conditions, diseases, or disorders disclosed elsewhere herein (see, e.g., methods of treatment herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide dimer or a pharmaceutically acceptable salt or solvate thereof for treating various conditions, diseases, or disorders disclosed herein.

[0235] The compounds described herein include isotopically labeled compounds, which are identical to the compounds described in the various formulas and structures presented herein, except for the fact that one or more atoms are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, for example, 3 H and 14 Compounds incorporating radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. Additionally, deuterium, i.e. 2 Substitution with isotopes such as H may offer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0236] The hepcidin analogs of the present invention can be formulated as pharmaceutical compositions suitable for administration with or without storage, and typically comprising a therapeutically effective amount of at least one hepcidin analog of the present invention together with a pharmaceutically acceptable carrier, excipient, or vehicle.

[0237] In some embodiments, the hepcidin analog pharmaceutical composition of the present invention is in unit dosage form. In such form, the composition is divided into unit doses containing appropriate amounts of the active ingredient(s). The unit dosage form can be provided as a packaged preparation, the package containing discrete quantities of the preparation, for example, packaged tablets, capsules, or powders in vials or ampoules. The unit dosage form can also be, for example, a capsule, cachet, or tablet itself, or an appropriate number of any of these packaged forms. The unit dosage form can also be provided in single-dose injectable form, for example, a pen device containing a liquid-phase (typically aqueous) composition. The composition can be formulated for any suitable route and means of administration, for example, any one of the routes and means of administration disclosed herein.

[0238] In certain embodiments, the hepcidin analog or a pharmaceutical composition comprising a hepcidin analog is suspended in a sustained-release matrix. As used herein, a sustained-release matrix is ​​a matrix made of a material (usually a polymer) that is degradable by enzymatic hydrolysis, acid-base hydrolysis, or dissolution. When inserted into the body, enzymes and bodily fluids act on the matrix. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactides (polylactic acids), polyglycolides (polymers of glycolic acid), polylactide-co-glycolides (copolymers of lactic and glycolic acids), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicone. A preferred biodegradable matrix is ​​a matrix of any one of polylactide, polyglycolide, or polylactide-co-glycolide (a copolymer of lactic acid and glycolic acid).

[0239] In certain embodiments, the composition is administered parenterally, subcutaneously, or orally. In certain embodiments, the composition is administered orally, intracisternally, intravaginally, intraperitoneally, intrarectally, topically (such as by powders, ointments, drops, suppositories, or transdermal patches, including intravitreal, intranasal, and inhalation delivery), or buccally. As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intraarticular injection and infusion. Thus, in certain embodiments, the composition is formulated for delivery by any of these administration routes.

[0240] In certain embodiments, pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders to be reconstituted immediately before use into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), carboxymethylcellulose and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0241] Injectable depot forms include those made by forming microencapsule matrices of hepcidin analogs in one or more biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), poly(anhydrides), and (poly)glycols, such as PEG. The release rate of the hepcidin analogs can be controlled depending on the ratio of peptide to polymer and the properties of the particular polymer used. Depot injectable formulations can also be prepared by entrapping the hepcidin analogs in liposomes or microemulsions that are compatible with body tissues.

[0242] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0243] The hepcidin analogs of the present invention can also be administered in liposomes or other lipid-based carriers. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes may be used. The compositions of the present invention in liposome form may contain stabilizers, preservatives, excipients, etc. in addition to the hepcidin analogs of the present invention. In certain embodiments, the lipids include phospholipids, including both natural and synthetic phosphatidylcholines (lecithins) and serine. Methods for forming liposomes are known in the art.

[0244] Pharmaceutical compositions used in the present invention suitable for parenteral administration may comprise sterile aqueous solutions and / or suspensions of the peptide inhibitor made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, or the like.

[0245] In some embodiments, the present invention provides pharmaceutical compositions for oral delivery. The compositions and hepcidin analogs of the present invention can be prepared for oral administration according to any of the methods, techniques, and / or delivery vehicles described herein. Furthermore, those skilled in the art will understand that the hepcidin analogs of the present invention can be modified or integrated into systems or delivery vehicles not disclosed herein but known in the art and suitable for use in oral delivery of peptides.

[0246] In certain embodiments, formulations for oral administration may include adjuvants (e.g., resorcinol and / or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and / or enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF), or trasylol) to inhibit enzymatic degradation. In certain embodiments, solid dosage forms of hepcidin analogs for oral administration may be mixed with at least one additive such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. These dosage forms may also contain other type(s) of additives, such as inert diluents, lubricants, e.g., magnesium stearate, parabens, preservatives, e.g., sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants, e.g., cysteine, disintegrating agents, binders, thickeners, buffers, pH adjusters, sweetening agents, flavoring agents, or perfuming agents.

[0247] In certain embodiments, oral dosage forms or unit doses suitable for use with the hepcidin analogs of the present invention may include a mixture of the hepcidin analog with non-drug ingredients or excipients, and other non-recyclable materials that may be considered ingredients or packaging. Oral compositions may include at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, oral dosage forms containing an effective amount of a hepcidin analog are provided, and the dosage forms include at least one of pills, tablets, capsules, gels, pastes, beverages, syrups, ointments, and suppositories. In some cases, oral dosage forms are provided that are designed and configured to achieve delayed release of the hepcidin analog in the small intestine and / or colon of a subject.

[0248] In one embodiment, oral pharmaceutical compositions containing a hepcidin analog of the present invention include an enteric coating designed to delay release of the hepcidin analog in the small intestine. In at least some embodiments, pharmaceutical compositions are provided that include a hepcidin analog of the present invention and a protease inhibitor, such as aprotinin, in a delayed-release formulation. In some cases, the pharmaceutical compositions of the present invention include an enteric coating that is soluble in gastric juice at a pH of about 5.0 or higher. In at least one embodiment, pharmaceutical compositions are provided that include an enteric coating comprising a polymer with a dissociable carboxylic acid group, such as derivatives of cellulose, including hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate, and similar derivatives of cellulose and other carbohydrate polymers.

[0249] In one embodiment, pharmaceutical compositions containing the hepcidin analogs of the present invention are provided with an enteric coating, which is designed to protect and release the pharmaceutical composition in a controlled manner within the subject's lower digestive system and avoid systemic side effects. In addition to enteric coatings, the hepcidin analogs of the present invention can be encapsulated, coated, engaged, or otherwise associated with any suitable oral drug delivery system or component. For example, in some embodiments, the hepcidin analogs of the present invention are provided in a lipid carrier system, including at least one of polymer hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0250] To overcome peptide degradation in the small intestine, some embodiments of the present invention include a hydrogel polymer carrier system containing the hepcidin analog of the present invention, whereby the hydrogel polymer protects the hepcidin analog from proteolytic degradation in the small intestine and / or colon. The hepcidin analog of the present invention can be further formulated for use with a carrier system designed to increase dissolution kinetics and enhance intestinal absorption of the peptide. These methods include the use of liposomes, micelles, and nanoparticles to increase gastrointestinal permeation of the peptide.

[0251] Various bioresponsive systems can be combined with one or more hepcidin analogs of the present invention to provide pharmaceuticals for oral delivery. In some embodiments, the hepcidin analogs of the present invention are used in combination with bioresponsive systems, such as hydrogels and mucoadhesive polymers with hydrogen-bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan, and alginate) to provide therapeutic agents for oral administration. Other embodiments include methods for optimizing or extending the drug residence time of the hepcidin analogs disclosed herein, in which the surface of the hepcidin analog is modified to include mucoadhesive properties through hydrogen bonding, polymers with linked mucin, or / and hydrophobic interactions. These modified peptide molecules can demonstrate increased drug residence time within a subject, in accordance with a desired feature of the present invention. Furthermore, targeted mucoadhesive systems can specifically bind to receptors on the surface of enterocytes and M cells, thereby further increasing the uptake of particles containing the hepcidin analog.

[0252] Another embodiment includes a method for oral delivery of the hepcidin analogs of the present invention, in which the hepcidin analogs are provided to a subject in combination with a permeation enhancer that promotes transport of the peptide across the intestinal mucosa by increasing paracellular or transcellular permeation. For example, in one embodiment, a permeation enhancer is combined with the hepcidin analog, the permeation enhancer comprising at least one of a long-chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent. In one embodiment, a permeation enhancer comprising sodium N-[hydroxybenzoyl]amino]caprylate is used to form a weak non-covalent association with the hepcidin analogs of the present invention, which favors membrane transport and further dissociation upon reaching the blood circulation. In another embodiment, the hepcidin analogs of the present invention are conjugated to oligoarginine, thereby increasing the cellular penetration of the peptide into various cell types. Additionally, in at least one embodiment, a non-covalent bond is provided between the peptide inhibitors of the present invention and a penetration enhancer selected from the group consisting of cyclodextrins (CDs) and dendrimers, which reduces peptide aggregation and increases the stability and solubility of the hepcidin analog molecule.

[0253] Another embodiment of the present invention provides a method for treating a subject with a hepcidin analog of the present invention having an increased half-life. In one aspect, the present invention provides a hepcidin analog having a half-life of at least several hours to a day in vitro or in vivo (e.g., when administered to a human subject) sufficient for once-daily (qd) or twice-daily (bid) administration of a therapeutically effective amount. In another embodiment, the hepcidin analog has a half-life of 3 days or more sufficient for once-weekly (qw) administration of a therapeutically effective amount. In yet another embodiment, the hepcidin analog has a half-life of 8 days or more sufficient for once-every-two-weekly (biw) or once-monthly administration of a therapeutically effective amount. In another embodiment, the hepcidin analog is derivatized or modified so that it has a longer half-life compared to a non-derivatized or unmodified hepcidin analog. In another embodiment, the hepcidin analog contains one or more chemical modifications to increase its serum half-life.

