Conjugated hepcidin mimetic

Abstract

The present invention provides hepcidin analogs and related pharmaceutical compositions, and their use in the treatment of polycythemia vera.

Description

[Technical Field]

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

[0002] Sequence List This application includes a sequence listing, which is filed 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 specific hepcidin peptide analogs comprising both peptide monomers and peptide dimers, as well as their conjugates and derivatives, and to the use of peptide analogs in the treatment and / or prevention of polycythemia vera (PV). [Background technology]

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

[0005] Abnormal hepcidin activity is associated with iron overload disorders, including hereditary hemochromatosis (HH) and iron-loading anemia. Hereditary hemochromatosis is a hereditary iron overload disorder caused primarily by hepcidin deficiency or, in some cases, hepcidin resistance. This can lead to excessive absorption of iron from the diet and the development of iron overload. Clinical manifestations of HH may include liver disease (e.g., cirrhosis and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is regular phlebotomy, which is very burdensome for patients. Iron-loading anemia is a hereditary anemia accompanied by severe iron overload and ineffective red blood cell formation, such as β-thalassemia. Complications due to iron overload are the main cause of morbidity and mortality in these patients. Hepcidin deficiency is the main cause of iron overload in non-transfusion patients and a cause of iron overload in transfusion patients. The current treatment for iron overload in these patients is iron chelation, which is very burdensome, sometimes ineffective, and frequently accompanied by side effects. Hepcidin has many limitations that restrict its use as a drug, including difficulties in the synthesis process, partly due to protein aggregation and deposition during folding, which then leads to the high cost of the product.

[0006] U.S. Patents US9,822,157 and US10,030,061 describe novel hepcidin analogs and their uses for the treatment of iron overload disorders, including hereditary hemochromatosis and iron-loading anemia.

[0007] PCT patent publication WO15200916 describes additional novel hepcidin analogs and their uses for the treatment of iron overload diseases.

[0008] PCT patent publication WO17117411 describes additional novel hepcidin analogs with improved in vivo half-lives and their uses for the treatment of iron overload diseases.

[0009] The PCT application publication WO18048944 describes additional novel hepcidin analogs and their use for preventing iron overload and / or treating reduced serum iron levels in a subject.

[0010] The PCT application publication WO18128828 describes additional novel hepcidin analogs and their use for treating hepcidin-related disorders, including the prevention and treatment of iron overload diseases such as hemochromatosis, iron-loaded anemia such as thalassemia, and diseases associated with ineffective or enhanced erythropoiesis.

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

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

[0013] Polycythemia vera (PV) is a chronic and progressive myeloproliferative disorder characterized by symptoms due to increased bone marrow, erythrocyte, and megakaryocyte proliferation / accumulation, and is characterized as a myeloproliferative neoplasm by the World Health Organization (WHO) (Arber et al., 2016, 127(20):2391 - 405). Diagnosis is defined by two criteria: the first criterion is an increase in erythrocyte mass, a bone marrow biopsy showing trilineage cell excess, and the presence of the JAK2V617F or JAK2 exon 12 mutation, and the second criterion incorporates polycythemia, bone marrow biopsy confirmation, and subnormal serum erythropoietin levels (Arber et al., 2016, 127(20):2391 - 405).

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

[0015] Since PV is a disease characterized by increased erythropoiesis, animal models have shown that high doses of hepcidin mimics can improve 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 mini-hepcidin significantly reduces splenomegaly and normalizes hematocrit. These studies indicate that the drug-like mini-hepcidin has the potential as a future therapeutic agent for non-transfusion β-thalassemia and PV (Casu et al., Blood. 2016;128(2):265-276).

[0016] There is clearly a need for new therapeutic agents and methods for treating and preventing PV, including in high-risk patients or those in whom phlebotomy is not acceptable. The present invention addresses such a need for treating PV.

Summary of the Invention

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

[0018] In a more specific embodiment, the present invention provides a method for treating polycythemia vera in a subject requiring treatment for polycythemia vera, 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 is given by formula (I): R1-XY-R2(I)(Sequence ID 1) A peptide containing, 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 either 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. If Y does not exist, then 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)(Sequence 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 either 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 either 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 of formula I above is optionally PEGylated at R1, X, or Y, and the amino acid side chains of the peptide are optionally conjugated to a lipophilic substituent or polymer moiety. In a related embodiment, formula II is shown above, where X3 is D-Lys.

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

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

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

[0023] In certain embodiments, the peptide analog of the present invention comprises an amidated C-terminal residue.

[0024] In certain embodiments, the hepcidin analog or dimer of the present 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 with this sequence.

[0025] In certain embodiments, the hepcidin analog or dimer of the present 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 with this sequence.

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

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

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

[0029] In another embodiment, the present invention provides a method for conjugating ferroportin or for 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 further embodiments, the present invention includes a method for treating polycythemia vera in a subject requiring treatment for polycythemia vera, comprising providing the subject with an effective amount of the hepcidin analog or pharmaceutical composition of the present invention. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, oral, parenteral, rectal, vaginal, or topical administration routes. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral or subcutaneous administration routes. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject up to twice or about twice a day, up to once or about once a day, up to once or about once every two days, up to once or about once a week, or up to once or about once a month.