[0254] When used in at least one of the treatment or delivery systems described herein, the hepcidin analogs of the present invention may be used in pure form or in a pharmaceutically acceptable salt form, if such form exists.

[0255] Dosage The total daily dosage of the hepcidin analogs and compositions of the present invention can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including: a) the disorder being treated and the severity of the disorder, b) the activity of the specific compound used, c) the specific composition used, the patient's age, weight, general health, sex, and diet, d) the administration time, route of administration, and excretion rate of the specific hepcidin analog used, e) the duration of treatment, and f) drugs used in combination or simultaneously with the specific hepcidin analog used, as well as similar factors well known in the medical field.

[0256] In certain embodiments, the total daily dose of a hepcidin analog of the present invention administered to a human or other mammalian host in a single or divided dose can be, for example, 0.0001 to 300 mg / kg body weight per day, or 1 to 300 mg / kg body weight per day. In certain embodiments, the dosage of a hepcidin analog of the present invention ranges from about 0.0001 to about 100 mg / kg body weight per day, e.g., about 0.0005 to about 50 mg / kg body weight per day, e.g., about 0.001 to about 10 mg / kg body weight per day, e.g., about 0.01 to about 1 mg / kg body weight per day, administered in one or more doses, e.g., 1 to 3 doses.

[0257] In certain embodiments, the total dose is, for example, about 10 mg to about 100 mg for a human patient, or about 10 mg to about 70 mg, about 10 mg to about 60 mg, about 20 mg to about 50 mg, about 20 mg to about 40 mg, about 30 mg, about 25 mg, about 20 mg, about 15 mg, or about 10 mg. In certain embodiments, the hepcidin analog is provided to the subject once a week. In another specific embodiment, the hepcidin analog is provided to the subject twice a week, for example, for a human patient.

[0258] In more specific embodiments, the total dosage is about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg once or twice weekly for a human patient. In more specific embodiments, the total dosage is about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg once or twice weekly for a human patient.

[0259] In various embodiments, the hepcidin analogs of the present invention can be administered continuously (e.g., by intravenous administration or another continuous drug administration method) or can be administered to a subject at intervals, typically at regular intervals, including, for example, once daily, twice daily, every other day, every third day, every fourth day, every fifth day, or every sixth day, once or twice weekly, once or twice monthly, etc., depending on the desired dosage and pharmaceutical composition selected by one of skill in the art for a particular subject.

[0260] Such regular hepcidin analog administration regimens of the present invention may be advantageously interrupted for a period of time in certain situations, such as during chronic long-term administration, to allow the subject receiving the drug to reduce drug levels or discontinue drug administration, often referred to as taking a "drug holiday." Drug holidays are useful, for example, to maintain or restore drug sensitivity, particularly during long-term chronic treatment, or to reduce undesirable side effects of long-term chronic treatment of a subject with the drug. The timing of the drug holiday depends on the timing of the regular administration regimen and the purpose of taking the holiday (e.g., to restore drug sensitivity and / or to reduce undesirable side effects of continued, long-term administration). In some embodiments, the drug holiday can be a reduction in the drug dosage (e.g., to fall below the therapeutically effective amount for a specific interval). In other embodiments, drug administration is stopped for a certain interval before administration is resumed using the same or a different administration regimen (e.g., a lower or higher dose and / or administration frequency). Thus, drug holidays of the present invention can be selected from a wide range of durations and administration regimens. Exemplary drug holidays are 2 days or more, 1 week or more, or 1 month or more, up to about 24 months.Thus, for example, the regular daily administration regimen using the peptide, peptide analogue or dimer of the present invention can be interrupted by a drug holiday of 1 week, 2 weeks, or 4 weeks, and then the previous regular administration regimen (for example, daily or once a week administration regimen) is resumed.It is contemplated that various other drug holiday regimens are useful for administering the hepcidin analogue of the present invention.

[0261] Thus, the hepcidin analog may be delivered by a dosing regime comprising two or more dosing periods separated by respective rest periods.

[0262] During each administration phase, the hepcidin analog is administered to the recipient subject in a therapeutically effective amount according to a predetermined administration pattern. The administration pattern can include continuously administering the drug to the recipient subject over the duration of the administration phase. Alternatively, the administration pattern can include administering multiple doses of the hepcidin analog to the recipient subject, the administrations being spaced apart by administration intervals.

[0263] The dosing pattern can include at least 2 doses per dosing period, at least 5 doses per dosing period, at least 10 doses per dosing period, at least 20 doses per dosing period, at least 30 doses per dosing period, or more.

[0264] The dosing intervals can be regular dosing intervals, which can be as set forth above, including once daily, twice daily, once every 2, 3, 4, 5 or 6 days, once or twice weekly, once or twice monthly, or regular and less frequent dosing intervals, depending on the particular dosage form, bioavailability and pharmacokinetic profile of the hepcidin analogs of the present invention.

[0265] The administration period may have a duration of at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months or longer.

[0266] If the dosing pattern includes multiple doses, the duration of the following drug holiday will be longer than the dosing interval used in that dosing pattern. If the dosing intervals are irregular, the duration of the drug holiday may be longer than the average interval between doses over the course of a dosing period. Alternatively, the duration of the drug holiday may be longer than the longest interval between successive doses during a dosing period.

[0267] The duration of the drug holiday may be at least twice the relevant dosing interval (or the average thereof), at least three times, at least four times, at least five times, at least ten times, or at least twenty times the relevant dosing interval or the average thereof.

[0268] Within these constraints, the withdrawal period may have a duration of at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months or longer, depending on the administration pattern during the previous administration period.

[0269] The dosing regime comprises at least two dosing periods, each separated by a rest period. Thus, the dosing regime may comprise at least three, at least four, at least five, at least 10, at least 15, at least 20, at least 25, or at least 30 dosing periods or more, each separated by a rest period.

[0270] The sequential administration phases may utilize the same administration pattern, although this may not always be desirable or necessary. However, when other drugs or active agents are administered in combination with the hepcidin analogs of the present invention, the same combination of drugs or active agents is typically given in the sequential administration phases. In certain embodiments, the recipient subject is a human.

[0271] In some embodiments, the present invention provides compositions and medicaments comprising at least one hepcidin analog disclosed herein. In some embodiments, the present invention provides methods for producing medicaments comprising at least one hepcidin analog disclosed herein for treating iron metabolism disorders, such as iron overload disorders. In some embodiments, the present invention provides methods for producing medicaments comprising at least one hepcidin analog disclosed herein for treating diabetes (type I or type II), insulin resistance, or impaired glucose tolerance. Also provided are methods for treating iron metabolism disorders in a subject, such as a mammalian subject, preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount. Also provided are methods for treating diabetes (type I or type II), insulin resistance, or impaired glucose tolerance in a subject, such as a mammalian subject, preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount. Also provided are methods for treating diabetes (type I or type II), insulin resistance, or impaired glucose tolerance in a subject, such as a mammalian subject, preferably a human subject, comprising administering to the subject at least one hepcidin analog or composition disclosed herein. In some embodiments, the hepcidin analog or composition is administered in a therapeutically effective amount.

[0272] In some embodiments, the present invention provides processes for producing the hepcidin analogs or hepcidin analog compositions (eg, pharmaceutical compositions) disclosed herein.

[0273] In some embodiments, the present invention provides a device for delivering a hepcidin analog to a subject, the device comprising at least one hepcidin analog of the present invention, or a pharmaceutically acceptable salt or solvate thereof.

[0274] In some embodiments, the present invention provides methods for binding to or inducing the internalization and degradation of ferroportin, comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0275] In some embodiments, the present invention provides kits comprising at least one hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) disclosed herein packaged with reagents, devices, instructions, or a combination thereof.

[0276] In some embodiments, the present invention provides methods of administering a hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) of the present invention to a subject via an implant or osmotic pump, via a cartridge or micropump, as is well known in the art, or by other means recognized by one of skill in the art. In some embodiments, the present invention provides complexes comprising at least one hepcidin analog disclosed herein that binds to ferroportin, preferably human ferroportin, or an antibody, e.g., an antibody that specifically binds to a hepcidin analog disclosed herein, Hep25, or a combination thereof.

[0277] In some embodiments, the hepcidin analogs of the invention have a measured activity (e.g., EC50) of less than 500 nM in an Fpn internalization assay. Those skilled in the art will understand that the function of a hepcidin analog depends on the tertiary structure and binding surface of the presented hepcidin analog. Therefore, minor modifications can be made to the sequence encoding the hepcidin analog that do not affect folding or are not on the binding surface and maintain function. In other embodiments, the present invention provides hepcidin analogs having 85% or greater (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity or homology to the amino acid sequence of any of the hepcidin analogs described herein that exhibit activity (e.g., hepcidin activity) or alleviate symptoms of a disease or indication in which hepcidin is implicated.

[0278] In other embodiments, the present invention provides hepcidin analogs having 85% or greater (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) identity or homology to the amino acid sequence of any hepcidin analog presented herein or a peptide according to any one of the formulas or hepcidin analogs described herein.

[0279] In some embodiments, the hepcidin analogs of the present invention may include functional fragments or variants thereof having up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to one or more of the specific peptide analog sequences described herein.

[0280] In addition to the methods described in the examples herein, the hepcidin analogue of the present invention can be produced by methods known in the art, including chemical synthesis, biosynthesis, or in vitro synthesis using recombinant DNA methods, and solid-phase synthesis.For example, see Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol.12, JK Setlow ed., Plenum Press, NY, pp.1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce, Rockford, IL, which are incorporated herein by reference.The hepcidin analogue of the present invention can be purified by using protein purification techniques known in the art, such as reversed-phase high-performance liquid chromatography (HPLC), ion-exchange chromatography or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem. 12(16):3121-3126 and Scopes (1982) Protein Purification, Springer-Verlag, NY, which are incorporated herein by reference. Alternatively, the hepcidin analogs of the present invention can be produced by recombinant DNA techniques known in the art. Thus, polynucleotides encoding the polypeptides of the present invention are contemplated herein. In certain preferred embodiments, the polynucleotide is isolated. As used herein, an "isolated polynucleotide" refers to a polynucleotide that is in an environment different from the environment in which the polynucleotide naturally occurs. [Example]

[0281] The following examples illustrate specific embodiments of the present invention. The following examples were carried out using standard techniques that are well known and routine to those skilled in the art, unless otherwise specified in detail. It should be understood that these examples are for illustrative purposes only and are not intended to be completely limiting on 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)

[0282] K(): In the peptide sequences provided herein, when a compound or chemical group is presented in parentheses immediately after a lysine residue, it is understood that the compound or chemical group in parentheses is the side chain conjugated to the lysine residue.Thus, for example, K-[(PEG8)]- indicates that a PEG8 moiety is conjugated to the side chain of this lysine, but is in no way limited thereto.

[0283] Palm: indicates palmitic acid (palmitoyl) conjugation.

[0284] As used herein, "C()" refers to a 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: The sequence of other peptides may optionally be written in the same manner.

[0285] Example 1 Synthesis of peptide analogues Unless otherwise specified, the reagents and solvents used below were commercially available as standard laboratory reagents or analytical grade and were used without further purification.

[0286] 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, while wang and trityl resins were also used to generate C-terminal acids. The side chain protecting groups were as follows: Glu, Thr, and Tyr: t-butyl; Trp and Lys: t-Boc (t-butyloxycarbonyl); Arg: N-gamma-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; His, Gln, Asn, and Cys: trityl. Acm (acetamidomethyl) was also used as a Cys protecting group for selective disulfide bridge formation. For coupling, a 4- to 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]. To improve coupling efficiency in difficult regions, HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) was used instead of HBTU. Removal of the Fmoc protecting group was achieved by treatment with a DMF / piperidine (2:1) solution.

[0287] Procedure for peptide cleavage from the resin Side chain deprotection and cleavage of peptide analogs of the present invention (e.g., Compound No. 2) were achieved by stirring the dried resin for 2-4 hours in a solution containing trifluoroacetic acid, water, ethanedithiol, and triisopropylsilane (90:5:2.5:2.5). 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 aqueous acetonitrile (1:1) containing 0.1% TFA (trifluoroacetic acid), and the resulting solution was filtered. Electrospray ionization mass spectrometry (ESI-MS) was used to assess the quality of the linear peptide.

[0288] Peptide purification procedure Purification of the peptides of the present invention (e.g., Compound No. 2) was achieved using reversed-phase high-performance liquid chromatography (RP-HPLC). Analytical 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).

[0289] Peptide oxidation procedure Method A (Single Disulfide Oxidation). Oxidation of unprotected peptides of the present invention was achieved by adding iodine in MeOH (1 mL per 1 mg) dropwise to the peptide in solution (ACN:HO, 7:3, 0.5% TFA). After stirring for 2 minutes, ascorbic acid was added in small portions until the solution was clear, and the sample was immediately loaded onto an HPLC for purification.

[0290] Method B (Selective Oxidation of Two Disulfides). Selective oxidation was often performed when two or more disulfides were present. Oxidation of the free cysteine ​​was achieved with 1 mg / 10 mL of peptide in a pH 7.6 NH4CO3 solution. After stirring for 24 hours and prior to purification, the solution was acidified to pH 3 with TFA, followed by lyophilization. The resulting single oxidized peptide (containing the ACM-protected cysteine) was then oxidized / selectively deprotected using an iodine solution. The peptide (1 mg per 2 mL) was dissolved in MeOH / H2O 80:20, and iodine dissolved in the reaction solvent was added to the reaction at room temperature (final concentration: 5 mg / mL). The solution was stirred for 7 minutes, after which ascorbic acid was added in small portions until the solution became clear. The solution was then loaded directly onto an HPLC column.

[0291] Method C (Natural Oxidation). When two or more disulfides were present and selective oxidation was not possible, native oxidation was performed. Native oxidation was achieved using 100 mM NH4CO3 (pH 7.4) 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 and prior to RP-HPLC purification, the solution was acidified to pH 3 with TFA, followed by lyophilization.

[0292] Cysteine ​​oxidation procedure to generate dimers. Oxidation of unprotected peptides of the present invention was achieved by adding iodine in MeOH (1 mL per 1 mg) dropwise to the peptide in solution (ACN:HO, 7:3, 0.5% TFA). After stirring for 2 minutes, ascorbic acid was added in small portions until the solution was clear, and the sample was immediately loaded onto HPLC for purification.

[0293] Dimerization procedure. Glyoxylic acid (DIG), IDA, or Fmoc-β-Ala-IDA was preactivated as its N-hydroxysuccinimide ester by treating 1 equivalent (abbreviated "eq") of the acid with 2.2 equivalents of both N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) in N-methylpyrrolidone (NMP) at a final concentration of 0.1 M. For the PEG13 and PEG25 linkers, these chemicals were purchased and preformed as the activated succinimide ester. Approximately 0.4 equivalents of the activated ester were added slowly in small increments to the peptide (1 mg / mL) in NMP. After stirring the solution for 10 min, two to three additional aliquots of approximately 0.05 equivalents of linker were slowly added. After stirring the solution for an additional 3 h, 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 followed by additional reverse-phase HPLC purification.

[0294] Those skilled in the art will appreciate that standard methods of peptide synthesis can be used to produce the compounds of the invention.

[0295] Linker Activation and Dimerization The peptide monomer subunits were linked to form hepcidin analog peptide dimers as described below.

[0296] 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 the precipitated dicyclohexylurea (DCU). The activated linker was kept in a sealed vial before use in dimerization. The nominal concentration of the activated linker was approximately 0.20 M.

[0297] Dimerization using PEG linkers did not involve a pre-activation step: commercially available pre-activated bifunctional PEG linkers were used.

[0298] 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, an activated linker (IDA or PEG13, PEG25) (0.48 equivalents relative to the monomer, 0.048 mmol) 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 required 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. This precipitation step was repeated twice. The crude dimer was then purified using reverse-phase HPLC (Luna C18 support, 10 μm, 100 A, 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 15 ml / min). Fractions containing pure product were then lyophilized on a lyophilizer.

[0299] Conjugation of half-life extending moieties Peptide conjugation was performed on the resin. Lys(ivDde) ​​was used as the key amino acid. After assembly of the peptide on the resin, selective deprotection of the ivDde group was performed using 2% hydrazine in DMF for 5 min (3 × 5 min). Linker activation and acylation were performed using HBTU, 1-2 equivalents of DIEA for 3 h, followed by Fmoc removal and subsequent second acylation with a fatty acid to yield the conjugated peptide.

[0300] Example 2 Peptide analogue activity Peptide analogs were tested in vitro for induction of internalization of human ferroportin protein. After internalization, the peptide is degraded. The assay measures the decrease in receptor fluorescence.

[0301] The cDNA encoding human ferroportin (SLC40A1) was cloned from the cDNA clone of Origene (NM_014585). The DNA encoding ferroportin was amplified by PCR using primers encoding terminal restriction sites but without termination codons for subcloning. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker so that the ferroportin reading frame was fused in frame with the GFP protein. The fidelity of the DNA encoding the protein was confirmed by DNA sequencing. HEK293 cells were used for transfection of the ferroportin GFP receptor expression plasmid. Cells were grown according to standard protocols in growth medium and transfected with the plasmid using Lipofectamine (manufacturer's protocol, Invitrogen). Cells stably expressing ferroportin-GFP were selected using G418 in the growth medium (only cells that had taken up the cDNA expression plasmid survive) and sorted several times in a Cytomation MoFlo™ cell sorter to obtain GFP-positive cells (488 nm / 530 nm). Cells were expanded and frozen in aliquots.

[0302] To determine the activity of hepcidin analogs (compounds) on human ferroportin, cells were incubated in 96-well plates in standard medium without phenol red. Compounds were added to the desired final concentration for at least 18 hours in an incubator. After incubation, residual GFP fluorescence was determined either by total cell GFP fluorescence (Envision plate reader, 485 / 535 filter pair) or by a Beckman Coulter Quanta™ flow cytometer (expressed as the geometric mean of fluorescence intensity at 485 nm / 525 nm). Compounds were added to the desired final concentration for at least 18 hours but less than 24 hours in an incubator.

[0303] In certain experiments, the reference compounds included native hepcidin, minihepcidin, and R1-minihepcidin, an analog of minihepcidin. The "RI" in RI-minihepcidin stands for Retro Inverse. Retro-inverse peptides are peptides with the reverse sequence at all D amino acids. An example is Hy-Glu-Thr-His-NH2 becoming Hy-DHis-DThr-DGlu-NH2. The EC values ​​of these reference compounds for ferroportin degradation were: 50 was determined according to the activity assay described above. These peptides served as control standards. [Table 8]

[0304] Potency EC determined for various peptide analogs of the invention 50 Values ​​(nM) and other activity data are provided in patents US 9,822,157 and US 10,030,061, which are incorporated herein by reference in their entirety.

[0305] Example 3 Hepcidin inhibition in polycythemia vera Hepcidin analogs of the present invention were tested for activity in polycythemia vera as described by Casuet., Blood. 2016;128(2):265-276.

[0306] Hepcidin, a 25-amino acid peptide, regulates whole-body iron homeostasis and is produced by the liver in response to plasma iron concentration and iron stores. Hepcidin inhibits the cellular iron exporter ferroportin (FPN-1), which is expressed on the surface of cells involved in iron absorption, recycling, and storage. Hepcidin mediates systemic iron restriction and exogenous administration of exogenous hepcidin mimics, as well as reduction of Hgb levels and splenomegaly in a mouse model of polycythemia vera (Casu et al., Blood. 2016;128(2):265-276).