[0032] In certain embodiments, the hepcidin analog is provided to the subject in doses of approximately 10 mg to approximately 100 mg, approximately 10 mg to approximately 80 mg, or approximately 10 mg to approximately 50 mg. In more specific embodiments, the hepcidin analog is provided to the subject in doses of approximately 20 mg to approximately 40 mg. In certain embodiments, the hepcidin analog is provided to the subject in doses of approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, or approximately 80 mg. In certain embodiments, the hepcidin analog is provided to the subject approximately once a week. In another specific embodiment, the hepcidin analog is provided to the subject approximately twice a week, for example, subcutaneously.

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

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

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

[0036] In any particular embodiment of the method of the present disclosure, polycythemia vera is polycythemia vera requiring phlebotomy or polycythemia vera requiring phlebotomy in a low-risk patient.

[0037] In any particular embodiment of the method of this disclosure, the subjects are low-risk patients with polycythemia vera, high-risk patients with polycythemia vera, symptomatic patients with polycythemia vera requiring phlebotomy, high-risk patients with polycythemia vera requiring phlebotomy, or low-risk patients with polycythemia vera requiring phlebotomy.

[0038] In any particular embodiment of the method of the present disclosure, the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies targeting a hematocrit of 45% or higher in the 24 weeks prior to administration of the hepcidin analog or peptide to the subject.

[0039] In any particular embodiment of the method of the present disclosure, the subject is administered about 5 mg to about 200 mg of a hepcidin analog or peptide, for example, 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 any particular embodiment of the method disclosed herein, the pharmaceutical composition is administered by subcutaneous injection.

[0041] In any particular embodiment of the method disclosed herein, the pharmaceutical composition is administered approximately weekly over a period of time.

[0042] In any particular embodiment of the method of this disclosure, the amount of hepcidin analog or peptide administered is increased over a period of time.

[0043] In any particular embodiment of the method of this disclosure, the subject is a mammal, for example, a human.

[0044] In any particular embodiment of the method of the present disclosure, the method reduces the subject's hematocrit level to 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 does not undergo phlebotomy during the course of treatment, for example, during treatment about once a week over a period of time. [Brief explanation of the drawing]

[0045] [Figure 1] The following describes the data obtained from Cobas Iron 2 analysis experiments for compound A and compound B. Graph A shows the serum iron levels and serum concentration of compound A at the indicated time after treatment with compound A. Graph B shows the serum iron levels and serum concentration of compound B at the indicated time after treatment with compound B. [Figure 2] The time profiles of hematocrit and RBC index in male cynomolgus monkeys after subcutaneous administration of compound A at vehicle (○) or doses of 1 (●), 3 (□), and 10 (■) mg / kg / dose, once a week for four doses, with a 28-day recovery period, are shown. Compound A induced changes in secondary hematological indicators of iron-restricted erythrogenesis (MCHC, mean corpuscular hemoglobin concentration, and MCH, mean corpuscular hemoglobin). Each point represents the mean ± SD of up to 6 monkeys (all groups) in the primary treatment and up to 2 monkeys (vehicle and compound A at 10 mg / kg) in the recovery period. Arrows indicate administration of compound A. [Figure 3] This study demonstrates that compound A induces significant changes in hematocrit (Hct) and secondary hematological indicators. Changes in Hct, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were evaluated after subcutaneous administration of compound A at vehicle (○) or doses of 0.6 (●), 2 (□), and 6 (■) mg / kg once a week for 13 consecutive weeks, followed by a 35-day recovery period. Arrows indicate the administration day, starting from day 1. Each point represents the mean ± SD of a maximum of 6 animals / sex during the primary phase and a maximum of 2 animals / sex during the recovery phase. [Figure 4]The bilirubin profile is consistent with iron-restricted erythropoiesis in iron-rich cynomolgus monkeys. Total bilirubin levels of male (left) and female (right) cynomolgus monkeys after subcutaneous administration of compound A at vehicle (○) or doses of 0.6 (●), 2 (□), and 6 (■) mg / kg once weekly for 13 consecutive weeks, followed by a 35-day recovery period. Arrows indicate administration days, starting from day 1. Each point represents the mean ± SD of a maximum of 6 monkeys / sex during the primary period and a maximum of 2 monkeys / sex during the recovery period. [Figure 5] The platelet profiles of male (left) and female (right) cynomolgus monkeys after subcutaneous administration of compound A at vehicle (○) or doses of 0.6 (●), 2 (□), and 6 (■) mg / kg once a week for 13 consecutive weeks, followed by a 35-day recovery period, are shown. Arrows indicate the administration day, starting from day 1. Each point represents the mean ± SD of a maximum of 6 monkeys / sex during the major phase and a maximum of 2 monkeys / sex during the recovery phase. [Figure 6] A diagram of the Phase II clinical trial design is provided. [Figure 7] This is a timeline showing the timing of therapeutic phlebotomy and treatment with compound A at the indicated dosage for 13 human PV patients. [Figure 8] This graph shows ferritin (ng / mL) levels in PV patients treated with compound A at the indicated time point. The number of subjects correlates with that shown in Table 9. [Figure 9] This graph shows the TSAT (%) in PV patients treated with compound A at the indicated time point. The number of subjects correlates with that shown in Table 9. [Figure 10] This graph shows the MCV(fL) in PV patients treated with compound A at the indicated time point. The number of subjects correlates with that shown in Table 9. [Figure 11] This graph shows MCH(pg) in PV patients treated with compound A at the indicated time point. The number of subjects correlates with that shown in Table 9. [Figure 12] This graph shows the hematocrit (%) of PV patients treated with compound A at the indicated time point. The number of subjects correlates with that shown in Table 9. [Figure 13] This graph shows the red blood cell count (10⁶ / uL) in PV patients treated with compound A at the indicated time point. The number of subjects correlates with that shown in Table 9. [Figure 14] This graph shows platelet counts (103 / uL) in PV patients before and after treatment with compound A at the indicated doses 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] This graph shows the percentage of reticulocytes (R) in PV patients before and after treatment with compound A at the indicated doses at the indicated time points. [Figure 16] This graph shows the white blood cell count ( / uL) in PV patients before and after treatment with compound A at the indicated dose at the indicated time point. [Figure 17] This shows the plasma concentrations of compound A in PV patients at various time points after administration of the indicated amount of compound A. A represents data for individual time points, and B represents the average over a specified time interval. [Modes for carrying out the invention]

[0046] This disclosure generally relates to the use of hepcidin analog peptides for the treatment and prevention of polycythemia vera (PV).

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

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

[0049] Hepcidin has many limitations that restrict its use as a drug, including difficulties in its synthesis process, partly due to protein aggregation and precipitation during folding, which subsequently leads to high product costs. This disclosure provides hepcidin analog peptides that possess hepcidin activity and also have other beneficial physical properties such as improved solubility, stability, and / or potency, so that hepcidin-like biological products can be manufactured at an affordable price and used for the treatment and prevention of polycythemia vera.

[0050] The present invention also generally relates to hepcidin analog peptides and methods for producing and using the same. In certain embodiments, the hepcidin analog exhibits one or more hepcidin activities. In certain embodiments, the present invention relates to hepcidin peptide analogs comprising one or more peptide subunits that form a cyclized structure by intramolecular bonding, for example, intramolecular disulfide bonds. In certain embodiments, the cyclized structure has increased potency and selectivity compared to non-cyclized hepcidin peptides and their analogs. In certain embodiments, the hepcidin analog peptides of the present invention exhibit an increased half-life, for example, when delivered orally, compared to hepcidin or conventional hepcidin analogs.

[0051] Definitions and Terms Unless otherwise defined herein, scientific and technical terms used in this application shall have meanings generally understood by those skilled in the art. Generally, the terms and techniques used in relation to 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 their respective meanings unless otherwise specified.

[0053] Throughout this specification, variations such as “comprise,” “comprises,” or “comprising” mean to include the integer (or component) or group of integers (or components) indicated, but not to exclude any other integer (or component) or group of integers (or components).

[0054] The singular forms "a," "an," and "the" include plural nouns unless otherwise specified by the context.

[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” may be used interchangeably and refer to either human or non-human animals. These terms include mammals such as humans, primates, domestic animals (e.g., cattle, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats). The term “mammal” refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, and domestic animals.

[0057] As used herein, the term "peptide" broadly refers to a sequence of two or more amino acids joined by a peptide bond. It should be understood that this term neither implies a polymer of amino acids of a specific length nor is it intended to mean, or distinguish, whether the polypeptide is produced using recombinant techniques, chemical synthesis, or enzymatic synthesis, or whether it occurs spontaneously.

[0058] When used herein, the term “peptide analog” broadly refers to peptide monomers and peptide dimers that possess one or more structural features and / or functional activity common to hepcidin or its functional domain. In certain embodiments, peptide analogs include peptides having substantial amino acid sequence identity with hepcidin, e.g., peptides comprising one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence of wild-type hepcidin, e.g., human hepcidin. In certain embodiments, peptide analogs include one or more additional modifications, such as conjugation to another compound. Any peptide monomer or peptide dimer of the present invention is encompassed by the term “peptide analog.” In certain cases, “peptide analog” may also be referred to herein as “hepcidin analog,” “hepcidin peptide analog,” or “hepcidin analog peptide.”

[0059] The terms “sequence identity,” “identity percentage,” “homology percentage,” or, for example, “sequences that are 50% identical,” as used herein, refer to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis across a comparison window. Thus, “sequence identity percentage” may be obtained by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present, 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 sequence identity percentage.

[0060] The calculation of sequence similarity or sequence identity between sequences (terms used interchangeably herein) can be performed as follows: To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (for example, gaps can be introduced in 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. Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

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

[0062] The comparison of sequences between two sequences and the determination of the identity percentage can be achieved using mathematical algorithms. In some embodiments, the identity percentage between two amino acid sequences is determined using the Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) algorithm, which is incorporated into the GAP program in the GCG software package, using a Blossum62 matrix or a PAM250 matrix, and any of the gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the identity percentage between two nucleotide sequences is determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix, and any of the gap weights 40, 50, 60, 70, or 80 and length weights 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum62 scoring matrix with a gap penalty of 12, a gap length penalty of 4, and a frameshift gap penalty of 5. The percentage of 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), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weighted 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 and identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) by Altschul, et al. (1990, J. Mol. Biol, 215:403-10). Using the NBLAST program, a BLAST nucleotide search can be performed with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. Using the XBLAST program, a BLAST protein search can be performed with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of the present invention. To obtain gapped alignment for comparative purposes, the gapped BLAST described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997) can be used. When using BLAST and gapped BLAST programs, you can use the default parameters of each program (e.g., XBLAST and NBLAST).