[0307] Constitutive activation of JAK2 leads to red blood cell-independent erythropoietin production, resulting in polycythemia. An approach to prevent the effects of mutant Jak2 is to induce iron restriction with hepcidin or a hepcidin-mimetic peptide. Low iron levels inhibit erythropoietin signaling downstream of Jak2, thus providing an override signal. When hepcidin-mimetic peptides were administered to transgenic mice expressing the human Jak2 gene with a polycythemia-causing mutation, increased erythropoiesis and hematocrit characteristic of polycythemia were reversed to the normal range (Casu et al., Blood. 2016;128(2):265-276).

[0308] Example 4 In vivo validation of peptide analogs of hepcidin Hepcidin analogs of the present invention were tested for in vivo activity to determine their ability to reduce free Fe2+ in serum.

[0309] In the PK-PD experiments, hepcidin analogs (Compound A (SEQ ID NO: 45) or Compound B (SEQ ID NO: 55)) or vehicle control were subcutaneously administered to cynomolgus monkeys (n=3 / group) at 2.44 mg / kg Compound A or 2.93 mg / kg Compound B. Serum samples were collected from the hepcidin analog-administered monkey groups at 0.5, 1, 2, 4, 8, 12, 24, 30, 36, 48, 60, 72, and 144 hours after dose administration. Plasma / serum iron content was measured using a colorimetric assay on a Cobas c 111 according to the manufacturer's instructions for the assay (Assay: IRON2: ACN661). Data from the Cobas Iron2 analysis are shown (as mean values ​​with SD) in Figure 1A for Compound A and Figure 1B for Compound B. Compound A induced a 5-fold reduction in serum iron 8 hours after administration. Compound B induced a maximal reduction in serum iron approximately 12 hours after administration, which was 12-fold lower than pre-administration levels.

[0310] These studies demonstrate that the hepcidin analogs of the present invention reduce serum iron levels for at least 60 hours when administered to cynomolgus monkeys. Both Compound A and Compound B had a concentration-dependent effect on serum iron reduction. There was also a delay in effect between the serum concentration of the hepcidin analog and its corresponding effect, i.e., the nadir of effect occurred at a time delayed from the peak serum concentration of the compound. For Compound A, serum concentrations ranging from 200 to 3200 ng / mL were effective, with 1000 to 3200 ng / mL having the maximum effect. For Compound B, serum concentrations from 25 to 125 ng / mL were effective, with 50 to 125 ng / mL having the maximum effect.

[0311] Example 5 Effectiveness of peptides that limit erythropoiesis The hepcidin analogs of the present invention were tested for their effectiveness in limiting erythropoiesis activity when administered to healthy cynomolgus monkeys. In a repeat-dose study, cynomolgus monkeys received four weekly doses of Compound A or vehicle control subcutaneously (SC) at three different doses: 0.6 mg / kg / dose, 2 mg / kg / dose, or 6 mg / kg / dose (n=6 / sex / high-dose or control group and n=3 / sex / low-dose or medium-dose group).

[0312] Compound A at 3 mg / kg / dose and above caused dose- and time-dependent reductions in pharmacologically mediated anemia, specifically, hematocrit (Hct) and hemoglobin (Hgb) (Table 7 and Figure 2). At day 29, Hgb levels in males treated with 1, 3, and 10 mg / kg Compound A were reduced by 0.1, 2.7, and 7.2 g / dL, respectively, compared with concurrent controls. Absolute Hct levels were reduced by 6% and 22% in the 3 mg / kg / dose and 10 mg / kg / dose male groups, respectively. Male animals exhibited a similar pharmacological response to females. After prolonged red blood cell (RBC) reduction, reticulocytosis occurred, with a statistically significant increase observed on day 29. After cessation of administration, RBC parameters returned to concurrent control values. [Table 9]

[0313] Secondary blood indices, including hematocrit and mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin per cell (MCH), which reflect RBC size and Hgb content, are shown in Figure 2. RBCs generated after Compound A-induced iron-restricted erythropoiesis were similar in size (MCV) to concurrent controls but showed a dose-dependent decrease in Hgb concentration per cell (MCHC and MCH) compared to concurrent controls. Upon recovery, vehicle control and 10 mg / kg Compound A-treated groups similarly demonstrated reversible hematological changes. The observed hematological findings are expected following excessive and persistent iron-restricted anemia in animals that were originally iron-replete.

[0314] Example 6 Effectiveness of peptides in restricting erythropoiesis In another repeat-dose study, Compound A or vehicle control was administered subcutaneously to cynomolgus monkeys at three different doses for 13 weeks, followed by a 5-week recovery period.

[0315] Cynomolgus monkeys (6 / sex / group) received 0 (0.9% saline), 0.6, 2, or 6 mg / kg / dose of Compound A subcutaneously once weekly (QW) for 3 months, a total of 13 doses (Days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85). One group of animals (4 / sex / group) was sacrificed on Day 92 (main), and the remaining animals (2 / sex / group) were sacrificed on Day 120 after a 35-day recovery period (recovery). Hematological samples were obtained twice before treatment initiation (Days -7 and -3) and on Days 27, 55, and 90 for main and recovery animals, corresponding to 5 days after the 4th, 8th, and 13th doses, and on Day 119 for recovery animals.

[0316] Hematological changes were consistent with the expected pharmacology of a hepcidin mimetic administered to iron-replete NHPs and included dose-dependent decreases in RBC parameters (RBC count, Hgb, and Hct) and increases in reticulocytes, as well as changes in RBC indices (decreases in MCHC and MCH) and RBC morphology (e.g., microcytosis and hypochromia) (data not shown). Consistent with Compound A-induced anemia, hematopoietic hypercellularity occurred in the bone marrow (femur, sternum), and extramedullary hematopoiesis and hemosiderin deposition were observed in the liver and spleen at doses 2 mg / kg and above (correlating with increased organ weights at 6 mg / kg / dose) (data not shown).

[0317] Hematological changes occurred consistent with the known erythropoietic pharmacology of hepcidin mimetics in inducing iron-deficiency anemia (Table 8). At day 90, Hgb levels were 0.6. Females treated with Compound A at 2 and 6 mg / kg had reductions of 1.3, 3.0, and 5.6 g / dL, respectively, compared to concurrent controls. Male animals developed a similar pharmacological response to females. Retinal cells increased in response to anemia in a dose-dependent manner at all dosing time points. After cessation of dosing, RBC parameters returned to concurrent control values. [Table 10]

[0318] In the same study, Compound A induced significant changes in secondary hematological indices, as shown in Figure 3. The dose-dependent decrease in MCHC and MCH is consistent with the expected RBC rheological changes associated with iron-restricted erythropoiesis. Hematological effects were similar to concurrent controls 35 days after treatment cessation.

[0319] Increases in total bilirubin (tBili) at doses above 2 mg / kg / day were also observed during treatment, which was associated with the destruction of RBCs associated with iron deficiency anemia (Figure 4) and was thought to be related to the preferential destruction of reticulocytes, the youngest red blood cells (Robinson & Koeppel, 1971). Consistent with the expected induction of iron deficiency anemia by Compound A, significant increases in platelet levels were observed in both male and female animals at the highest evaluated dose of 6 mg / kg / dose (Figure 5). After cessation of treatment at the recovery time, bilirubin levels had returned to normal levels, while platelet levels were beginning to rebound to within normal limits (compared to concurrent controls).

[0320] Example 7 Phase 2 Study of Compound A in Patients with Polycythemia Vera Requiring Phlebotomy Background: Patients with polycythemia vera (PV) are typically treated with periodic therapeutic phlebotomy (with or without concomitant cytoreductive therapy) to maintain a hematocrit below 45%. As a result, PV patients with a high need for phlebotomy are more likely to have a hematocrit above 45% between appointments. Meanwhile, the majority of PV patients are iron-deficient at diagnosis and worsen after repeated phlebotomy. PV patients may be symptomatic from iron deficiency (with cognitive impairment and fatigue even in the absence of anemia), and iron supplementation typically results in an increased rate of phlebotomy. Recent trials have shown that available therapies partially ameliorate PV-related symptoms by reversing iron deficiency. Therefore, symptomatic PV patients requiring phlebotomy represent an unmet therapeutic need. We hypothesize that hepcidin mimetics promote iron sequestration in splenic macrophages, reducing iron availability for malignant erythropoiesis and reducing the need for phlebotomy while reversing iron deficiency-related symptoms.

[0321] Compound A is a hepcidin mimetic in clinical trials for multiple blood disorders. In wild-type mice, repeated subcutaneous injections of Compound A transiently reduced serum iron and caused a dose-related decrease in hematocrit. A phase I study of Compound A as a single dose in 62 healthy subjects demonstrated a 65% reduction in serum iron concentration and a 70% reduction in transferrin saturation from baseline, without significant adverse events.

[0322] Objectives: The primary objective of this three-part Phase 2 clinical trial (outlined in Figure 6) was to demonstrate the efficacy (reduced phlebotomy requirements) and safety of Compound A in PV patients requiring phlebotomy. Secondary objectives were to determine the effect of Compound A on patient-reported outcomes and markers of iron metabolism.

[0323] Methods: Eligibility criteria included a diagnosis of PV (according to the 2016 WHO criteria) and at least three phlebotomies targeting a hematocrit of 45% or less in the 6 months prior to enrollment, with or without stable-dose cytoreductive therapy. Eligible patients were enrolled in a 28-week dose-finding portion of a phase 2 clinical trial. As outlined in Figure 7, patients received Compound A doses of 10, 20, 40, 60, and 80 mg administered subcutaneously once weekly, with individualized titration to maintain a hematocrit of less than 45%. Iron status was quantified by monitoring serum ferritin, serum iron, transferrin saturation (TSAT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH).