[0064] As used herein, the term “conservative substitution” refers to the substitution of one or more amino acids with other biologically similar residues. Examples include substitutions of amino acid residues with similar characteristics, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. See, for example, the table below. In some embodiments of the present invention, one or more Met residues are substituted with norleucine (Nle), a bioisomer of Met that, 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 by residues not typically 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 conserved substitution is the substitution of one or more Pro residues with bhPro, Leu, or D-Npc (isopithecotinic acid). For further information on phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et al., Science 247, 1306-1310, 1990. In the following scheme, conserved amino acid substitutions are grouped by their physicochemical properties: I: neutral, hydrophilic; II: acidic and amide; III: basic; IV: hydrophobic; V: bulky aromatic amino acids. [Table 1]

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

[0066] As used herein, the terms “amino acid” or “any amino acid” refer to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and unnatural amino acids. This includes both D-amino acids and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that combine to form peptide chains and various protein components. These are primarily L-stereoisomers, although several D-amino acids occur in bacterial coats and some antibiotics. Twenty “standard” natural amino acids are listed in the table above. “Non-standard” natural amino acids are pyrrollysine (found in methanogenic organisms and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Non-natural" or "non-natural" amino acids are non-proteinogenic amino acids (i.e., amino acids that are not naturally encoded, i.e., not found in the genetic code) that exist naturally or are chemically synthesized. More than 140 natural amino acids are known, and thousands more combinations are possible. Examples of "non-natural" amino acids include β-amino acids (β 3 and β 2 This includes homoamino acids, proline and pyruvate derivatives, trisubstituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methylamino acids. Unnatural 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 one or more groups or chemical moieties that do not exist naturally on the amino acid.

[0067] As will be apparent to those skilled in the art, the peptide sequences disclosed herein are presented from left to right, with the leftmost part of the sequence being the N-terminus of the peptide and the rightmost part being the C-terminus of the peptide. Among the sequences disclosed herein are sequences incorporating a "Hy-" moiety at the amino terminus (N-terminus) and sequences incorporating either an "-OH" moiety or an "-NH2" moiety at the carboxyl terminus (C-terminus). In such cases, unless otherwise indicated, the "Hy-" moiety at the N-terminus of the sequence represents 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 represents a hydroxyl group or an amino group corresponding to the presence of an amide (CONH2) group at the C-terminus, respectively. In each sequence of the present invention, the C-terminal "-OH" moiety may be substituted for the C-terminal "-NH2" moiety, and vice versa. It is further understood that the amino-terminus or carboxy-terminus may be bonded, for example, a covalent bond, particularly in situations where the amino-terminus or carboxy-terminus is bonded to a linker or another chemical part, such as a PEG part.

[0068] As used herein, the term "NH2" refers to the free amino group at the amino terminus of a polypeptide. As used herein, the term "OH" refers to the free carboxyl group at the carboxy terminus of a peptide. Furthermore, as used herein, the term "Ac" refers to acetyl protection through acylation of the C-terminus or N-terminus of a polypeptide.

[0069] As used herein, the term "carboxy" 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 proposed 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). Where the names and abbreviations of amino acids and aminoacyl residues used herein and in the appended claims differ from these proposals, they are to be clearly stated to the reader. Several abbreviations useful for describing the present invention are defined below in Table 1. [Table 3-1] [Table 3-2] [Table 3-3]

[0071] Throughout this specification, natural amino acids are designated by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arginine, etc.) unless they are referred to by their full names (e.g., alanine, arginine, etc.). For less common or naturally occurring amino acids, unless they are referred to by their full names (e.g., sarcosine, ornithine, etc.), commonly used three-letter or four-letter codes are used for their residues, such as Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (alpha-aminoisobutyric acid), Daba (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), pGlu (p- It contains logglutamic 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 In all sequences, isovaleric acid or an equivalent can be substituted. In some embodiments, the peptides of the present invention are conjugated with an acidic compound such as isovaleric acid, isobutyric acid, or valeric acid, and the presence of such a conjugation is referred to in acid form. Therefore, in some embodiments, instead of referring to the conjugation of isovaleric acid to the peptide by referring to isovaleroyl, for example, but not limited thereto, the present application may refer to such a conjugation as isovaleric acid.

[0073] As used herein, the term "L-amino acid" refers to a peptide in its "L" isomer form, and conversely, the term "D-amino acid" refers to a peptide in its "D" isomer form. In certain embodiments, the amino acid residues described herein are in their "L" isomer form, but any residue in its "D" isomer form can be substituted with any L-amino acid residue, as long as the desired function is retained by the peptide.

[0074] Unless otherwise indicated, the L-isomer forms of the relevant natural and unnatural amino acids having a chiral center are referred to. Where appropriate, the D-isomer forms of amino acids are indicated in the conventional style by the prefix "D" preceding the conventional three-letter code (e.g., Dasp, (D)Asp or D-Asp; Dphe, (D)Phe or D-Phe).

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

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

[0077] As used herein, the term "DRP" refers to a disulfide-rich peptide.

[0078] As used herein, the term “dimer” broadly refers to a peptide comprising two or more monomeric subunits. A particular dimer contains two DRPs. The dimers of the present invention include homodimers and heterodimers. The monomeric subunits of a dimer may be linked at their C-terminus or N-terminus, or via internal amino acid residues. Each monomeric subunit of a dimer may be linked via the same site, or each may be linked via different sites (e.g., C-terminus, N-terminus, or internal site).

[0079] When used herein, in relation to specific peptide sequences disclosed herein, parentheses, e.g., (__), represent side-chain conjugations, and square brackets, e.g., [__], represent non-natural amino acid substitutions or amino acids and conjugation side chains. 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 bound to the N-terminus of both 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 bound to the C-terminus of both peptides.

[0080] The terms “isostere replacement” or “isostere substitution” are used interchangeably herein and refer to any amino acid or other analogue moiety that has similar chemical and / or structural properties to a particular amino acid. In certain embodiments, an isostere substitution is a conservation substitution with a native or non-native amino acid.

[0081] As used herein, the term "cyclization" refers to a reaction in which part of a polypeptide molecule is linked to another part of a polypeptide molecule, thereby forming a ring closure, such as by a disulfide bridge or other similar linkage.

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

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

[0084] In the context of this invention, the term “solvate” refers to a defined stoichiometric complex formed between a solute (e.g., a hepcidin analog or a pharmaceutically acceptable salt thereof according to this invention) and a solvent. The solvent in this context may be, for example, water, ethanol, or another pharmaceutically acceptable species, typically a small organic molecule, such as water, ethanol, 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 the compound of the present invention that is water-soluble or oil-soluble or dispersible, suitable for the treatment of a disease without excessive toxicity, irritation, and allergic reactions, conforms to a reasonable benefit / risk ratio, and is effective for their intended use. Salts may be prepared separately during the final isolation and purification of the compound, or by reacting the amino group with a suitable acid. Typical acid addition salts include acetate, adipine, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, and mesitylene. Examples of amino acids include rufonates, methanesulfons, naphthylenesulfons, nicotinates, 2-naphthalenesulfons, oxalates, pamoates, pectins, persulfates, 3-phenylproprionate, picrates, pivalates, propions, succinates, tartrates, trichloroacetates, trifluoroacetates, phosphates, glutamates, bicarbonates, p-toluenesulfons, and undecanoates. Furthermore, the 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 bromide and phenethyl bromide. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Pharmaceutically acceptable salts are preferably salts selected from, for example, acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts.Examples of basic salts include salts in which the cation is selected from alkali metal cations such as sodium or potassium ions, alkaline earth metal cations such as calcium or magnesium ions, and substituted ammonium ions such as N(R1)(R2)(R3)(R4)+ 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 that form non-toxic salts. Typical examples include salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc. Hemisalts of acids and bases, such as hemisulfate salts and hemicalcium salts, can also be formed.

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

[0087] The terms “symmetric methylation” or “Arg-Me-sym,” as used herein, describe the symmetric methylation of two nitrogen atoms of the guanidine group of arginine. Furthermore, the terms “asymmetric methylation” or “Arg-Me-asym” describe the methylation of a single nitrogen atom of the guanidine group of arginine.

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

[0089] The term "alkyl" includes linear or branched, acyclic or cyclic saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Typical saturated linear alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Typical saturated branched alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Typical saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Typical unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl and cyclohexenyl.

[0090] As used herein, “therapeutic effective dose” of the peptide agonist of the present invention is intended to describe a sufficient amount of the peptide agonist to treat any hepcidin-related disease, including but not limited to any of the diseases and disorders described herein (e.g., iron metabolic disorders). In particular embodiments, the therapeutic effective dose achieves a desired benefit / risk ratio applicable to any medical treatment.

[0091] Hematocrit is the ratio of the volume of red blood cells to the volume of whole blood. The normal range of hematocrit differs between sexes, being approximately 45%–52% in men and approximately 37%–48% in women. The clinical goal of PV treatment is to achieve a hematocrit of less than 45%. In this specification, hematocrit may also be referred to as hematocrit level, and a numerical value of hematocrit level, such as 45, is understood to mean a hematocrit of 45%.

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

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

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

[0095] In some embodiments, the hepcidin analogs of the present invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99% of the ferroportin-binding ability shown by reference hepcidin. In some embodiments, the hepcidin analogs of the present invention exhibit a lower IC50 for binding to ferroportin (e.g., human ferroportin) compared to reference hepcidin. 50 (i.e., higher binding affinity). In some embodiments, the hepcidin analogs of the present invention exhibit an IC50% lower IC50% than reference hepcidin in ferroportin competitive binding assays, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000%. 50 It has.

[0096] In certain embodiments, the hepcidin analogs of the present invention exhibit increased hepcidin activity compared to the hepcidin reference peptide. In some embodiments, the activity is in vitro or in vivo activity, e.g., in vivo or in vitro activity as described herein. In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity greater than 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 that of the reference hepcidin. In certain embodiments, the hepcidin analogs of the present invention exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% of the activity of reference hepcidin.