[0324] Results: Efficacy data were available for 13 subjects enrolled in the trial: 7 / 13 with low-risk PV and 6 / 13 with high-risk PV; mean age 57.4 years (range 31-74 years); 6 received TP alone, 6 received hydroxyurea concomitantly, and 1 received interferon concomitantly; TP = 3-9 in the 24 weeks prior to enrollment; median time between TP = 42 days. Patient characteristics are shown in Table 9. [Table 11-1] [Table 11-2]

[0325] All subjects maintained hematocrits below 45% after appropriate dose adjustments. Mean baseline values ​​were serum ferritin = 14.2 ng / mL (5, 37), serum iron = 33.0 ug / dL (16.8, 107.8), and TSAT = 7.6% (4, 30). During treatment with Compound A, serum ferritin levels gradually increased toward normal, reflecting increased iron stores (Figure 8). TSAT (Figure 9) and serum iron values ​​fluctuated transiently but remained below the normal range, reflecting the pharmacodynamic effect of Compound A inhibiting iron release from intracellular stores. This was associated with increases in MCV (Figure 10) and MCH (Figure 11), as well as decreases in hematocrit and red blood cell count, both of which suggested normalization of iron distribution.

[0326] Eight subjects were treated with Compound A for ≥3 months (Figure 7). Three subjects were randomized. During the open-label dose-finding portion of the study, all subjects were phlebotomy-free, except for one subject who was non-adherent, missed scheduled treatment between weeks 4 and 9, and underwent phlebotomy at approximately week 13. Three subjects completed Part 1 (28 weeks) without TP, compared with the 3–5 TPs required for a similar period prior to study initiation. During the 28-week dose-finding period, hematocrit was continuously controlled below 45% in all but two subjects (Figure 12). Two subjects had hematocrits transiently above 45%, while one subject remained below 45% after phlebotomy, and both required dose escalation. Furthermore, red blood cell counts decreased (Figure 13), and MCV increased in all but two subjects. These findings suggest a redistribution of iron within erythropoiesis. Finally, before treatment, mean iron-related parameters were consistent with systemic iron deficiency, and serum ferritin gradually increased toward the normal range. The most frequent adverse event was injection site reaction (ISR), reported by three patients. Most reactions were grade 1-2 and transient, and no patients discontinued the drug.

[0327] These studies demonstrate that Compound A is well tolerated. At the end of the evaluable treatment period, compliant subjects demonstrated a significant decrease in hematocrit and absolute levels below 45% compared to pre-enrollment, as well as increased ferritin, suggesting an improvement in iron deficiency symptoms. Subjects' platelet counts generally remained stable over the course of treatment ( FIG. 14 ). Subjects' reticulocyte % showed an upward trend over the course of treatment ( FIG. 15 ), although no similar increase in mature red blood cells was observed (data not shown). Subjects' white blood cell counts generally remained stable over the course of treatment, suggesting that treatment did not induce an inflammatory response ( FIG. 16 ). There appeared to be no progression of PV disease, as evidenced by the lack of platelet and white blood cell increases.

[0328] Plasma concentrations of Compound A were measured at various times after subcutaneous administration of 10 mg to 80 mg in patients with PV. Concentrations increased dose-dependently and varied based on the dose and time of sampling. Compound A concentrations ranged from undetectable (<2 ng / mL) to 866 ng / mL (Figures 17A and 17B).

[0329] Conclusions: These results support the use of hepcidin mimetics, such as Compound A, in the treatment of patients with PV, including low-risk patients with a high need for therapeutic phlebotomy. Compound A and other hepcidin analogs are hypothesized to promote iron sequestration in splenic macrophages, reducing iron availability for malignant erythropoiesis and reducing the need for phlebotomy while reversing iron deficiency-related symptoms. These studies demonstrate that Compound A is an effective agent for the treatment of PV, reversing iron deficiency and eliminating the need for TP in patients with PV. Elimination of the need for TP over a 7-month period in patients with TP-dependent PV is significant and unexpected. These results demonstrate that Compound A is an effective agent for controlling hematocrit, reversing iron deficiency, and eliminating therapeutic phlebotomy in both low- and high-risk patients. These results also establish the dosing regimen, route of administration, and methods for monitoring and adjusting the dose throughout treatment.

[0330] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and / or non-patent publications referred to in this application and / or listed in this application data sheet are hereby incorporated by reference in their entirety.