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

[0098] In some embodiments, the peptides or peptide dimers of the present invention exhibit in vivo activity of 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% to induce a reduction in free plasma iron in an individual, similar to reference hepcidin, and the activity is measured according to the methods described herein.

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

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

[0101] In some embodiments, the hepcidin analogs of the 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, the in vitro activity is measured by the dose-dependent loss of fluorescence of cells engineered to express ferroportin fused to green fluorescent protein, as described in Nemeth et al. (2006) Blood 107:328-33. An aliquot of cells is incubated for 24 hours with a gradient concentration of a reference preparation of Hep25 or mini-hepcidin. As provided herein, the EC 50 value is 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. The EC 50 of the Hep25 preparation in this assay ranges from 5-15 nM, and in certain embodiments, preferred hepcidin analogs of the invention have an EC 50 value of about 1,000 nM or less in an in vitro activity assay. In certain embodiments, the hepcidin analogs of the invention have an EC 50 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). In some embodiments, the hepcidin analog or biotherapeutic composition (e.g., any one of the pharmaceutical compositions described herein) has an EC 50 value of about 1 nM or less.

[0102] Other methods known in the art may be used to calculate the hepcidin activity and in vitro activity of the hepcidin analogs according to the present invention. For example, in certain embodiments, the in vitro activity of the hepcidin analog or reference peptide is measured by their ability to internalize cellular ferroportin, which is determined by immunohistochemistry or flow cytometry using an antibody that recognizes the extracellular epitope of ferroportin. Alternatively, in certain embodiments, the in vitro activity of the hepcidin analog or reference peptide is measured by their dose-dependent ability to inhibit iron efflux from ferroportin-expressing cells pre-filled with radioactive or stable isotopes 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, proteolytic rate) compared to reference hepcidin. In certain embodiments, the stability of the hepcidin analogs of the present invention is increased by at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 times compared to reference hepcidin, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500%. In some embodiments, the stability is the stability described herein. In some embodiments, stability is plasma stability, which is optionally measured, for example, according to the method described herein. In some embodiments, stability is stability when delivered orally.

[0104] In certain embodiments, the hepcidin analog of the present invention exhibits a longer half-life than reference hepcidin. In certain embodiments, the hepcidin analog of the present invention exhibits 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 given conditions (e.g., temperature, pH), The half-lives are at least about two weeks, at least about three weeks, at least about one month, at least about two months, at least about three months or more, or any intermediate half-lives or intermediate ranges, about five minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about twelve hours, about eighteen hours, about one day, about two days, about four days, about seven days, about ten days, about two weeks, about three weeks, about one month, about two months, about three months or more, or any intermediate half-lives or intermediate ranges. In some embodiments, the half-life of the hepcidin analog of the present invention is extended by conjugation to one or more lipophilic substituents or half-life extending portions, for example, any of the lipophilic substituents or half-life extending portions disclosed herein. In some embodiments, the half-life of the hepcidin analog of the present invention is extended by conjugation to one or more polymer moieties, for example, any of the polymer moieties or half-life-extending moieties disclosed herein. In certain embodiments, the hepcidin analog of the present invention has the above half-life under given 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, the hepcidin analog of the present invention, comprising a conjugated half-life extension portion, has an increased serum half-life after oral, intravenous, or subcutaneous administration compared to the same analog lacking the conjugated half-life extension portion. In certain embodiments, the serum half-life of the hepcidin analog of the present 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, the hepcidin analog of the present invention, comprising a conjugated half-life extension portion, results in a decrease in serum iron concentration after oral, intravenous, or subcutaneous administration to a subject. In certain embodiments, the subject's serum iron concentration decreases to less than 10%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90% of the serum iron concentration without administration of the hepcidin analog to the subject. In certain embodiments, the decreased 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 target serum iron concentration is reduced to less than 20% approximately 4 hours or 10 hours after administration to the target, for example, intravenously, or orally, or subcutaneously. In another embodiment, the target serum iron concentration is reduced to less than 50% or 60% approximately 24 to 30 hours after administration, for example, intravenously, or or subcutaneously.

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

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

[0109] In some embodiments, the present invention provides hepcidin analogs described herein, wherein the hepcidin analog exhibits improved solubility or improved aggregation properties compared to reference hepcidin. Solubility can be determined by any preferred method known in the art. In some embodiments, preferred 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 (Phos / Citrate) pH 5.0, citrate phosphate (Phos 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, for example, fluorescent dyes for measuring visible precipitation, dynamic light scattering, circular dichroism, and surface hydrophobicity to detect aggregation or fibrillation. In some embodiments, improved solubility means that the peptide (e.g., the hepcidin analog of the present invention) is more soluble in a given liquid than reference hepcidin.

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

[0111] In certain embodiments, the present invention provides hepcidin analogs that exhibit reduced aggregation, where the aggregation of peptides 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 lower than reference hepcidin in certain solutions or buffers, for example, water or buffers known in the art or disclosed herein.

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

[0113] In some embodiments, the hepcidin analogs of the present invention are produced by synthesis. In other embodiments, the hepcidin analogs of the present invention are produced by recombinant synthesis.