[0331] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. The present invention includes the following aspects. [Section 1] 1. A method for treating polycythemia vera in a subject in need thereof, comprising administering to the subject an effective amount of a hepcidin analog or a pharmaceutically acceptable salt or solvate thereof, wherein the hepcidin analog comprises a peptide comprising or consisting of Formula I; R1-XY-R2(I) (SEQ ID NO: 1) During the ceremony, R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C20 alkanoyl, or pGlu; R2 is NH2 or OH; X is a peptide sequence having formula II, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10(II) (Sequence number 2) 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, or D-His; 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 a peptide sequence having formula III, Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15(III) (SEQ ID NO: 3) 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 or consisting of Formula I is optionally PEGylated at R1, X or Y; The foregoing methods, wherein the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer moieties. [Section 2] Item 1, the method of claim 1, wherein R1 is hydrogen, isovaleric acid, isobutyric acid, or acetyl. [Section 3] X is a peptide sequence having formula IV, X1-Thr-His-X4-X5-X6-X7-X8-Phe-X10(IV) (SEQ ID NO: 4) 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; Item 10. The method of item 1, wherein X10 is Lys or absent. [Section 4] X is a peptide sequence having the formula V, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10(V) (SEQ ID NO: 5) 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; Item 3. The method of item 1 or 2, wherein X10 is Lys or absent. [Section 5] The peptide is according to formula VI or R1-XY-R2(VI) (SEQ ID NO: 6) or a pharmaceutically acceptable salt thereof, wherein: R1 is hydrogen, isovaleric acid, isobutyric acid, or acetyl; R2 is -NH2 or -OH; X is a peptide sequence having formula VII, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10(VII) (SEQ ID NO: 7) 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 a peptide sequence having formula VIII; Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10(VIII) (SEQ ID NO: 8) 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 comprises a disulfide bond between two Cys; the peptide of formula I is optionally PEGylated at R1, X, or Y; the side chains of the amino acids of said peptides are optionally conjugated to lipophilic substituents or polymer moieties; Item 2. The method according to item 1, wherein Ida is iminodiacetic acid, pGlu is pyroglutamic acid, bhAsp is β-homoaspartic acid, and bhPro is β-homoproline. [Section 6] The peptide has the following sequence: DTHFPICIFGPRSKGWVC (SEQ ID NO: 9), DTHFPCIIFGPRSKGWVCK (SEQ ID NO: 10), DTHFPCIIFEPRSKGWVCK (SEQ ID NO: 11), DTHFPCIIFGPRSKGWACK (SEQ ID NO: 12), DTHFPCIIFGPRSKGWVCKK (SEQ ID NO: 13), DTHFPCIIFVCHRPKGCYRRVCR (SEQ ID NO: 14), DTHFPCIKFGPRSKGWVCK (SEQ ID NO: 15), DTHFPCIKFKPRSKGWVCK (SEQ ID NO: 16), DTHFPCIIFGPRSRGWVCK (SEQ ID NO: 17), DTHFPCIKFGPKSKGWVCK (SEQ ID NO: 18), DTHFPCIKFEPRSKGCK (SEQ ID NO: 19), DTHFPCIKFEPKSKGWECK (SEQ ID NO: 20), DTHFPCIKFEPRSKKCK (SEQ ID NO: 21), DTHFPCIKFEPRSKGCKK (SEQ ID NO: 22), DTHFPCIKFKPRSKGCK (SEQ ID NO: 23), DTHFPCIKFEPKSKGCK (SEQ ID NO: 24), DTHFPCIKF (SEQ ID NO: 25), DTHFPCIIF (SEQ ID NO: 26) or DTKFPCIIF (SEQ ID NO: 27), Item 1 or 2. The method of claim 1 or 2, wherein the peptide is optionally PEGylated at R1, X, or Y, and the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer moieties. [Section 7] The hepcidin analog has the following sequence: Isovaleric acid-DTHFPICIFGPRSKGWVC-NH2 (SEQ ID NO: 9), Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH2 (SEQ ID NO: 10), Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH2 (SEQ ID NO: 11), Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH2 (SEQ ID NO: 12), Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH2 (SEQ ID NO: 13), Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH2 (SEQ ID NO: 14), Isovaleric acid-DTHFPCI(K(PEG))FGPRSKGWVCK-NH (SEQ ID NO: 28), Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH2 (SEQ ID NO: 16), Isovaleric acid-DTHFPICIFGPRS(K(PEG))GWVC-NH (SEQ ID NO: 29), Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH2 (SEQ ID NO: 30), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG))-NH2 (SEQ ID NO: 31), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG))-NH (SEQ ID NO: 32), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH2 (SEQ ID NO: 33), Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 34), Isovaleric acid-DTHFPCIKF)K(Palm))PRSKGWVCK-NH2 (SEQ ID NO: 35), Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH2 (SEQ ID NO: 36), Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH2 (SEQ ID NO: 37), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))NH2 (SEQ ID NO: 38), Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 39), Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40), Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH2 (SEQ ID NO: 41), Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH2 (SEQ ID NO: 42), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH2 (SEQ ID NO: 43), Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG))-NH2 (SEQ ID NO: 44), Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45), Isovaleric acid-DTHFPCIKF-K(isoGlu-Palm)-PRSKGCK-NH2 (SEQ ID NO: 46), Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47), Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGWECK-NH2 (SEQ ID NO: 20), Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48), Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))CK-NH2 (SEQ ID NO: 21), Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH2 (SEQ ID NO: 49), Isovaleric acid-DTHFPCI-K(Dapa-Palm)-FEPRSKGCK-NH2 (SEQ ID NO: 50), Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGCK-NH2 (SEQ ID NO: 23), Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH2 (SEQ ID NO: 24), Isovaleric acid-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH2 (SEQ ID NO: 51), Isovaleric acid-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH2 (SEQ ID NO: 52), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))K-NH2 (SEQ ID NO: 53), Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))-NH2 (SEQ ID NO: 54), Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH2 (SEQ ID NO: 55), Isovaleric acid-DTHFPCIKF-NH2 (SEQ ID NO: 25), Hy-DTHFPCIKF-NH2 (SEQ ID NO: 25), Isovaleric acid-DTHFPCIIF-NH2 (SEQ ID NO: 26), Hy-DTHFPCIIKF-NH2 (SEQ ID NO: 26), Isovaleric acid-DTKFPCIIF-NH2 (SEQ ID NO: 27) or Hy-DTKFPCIIF-NH2 (SEQ ID NO: 27) The method according to item 1 or 2, comprising one of the following: [Section 8] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH2 (SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof. [Section 9] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH2 (SEQ ID NO: 11) or a pharmaceutically acceptable salt thereof. [Section 10] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH2 (SEQ ID NO: 28) or a pharmaceutically acceptable salt thereof. [Section 11] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH2 (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof. [Section 12] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH2 (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof. [Section 13] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 34) or a pharmaceutically acceptable salt thereof. [Section 14] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKF(K(Palm))PRSKGWVCK-NH2 (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof. [Section 15] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH2 (SEQ ID NO: 36) or a pharmaceutically acceptable salt thereof. [Section 16] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))-NH2 (SEQ ID NO: 38) or a pharmaceutically acceptable salt thereof. [Section 17] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH2 (SEQ ID NO: 39) or a pharmaceutically acceptable salt thereof. [Section 18] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof. [Section 19] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH2 (SEQ ID NO: 41) or a pharmaceutically acceptable salt thereof. [Section 20] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH2 (SEQ ID NO: 42) or a pharmaceutically acceptable salt thereof. [Section 21] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH2 (SEQ ID NO: 43) or a pharmaceutically acceptable salt thereof. [Section 22] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH2 (SEQ ID NO: 44) or a pharmaceutically acceptable salt thereof. [Section 23] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof. [Section 24] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof. [Section 25] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof. [Section 26] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof. [Section 27] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(Dapa-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 50) or a pharmaceutically acceptable salt thereof. [Section 28] Item 3. The method of item 1 or 2, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH2 (SEQ ID NO: 24) or a pharmaceutically acceptable salt thereof. [Section 29] The hepcidin analog is [ka] Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40), [ka] Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45), [ka] Isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46), [ka] Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) and [ka] Item 3. The method of item 1 or 2, wherein the amino acid is selected from the group consisting of isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48), (wherein the amino acid is an L-amino acid), and pharmaceutically acceptable salts thereof. [Section 30] Item 30. The method of any one of items 1 to 29, wherein the hepcidin analog is administered to the subject in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, or diluents. [Section 31] 31. The method of claim 30, wherein the pharmaceutical composition is provided to the subject by oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal or topical administration route. [Section 32] 32. The method of claim 31, wherein the pharmaceutical composition is provided to the subject by oral or subcutaneous administration. [Section 33] Item 33. The method of any one of items 1 to 32, wherein the hepcidin analog or pharmaceutical composition is provided to the subject at most twice a week or at most once a week. [Section 34] Item 34. The method according to any one of Items 1 to 33, wherein the hepcidin analog is provided to the subject at a dose of about 10 mg to about 100 mg, about 10 mg to about 70 mg, about 10 mg to about 60 mg, about 20 mg to about 50 mg, about 20 mg to about 40 mg, about 80 mg, about 70 mg, about 60 mg, about 50 mg, about 40 mg, about 30 mg, about 25 mg, about 20 mg, about 15 mg, or about 10 mg. [Section 35] Item 34. The method according to any one of Items 1 to 33, wherein the peptide or the pharmaceutical composition is provided to the subject at a dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, or about 40 mg. [Section 36] Item 34. The method according to any one of Items 1 to 33, wherein the peptide or pharmaceutical composition is provided to the subject about once a week at a dose of about 15 mg, about 20 mg, about 25 mg, about 30 mg, or about 40 mg. [Section 37] Item 34. The method according to any one of Items 1 to 33, wherein the peptide or pharmaceutical composition is provided to the subject about twice a week at a dose of about 15 mg, about 20 mg, about 25 mg, about 30 mg, or about 40 mg. [Section 38] administering to the subject an effective amount of a hepcidin analog, wherein the hepcidin analog comprises: [ka] Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof; [ka] Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof; [ka] Isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof; [ka] Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof, and [ka] Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof; wherein the amino acid is an L-amino acid; Optionally, the hepcidin analog comprises a disulfide bond between two Cys amino acids; the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject about once a week at a dose of about 5 mg to about 200 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg; the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject by subcutaneous administration; the subject is a human; Optionally, the hepcidin analog or a pharmaceutically acceptable salt thereof is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. [Section 39] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 10 mg. [Section 40] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 15 mg. [Section 41] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 20 mg. [Section 42] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 25 mg. [Section 43] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 30 mg. [Section 44] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 40 mg. [Section 45] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 50 mg. [Section 46] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 60 mg. [Section 47] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 70 mg. [Section 48] Item 39. The method of any one of items 1 to 33 or 38, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dosage of about 80 mg. [Section 49] Item 49. The method according to any one of Items 38 to 48, wherein the hepcidin analog is isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof. [Section 50] Item 49. The method of any one of Items 38 to 48, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof. [Section 51] Item 49. The method according to any one of Items 38 to 48, wherein the hepcidin analog is isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof. [Section 52] Item 49. The method according to any one of Items 38 to 48, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof. [Section 53] Item 49. The method according to any one of Items 38 to 48, wherein the hepcidin analog is isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof. [Section 54] Item 54. The method according to any one of Items 1 to 53, wherein the polycythemia vera is polycythemia vera requiring phlebotomy. [Section 55] Item 55. The method according to any one of Items 1 to 54, wherein the polycythemia vera is polycythemia vera requiring phlebotomy in a low-risk patient. [Section 56] Item 55. The method according to any one of Items 1 to 54, wherein the subject is a low-risk polycythemia vera patient or a high-risk polycythemia vera patient. [Section 57] Item 57. The method according to any one of Items 1 to 56, wherein the subject is a symptomatic polycythemia vera patient requiring phlebotomy. [Section 58] Item 55. The method according to any one of Items 1 to 54, wherein the subject is a low-risk patient with polycythemia vera requiring phlebotomy or a high-risk patient with polycythemia vera requiring phlebotomy. [Section 59] Item 59. The method according to any one of items 1 to 58, wherein the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies to achieve a hematocrit of 45% or less in the 24 weeks prior to administering the pharmaceutical composition to the subject. [Section 60] Item 60. The method according to any one of Items 1 to 33, 38, and 49 to 59, wherein about 5 mg to about 200 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 61] Item 60. The method according to any one of Items 1 to 33, 38, and 49 to 59, wherein about 10 mg to about 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 62] Item 60. The method according to any one of Items 1 to 33, 38, and 49 to 59, wherein about 20 mg to about 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 63] Item 60. The method according to any one of items 1 to 33, 38, and 49 to 59, wherein about 20 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 64] Item 60. The method according to any one of items 1 to 33, 38, and 49 to 59, wherein the subject is administered about 40 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof. [Section 65] Item 60. The method according to any one of items 1 to 33, 38, and 49 to 59, wherein about 80 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 66] Item 60. The method according to any one of items 1 to 33, 38, and 49 to 59, wherein about 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 67] Item 60. The method according to any one of items 1 to 33, 38, and 49 to 59, wherein about 120 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject. [Section 68] Item 68. The method according to any one of Items 1 to 67, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered by subcutaneous injection. [Section 69] Item 69. The method according to any one of Items 1 to 68, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof, or pharmaceutical composition is administered approximately weekly for a certain period of time. [Section 70] Item 70. The method according to any one of items 1 to 69, wherein the dosage of the hepcidin analog or a pharmaceutically acceptable salt thereof is increased over a period of time. [Section 71] Item 69. The method of any one of items 1 to 69, further comprising determining the subject's hematocrit at one or more time points after administration of the hepcidin analog or its pharmaceutically acceptable salt; and maintaining or adjusting the amount of the hepcidin analog or its pharmaceutically acceptable salt administered to the subject, wherein the amount is increased if the subject's determined hematocrit is greater than 45, decreased if the subject's determined hematocrit is either less than 37.5 or less than 40, and maintained if the subject's determined hematocrit is between 37.5 and 45 or between 40 and 44. [Section 72] Item 72. The method according to any one of Items 1 to 71, wherein the subject is a mammal. [Section 73] Item 73. The method according to any one of Items 1 to 72, wherein the subject is a human. [Section 74] 74. The method of paragraph 72 or paragraph 73, wherein the subject is treated with cytoreductive therapy, optionally with hydroxyurea. [Section 75] Item 75. The method of any one of items 1 to 74, wherein the method reduces the subject's hematocrit level to 45% or less. [Section 76] 76. The method of any one of paragraphs 1 to 75, wherein the method results in a reduction in hematocrit of at least 3%. [Section 77] Item 77. The method of any one of items 1 to 76, wherein the method results in an increase in serum ferritin in the subject. [Section 78] Item 78. The method according to any one of items 1 to 77, wherein the method does not substantially alter the platelet count in the subject. [Section 79] Item 79. The method according to any one of items 1 to 78, wherein the method does not substantially increase white blood cells or leukocytes in the blood or serum of the subject. [Section 80] 80. The method of any one of paragraphs 1 to 79, wherein the subject remains phlebotomy-free during the course of treatment, for example, about once per week, about once every two weeks, or about once per month over a period of time. [Section 81] Item 81. The method of any one of items 1 to 80, wherein the method comprises administering an effective amount of the hepcidin analog or a pharmaceutically acceptable salt thereof multiple times over a period of time, and optionally, the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject about once a week over the period of time. [Section 82] The hepcidin analog is [ka] Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH2 (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof; [ka] Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH2 (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof; [ka] Isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH2 (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof; [ka] Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH2 (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof, and [ka] Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH2 (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof; wherein the amino acid is an L-amino acid; Optionally, the hepcidin analog comprises a disulfide bond between two Cys amino acids; the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject at a dosage of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg about once per week for said period of time; the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject by subcutaneous administration; the subject is a human; Optionally, the hepcidin analog or a pharmaceutically acceptable salt thereof is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent; 82. The method of claim 81, further comprising determining the subject's hematocrit after one or more of the multiple administrations of the hepcidin analog or pharmaceutically acceptable salt thereof, and maintaining or adjusting the amount of the hepcidin analog or pharmaceutically acceptable salt thereof subsequently administered to the subject, wherein the subsequent dose is increased if the subject's determined hematocrit is above an acceptable range based on the subject's gender and pregnancy status, decreased if the subject's determined hematocrit is below the acceptable range, and the subsequent dose is the same as the previously administered amount if the subject's determined hematocrit is within the acceptable range.