[0114] The various hepcidin analog monomers and dimer peptides of the present invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may include, but are not limited to, modified amino acids, non-natural or non-natural amino acids. In certain embodiments, the modified amino acids include natural amino acids that have been chemically modified to include one or more groups or chemical moieties that are not naturally present on the amino acid. The hepcidin analogs of the present invention may further include D-amino acids. Furthermore, 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 substituted with non-natural or non-natural amino acids, or D-amino acids.

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

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

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

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

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

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

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

[0122] Peptide monomer hepcidin analog In a specific embodiment, the present invention provides a method for treating polycythemia vera in a subject requiring treatment for polycythemia vera, comprising administering to the subject a pharmaceutical composition comprising a hepcidin analog or a pharmaceutically acceptable salt thereof, or an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof.

[0123] In a more specific embodiment, the present invention provides a method for treating polycythemia vera in a subject requiring treatment for polycythemia vera, 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 is given by formula (I): R1-XY-R2(I)(Sequence ID 1) A peptide containing, 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 either 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. If Y does not exist, then 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)(Sequence 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 either 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 either 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 of formula I above is optionally PEGylated at R1, X, or Y, and the amino acid side chains of the peptide are optionally conjugated to a lipophilic substituent or polymer moiety.

[0125] In the relevant embodiments, formula II is shown above for X1, X2, X4, X5, X6, X7, X8, X9, and X3, where 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 either Phe or Dpa. The X5 is either 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 either Lys or absent.

[0128] In another embodiment, X is a peptide sequence having 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 either Phe or Dpa. The X5 is either Pro or bhPro. X8 is Ile, Lys, Glu, Phe, Gln, or Arg. X10 is either Lys or absent.

[0129] In certain embodiments, the peptide follows formula VI, R 1 -XYR 2 (VI)(Sequence ID 6) or a pharmaceutically acceptable salt thereof, in the formula, R 1 These are 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 either Phe or Dpa. The X5 is either Pro or bhPro. X8 is Ile, Lys, Glu, Phe, Gln, or Arg. X10 is either Lys or absent. Y is a peptide sequence having formula VIII, Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10(VIII)(Sequence No. 8) During the ceremony, Y1 is Gly, Glu, Val, or Lys. Y3 is either Arg or Lys. Y5 is either Arg or Lys. Y6 is Gly, Ser, Lys, Ile, or Arg. Y7 is either Trp or absent. Y8 is Val, Thr, Asp, Glu, or absent. Y10 is either Lys or absent. The peptide contains a disulfide bond between two Cys molecules. The peptide in formula I above can be optionally selected as R 1 It is PEGylated in X or Y, The amino acid side chains 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 further specific embodiments, 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 (SEQ ID NO: 15) DTHFPCIKFKPRSKGWVCK (Sequence ID 16), DTHFPCIIFGPRSRGWVCK (Sequence ID 17), DTHFPCIKFGPKSKGWVCK (Sequence ID 18), DTHFPCIKFEPRSKGCK (Sequence ID 19), DTHFPCIKFEPKSKGWECK (Sequence No. 20), DTHFPCIKFEPRSKKCK (Sequence ID 21), DTHFPCIKFEPRSKGCKK (Sequence ID 22), DTHFPCIKFKPRSKGCK (Sequence ID 23), DTHFPCIKFEPKSKGCK (Sequence No. 24), DTHFPCIKF (Sequence ID 25), DTHFPCIIF (Sequence ID 26) or Includes one of the DTKFPCIIF(SEQ ID NO: 27), The above peptides are optionally PEGylated at R1, X, or Y, and the amino acid side chains of the peptides 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(PEG8))FGPRSKGWVCK-NH2 (SEQ ID NO: 28), Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH2 (SEQ ID NO: 16) Isovaleric acid-DTHFPICIFGPRS(K(PEG8))GWVC-NH2 (SEQ ID NO: 29), Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH2 (SEQ ID NO: 30), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH2 (SEQ ID NO: 31), Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG4))-NH2 (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(PEG8))-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 (Sequence ID 26), Isovaleric acid-DTKFPCIIF-NH2 (SEQ ID NO: 27) or Contains one of the Hy-DTKFPCIIF-NH2 (SEQ ID NO: 27).

[0132] In more specific embodiments, 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 more specific embodiments, the hepcidin analog or peptide is isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH2 (SEQ ID NO: 28).

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

[0136] In more specific embodiments, 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 more specific embodiments, 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 more specific embodiments, 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 more specific embodiments, 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 specific 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), and any pharmaceutically acceptable salt of the above (wherein the formula, the amino acid is an L-amino acid).

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

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

[0156] In certain embodiments, the present invention includes polypeptides comprising amino acid sequences shown herein or any amino acid sequences having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity with any of these amino acid sequences. In related embodiments, the present invention includes a dimer comprising two polypeptides, each comprising an amino acid sequence described herein or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity with any of these amino acid sequences.