Claims

1. A pharmaceutical product comprising a hepcidin analog or a pharmaceutically acceptable salt or solvate thereof, for use in a method for treating polycythemia vera in a subject requiring treatment for polycythemia vera, wherein the method comprises administering an effective amount of the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof to the subject, wherein the hepcidin analog comprises formula I or a peptide comprising formula I. R1-X-Y-R2(I) (Sequence ID 1) During the ceremony, R1 is C1-C20 alkanoyl, hydrogen, C1-C6 alkyl, C6-C12 aryl, or pGlu. R2 is NH ２ or OH, X is a peptide sequence having formula II, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (II) (SEQ ID NO: 2) 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, or D-His. X4 is Phe, Ala, Dpa, or D-Phe, X5 is Pro, Gly, Arg, Lys, Ala, D-Pro, or bhPro. X6 is Cys, Ile, Arg, Lys, D-Ile, or D-Cys. X7 is Ile, Cys, Leu, Val, Phe, D-Ile or D-Cys, X8 is Lys, Ile, Arg, Phe, Gln, Glu, Val, Leu, or D-Ile. X9 is Phe or bhPhe, X10 is Lys, Phe, or absent. If Y does not exist, then X7 is Ile, and Y is a peptide sequence having formula III, Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15 (III) (Sequence ID 3) During the ceremony, Y1 is Pro, Gly, Cys, Ala, Phe, Glu, Lys, D-Pro, Val, Ser, or absent. Y2 is absent, Pro, Ala, Cys, or Gly. 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 a non-natural amino acid, Gly, Ser, Lys, Ile, Ala, Pro, Val, or absent. Y7 is absent, Trp, Lys, Gly, Ala, Ile, or Val. Y8 is Cys, Val, Thr, Gly, Met, Tyr, Ala, Glu, Lys, Asp, Arg, or absent. Y9 is absent, Cys, or Tyr. Y10 is Lys, Met, Arg, Tyr, or absent. Y11 is absent, Arg, Met, Cys, or Lys. Y12 is absent, Arg, Lys, or Ala. Y13 is nonexistent, Arg, Cys, Lys, or Val. Y14 is absent, Arg, Lys, Pro, Cys, or Thr, and Y15 is absent, Thr, or Arg. The peptide containing or consisting of formula I is optionally PEG-modified at R1, X, or Y. The pharmaceutical product wherein the side chains of the amino acids of the peptide are optionally conjugated to a lipophilic substituent or polymer moiety.

2. The pharmaceutical product according to claim 1, wherein R1 is isovaleric acid, hydrogen, isobutyric acid, or acetyl.

3. (a) X is a peptide sequence having formula IV, X1-Thr-His-X4-X5-X6-X7-X8-Phe-X10 (IV) (SEQ ID NO: 4) During the ceremony, X1 is Asp, Ida, pGlu, bhAsp or absent, X4 is either Phe or Dpa, X5 is either Pro or bhPro, X6 is Cys, Ile, or Arg. X7 is Ile, Cys, Leu, or Val. X8 is Lys, Ile, Glu, Phe, Gln, or Arg, and X10 is Lys or absent, (b) X is a peptide sequence having formula V, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10 (V) (SEQ ID NO: 5) During the ceremony, X1 is Asp, Ida, pGlu, bhAsp or absent, X4 is either Phe or Dpa, X5 is either Pro or bhPro, X8 is Lys, Ile, Glu, Phe, Gln, or Arg, and X10 is Lys or absent, (c) The peptide conforms to formula VI, R １ -X-Y-R ２ (VI) (Sequence No. 6) or a pharmaceutically acceptable salt thereof, in the formula, R １ However, these are isovaleric acid, hydrogen, isobutyric acid, or acetyl, R ２ However, -NH ２ Or it is -OH, X is a peptide sequence having formula VII, X1-Thr-His-X4-X5-Cys-Ile-X8-Phe-X10 (VII) (SEQ ID NO: 7) During the ceremony, X1 is Asp, Ida, pGlu, bhAsp or absent, X4 is either Phe or Dpa, X5 is either Pro or bhPro, X8 is Lys, Ile, Glu, Phe, Gln, or Arg, and X10 is absent or Lys, Y is a peptide sequence having formula VIII, Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10(VIII)(Sequence ID 8) During the ceremony, Y1 is Lys, Gly, Glu, or Val. Y3 is either Arg or Lys, Y5 is either Lys or Arg. Y6 is a non-natural amino acid, Gly, Ser, Lys, Ile, or Arg. Y7 is absent or Trp, Y8 is absent, Val, Thr, Asp, or Glu. Y10 is Lys or does not exist. The peptide contains a disulfide bond between two Cys molecules. The peptide of formula I is optionally R １ , PEG formatted in X or Y, The amino acid side chains of the peptide are optionally conjugated to lipophilic substituents or polymer moieties. The pharmaceutical product according to claim 1, wherein Ida is iminodiacetic acid, pGlu is pyroglutamic acid, bhAsp is β-homoaspartic acid, and bhPro is β-homoproline.

4. (a) The hepcidin analog has the following sequence: Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH2 (SEQ ID NO: 55), Isovaleric acid-DTHFPICIFGPRSKGWVC-NH ２ (Sequence No. 9) Iso-valeric acid-DTHFPCIIFGPRSKGWVCK-NH ２ (SEQ ID NO: 10), Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH ２ (Sequence No. 11) Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH ２ (Sequence No. 12) Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH ２ (Sequence No. 13) Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH ２ (Sequence No. 14) Isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH ２ (Sequence No. 28) Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH ２ (Sequence No. 16) Isovaleric acid-DTHFPICIFGPRS(K(PEG8))GWVC-NH ２ (Sequence No. 29) Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH ２ (Sequence No. 30) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH ２ (Sequence No. 31) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG4))-NH ２ (Sequence No. 32) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH ２ (Sequence No. 33) Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH ２ (Sequence No. 34) Isovaleric acid-DTHFPCIKF)K(Palm))PRSKGWVCK-NH ２ (Sequence No. 35) Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH ２ (Sequence No. 36) Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH ２ (Sequence No. 37) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))NH ２ (Sequence No. 38) Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH ２ (Sequence No. 39) Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH ２ (Sequence No. 40) Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH ２ (Sequence No. 41) Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH ２ (Sequence No. 42) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH ２ (Sequence No. 43) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH ２ (Sequence No. 44) Isovaleric acid-DTHFPCI (K(isoGlu-Palm))FEPRSKGCK-NH ２ (Sequence No. 45) Isovaleric acid-DTHFPCIKF-K (isoGlu-Palm)-PRSKGCK-NH ２ (Sequence No. 46) Isovaleric acid-DTHFPCIKFEP (K(isoGlu-Palm))SKGCK-NH ２ (Sequence No. 47) Isovaleric acid-DTHFPCIKFEP (K(isoGlu-Palm))SKGWECK-NH ２ (Sequence No. 20) Iso-glucosamine-DTHFPCIKFEPRS (K(isoGlu-Palm))GCK-NH ２ (Allocation number 48), Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))CK-NH ２ (Sequence No. 21) Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH ２ (Sequence No. 49) Isovaleric acid-DTHFPCI-K(Dap-Palm)-FEPRSKGCK-NH ２ (Sequence No. 50) Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGCK-NH ２ (Sequence No. 23) Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH ２ (Sequence No. 24) Isovaleric acid-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH ２ (Sequence No. 51) Isovaleric acid-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH ２ (Sequence No. 52) Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))K-NH ２ (Sequence No. 53) Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))-NH ２ (Sequence No. 54) Isovaleric acid-DTHFPCIKF-NH ２ (Sequence No. 25) Hy-DTHFPCIKF-NH ２ (Sequence No. 25) Isovaleric acid-DTHFPCIIF-NH ２ (Sequence No. 26) Hy-DTHFPCIIKF-NH ２ (Sequence No. 26) Isovaleric acid-DTKFPCIIF-NH ２ (Sequence ID 27) or Hy-DTKFPCIIF-NH ２ (Sequence No. 27) Includes one of the following, or (b) The peptide has the following sequence: DTHFPICIFGPRSKGWVC (Sequence ID 9), DTHFPCIIFGPRSKGWVCK (Sequence ID 10), DTHFPCIIFEPRSKGWVCK (Sequence ID 11), DTHFPCIIFGPRSKGWACK (Sequence ID 12), DTHFPCIIFGPRSKGWVCKK (Sequence ID 13), DTHFPCIIFVCHRPKGCYRRVCR (Sequence ID 14), DTHFPCIKFGPRSKGWVCK (Sequence ID 15), DTHFPCIKFKPRSKGWVCK (Sequence ID 16), DTHFPCIIFGPRSRGWVCK (SEQ ID NO: 17), DTHFPCIKFGPKSKGWVCK (Sequence ID 18), DTHFPCIKFEPRSKGCK (Sequence ID 19), DTHFPCIKFEPKSKGWECK (Sequence ID 20), DTHFPCIKFEPRSKKKCK (Sequence ID 21), DTHFPCIKFEPRSKGCKK (Sequence ID 22), DTHFPCIKFKPRSKGCK (Sequence ID 23), DTHFPCIKFEPKSKGCK (Sequence ID 24), DTHFPCIKF (Sequence ID 25), DTHFPCIIF (Sequence ID 26) or It includes one of the DTKFPCIIF (Sequence ID 27), The pharmaceutical product according to claim 1 or 2, wherein the peptide is optionally PEG-modified at R1, X, or Y, and the amino acid side chains of the peptide are optionally conjugated to a lipophilic substituent or polymer portion.

5. The aforementioned hepcidin analog is (a) Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH2 (SEQ ID NO: 55) or a pharmaceutically acceptable salt thereof (b) Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH ２ (SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof, (c) Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH ２ (SEQ ID NO: 11) or a pharmaceutically acceptable salt thereof, (d) Isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH ２ (SEQ ID NO: 28) or a pharmaceutically acceptable salt thereof, (e) Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH ２ (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof (f) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH ２ (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof, (g) Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH ２ (SEQ ID NO: 34) or a pharmaceutically acceptable salt thereof (h) Isovaleric acid-DTHFPCIKF(K(Palm))PRSKGWVCK-NH ２ (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof, (i) Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH ２ (SEQ ID NO: 36) or a pharmaceutically acceptable salt thereof, (j) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))-NH ２ (SEQ ID NO: 38) or a pharmaceutically acceptable salt thereof (k) Isovaleric acid-DTHFPCI (K(PEG3-Palm))FGPRSKGWVCK-NH ２ (SEQ ID NO: 39) or a pharmaceutically acceptable salt thereof, (l) Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH ２ (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof, (m) Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH ２ (SEQ ID NO: 41) or a pharmaceutically acceptable salt thereof, (n) Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH ２ (SEQ ID NO: 42) or a pharmaceutically acceptable salt thereof, (o) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH ２ (SEQ ID NO: 43) or a pharmaceutically acceptable salt thereof, (p) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH ２ (SEQ ID NO: 44) or a pharmaceutically acceptable salt thereof, (q) Isovaleric acid-DTHFPCI (K(isoGlu-Palm))FEPRSKGCK-NH ２ (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof, (r) Isovaleric acid-DTHFPCIKF (K(isoGlu-Palm))PRSKGCK-NH ２ (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof (s) Isovaleric acid-DTHFPCIKFEP (K(isoGlu-Palm))SKGCK-NH ２ (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof, (t) Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH ２ (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof, (u) Isovaleric acid-DTHFPCI(K(Dap-Palm))FEPRSKGCK-NH ２ (SEQ ID NO: 50) or a pharmaceutically acceptable salt thereof, (v) Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH ２ The pharmaceutical product according to claim 1 or 2, wherein the product is (Sequence ID 24) or a pharmaceutically acceptable salt thereof.

6. The aforementioned hepcidin analog is 【Chemistry 1】 Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH ２ (Sequence No. 40) 【Chemistry 2】 Isovaleric acid-DTHFPCI (K(isoGlu-Palm))FEPRSKGCK-NH ２ (Sequence No. 45) 【Transformation 3】 Isovaleric acid-DTHFPCIKF (K(isoGlu-Palm))PRSKGCK-NH ２ (Sequence No. 46) 【Chemistry 4】 Isovaleric acid-DTHFPCIKFEP (K(isoGlu-Palm))SKGCK-NH ２ (Sequence ID 47) and 【Transformation 5】 Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH ２ A pharmaceutical agent according to claim 1 or 2, selected from the group consisting of (Sequence ID 48), (wherein the formula the amino acid is an L-amino acid), and pharmaceutically acceptable salts thereof.

7. The hepcidin analog is administered to the subject in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may optionally be provided to the subject by oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, oral, parenteral, rectal, vaginal, or topical administration route. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition is optionally provided to the subject by an oral or subcutaneous administration route.

8. The pharmaceutical product according to any one of claims 1 to 7, wherein the hepcidin analog or pharmaceutical composition is provided to the subject up to twice a week or up to once a week.

9. (a) The hepcidin analog is provided to the subject in doses of approximately 10 mg to approximately 100 mg, approximately 10 mg to approximately 70 mg, approximately 10 mg to approximately 60 mg, approximately 20 mg to approximately 50 mg, approximately 20 mg to approximately 40 mg, approximately 80 mg, approximately 70 mg, approximately 60 mg, approximately 50 mg, approximately 40 mg, approximately 30 mg, approximately 25 mg, approximately 20 mg, approximately 15 mg, or approximately 10 mg. (b) The peptide or the pharmaceutical composition is provided to the subject in doses of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, or about 40 mg. (c) The peptide or the pharmaceutical composition is provided to the subject about once a week in a dose of about 15 mg, about 20 mg, about 25 mg, about 30 mg, or about 40 mg. (d) The peptide or the pharmaceutical composition is provided to the subject in doses of about 15 mg, about 20 mg, about 25 mg, about 30 mg, or about 40 mg, about twice a week. (e) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 10 mg. (f) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 15 mg. (g) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 20 mg. (h) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 25 mg. (i) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 30 mg. (j) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 40 mg. (k) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 50 mg. (l) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 60 mg. (m) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 70 mg, or (n) The pharmaceutical product according to any one of claims 1 to 8, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of about 80 mg.

10. The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject about once a week in doses of about 5 mg to about 200 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, or about 80 mg. The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject via a subcutaneous administration route. The subject is a human, Optional, (a) The hepcidin analog comprises a disulfide bond between two Cys amino acids, and / or (b) The hepcidin analog or a pharmaceutically acceptable salt thereof is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient or diluent, The pharmaceutical product according to claim 6.

11. The pharmaceutical product according to any one of claims 1 to 10, wherein the polycythemia vera is polycythemia vera requiring phlebotomy, and optionally, the subject is a patient with symptomatic polycythemia vera requiring phlebotomy.

12. (a) The polycythemia vera is polycythemia vera requiring phlebotomy in a low-risk patient, (b) The subject is a low-risk patient with polycythemia vera or a high-risk patient with polycythemia vera, (c) The pharmaceutical product according to any one of claims 1 to 11, wherein the subject is a low-risk patient having polycythemia vera requiring bloodletting, or a high-risk patient having polycythemia vera requiring bloodletting.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the subject is diagnosed with polycythemia vera and has undergone at least three phlebotomies in the 24 weeks prior to administering the pharmaceutical composition to the subject in order to target a hematocrit of 45% or less.

14. (a) about 5 mg to about 200 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof (b) about 10 mg to about 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof (c) about 20 mg to about 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof (d) about 20 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof (e) about 40 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof (f) About 80 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof (g) About 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof, (h) about 120 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof The pharmaceutical product according to any one of claims 1 to 8, 10, and 11 to 12, wherein the above is administered to the subject.

15. The hepcidin analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, is administered by subcutaneous injection. The pharmaceutical product according to any one of claims 1 to 14, wherein, optionally, the hepcidin analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, is administered approximately weekly over a certain period of time.

16. (a) The dose of the hepcidin analog or a pharmaceutically acceptable salt thereof is increased over a period of time, or (b) The pharmacopoeia according to any one of claims 1 to 15, further comprising: determining the hematocrit of the subject at one or more time points after administration of the hepcidin analog or a pharmaceutically acceptable salt thereof; and maintaining or adjusting the amount of the hepcidin analog or a pharmaceutically acceptable salt thereof administered to the subject, wherein the amount is increased if the determined hematocrit of the subject is greater than 45; the amount is decreased if the determined hematocrit of the subject is less than 37.5 or less than 40; and the amount is maintained if the determined hematocrit of the subject is between 37.5 and 45 or between 40 and 44.

17. The subject is a mammal, and optionally a human, and / or The pharmaceutical product according to any one of claims 1 to 16, wherein the subject is treated with cytoreductive therapy, and optionally with hydroxyurea.

18. The pharmaceutical product according to any one of claims 1 to 17, wherein the method comprises administering an effective amount of the hepcidin analog or a pharmaceutically acceptable salt thereof multiple times over a certain period of time, and optionally, the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject about once a week over the certain period of time.

19. The aforementioned hepcidin analog is 【Transformation 6】 Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH ２ (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof, 【Transformation 7】 Isovaleric acid-DTHFPCI (K(isoGlu-Palm))FEPRSKGCK-NH ２ (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof, 【Transformation 8】 Isovaleric acid-DTHFPCIKF (K(isoGlu-Palm))PRSKGCK-NH ２ (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof 【Chemistry 9】 Isovaleric acid-DTHFPCIKFEP (K(isoGlu-Palm))SKGCK-NH ２ (SEQ ID NO: 47) or its pharmaceutically acceptable salts and 【Chemistry 10】 Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH ２ (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof, Selected from the group consisting of (wherein the formula, the amino acid is an L-amino acid), Optionally, the hepcidin analog contains a disulfide bond between two Cys amino acids. The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in doses of approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, or approximately 80 mg, approximately once a week over the aforementioned period. The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject via a subcutaneous administration route. The subject is a human, Optionally, the hepcidin analog or a pharmaceutically acceptable salt thereof is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. The method further comprises determining the hematocrit of the subject after one or more administrations of the hepcidin analog or a pharmaceutically acceptable salt thereof, and maintaining or adjusting the amount of the hepcidin analog or a pharmaceutically acceptable salt subsequently administered to the subject, wherein if the determined hematocrit of the subject exceeds an acceptable range based on the subject's sex and pregnancy status, the next dose is increased; if the determined hematocrit of the subject falls below the acceptable range, the next dose is decreased; and if the determined hematocrit of the subject is within the acceptable range, the next dose is the same as the previously administered amount, as described in claim 18.

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