[0157] In certain embodiments, monomeric subunits may be dimerized either by a disulfide bridge between two cysteine ​​residues present in each peptide monomeric subunit, or by another preferred linker moiety, including the linker moiety described herein. Some of the monomeric subunits are shown to have both a C-terminus and / or N-terminus containing free amines. Therefore, to generate peptide dimerization inhibitors, monomeric subunits may be modified to eliminate the free amine at either the C-terminus or N-terminus, thereby allowing dimerization in the remaining free amine. For example, in some cases, the termini of one or more monomeric subunits are acylated with acylated organic compounds selected from the group consisting of 2-me-trifluorobutyl, trifluoropentyl, acetyl, octonyl, butyl, pentyl, hexyl, palmityl, trifluoromethylbutyrate, cyclopentanecarboxylic acid, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, tetrahedo-2H-pyran-4carboxylic acid, succinic acid, and glutaric acid. In some cases, the monomeric subunits include both free carboxyl and free amino termini, thereby allowing the user to selectively modify the subunits to achieve dimerization at the desired termini. 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 monomeric 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 an amine-functionalized side chain, as is commonly understood in the art. With respect to the N-terminal residue, it is generally understood that dimerization may be achieved via a free amine at the terminal residue or by using a suitable amino acid side chain having a free amine, as is commonly understood in the art.

[0159] Furthermore, it is understood that the side chains of one or more internal residues contained in the hepcidin analog peptide monomer of the present invention can be used for the purpose of dimerization. In such embodiments, the side chains are, in some embodiments, preferred native amino acids (e.g., Lys) or non-native amino acids that include a side chain suitable for conjugation to a preferred linker moiety, as defined herein.

[0160] The linker portion connecting the monomer subunits may include any structure, length, and / or size compatible with the teachings herein. In at least one embodiment, the linker portion is selected from a 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 molecular weights of approximately 400 Da to approximately 40,000 Da. Non-limiting examples of suitable linker portions are provided in Table 2. In certain embodiments, one of these linker portions, or the half-life extension portion, may be linked to a hepcidin analog. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0161] Those skilled in the art will understand that the C-terminus, N-terminus, and internal linker portions disclosed herein are non-limiting examples of preferred linker portions, and that the present invention may include any preferred linker portion.

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

[0163] In any particular embodiment of the hepcidin analog peptide dimer, 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 (e.g., Lys residues) contained in each peptide monomer subunit of the hepcidin analog peptide dimer are linked by a linker moiety.

[0165] In any particular embodiment of the hepcidin analog peptide dimer, the C-terminus, N-terminus, or internal amino acids (e.g., lysine side chains) of each peptide monomer subunit are linked by a linker moiety, and at least two cysteine ​​or Pen residues of the hepcidin analog peptide dimer are linked by disulfide crosslinks. In some embodiments, the peptide dimer has the general structure shown below. Non-limiting schematic examples of such hepcidin analogs are shown in the following figures: [ka]

[0166] Peptide analog conjugates In certain embodiments, the hepcidin analogs of the present invention, including both monomeric and dimeric forms, contain one or more conjugated chemical substituents, such as lipophilic substituents and polymeric substituents, collectively referred herein as half-life extension portions. While we do not wish to be bound by any particular theory, it is thought that the lipophilic substituents bind to albumin in the bloodstream, thereby preventing the hepcidin analog from enzymatic degradation and thus increasing its half-life. Furthermore, it is hypothesized that the polymeric substituents may increase the half-life, reduce clearance in the bloodstream, and, in some cases, enhance epithelial permeability and retention in the lamina propria. Those skilled in the art will be familiar with preferred techniques for preparing the compounds used in the context of the present invention. For non-limiting examples of suitable chemistry, see, for example, WO98 / 08871, WO00 / 55184, WO00 / 55119, Madsen et al (J.Med.Chem.2007,50,6126-32), and Knudsen et al.2000 (J.Med Chem.43,1664-1669).

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

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

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

[0170] In further embodiments of the present invention, or, in addition, the side chains of one or more amino acid residues in the hepcidin analog of the present invention may be conjugated to a polymer moiety or other half-life extension moiety to increase solubility and / or half-life and / or bioavailability, for example, in vivo (e.g., in plasma). Such modifications are also known to reduce the clearance of therapeutic proteins and peptides (e.g., renal clearance).

[0171] As used herein, “polyethylene glycol” or “PEG” refers to 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 its molecular weight, and as used herein, PEO, PEE, or POG refers to an oligomer or polymer of ethylene oxide. While the three names are chemically synonymous, PEG tends to refer to oligomers and polymers with molecular weights less than 20,000 g / mol, PEO to polymers with molecular weights greater than 20,000 g / mol, and POE to polymers with any molecular weight. PEG and PEO are liquid or low-melting-point solids, depending on their molecular weight. Throughout this disclosure, the three names are used without distinction. 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. PEGs and PEOs with different molecular weights have been found to be used 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), homopolymers or copolymers of PEG, 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). Monoactivated alkoxy-terminated polyalkylene oxides (POAs), such as monomethoxy-terminated polyethylene glycol (mPEG), prepared for the purpose of extending half-life, are also being considered, as are bisactivated polyethylene oxides (glycols) or other PEG derivatives. Suitable polymers vary substantially by weight from about 200 to about 40,000 and are typically selected for the purposes of the present invention. In certain embodiments, PEGs having molecular weights of 200 to 2,000 daltons or 200 to 500 daltons are used.Different forms of PEG may 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, homogeneous, or distinct. These are used in specific embodiments of the present invention.

[0173] PEG is also available in different forms: branched PEG has 3 to 10 PEG chains emanating from a central core group, star-shaped PEG has 10 to 100 PEG chains emanating from a central core group, and comb-shaped PEG typically has multiple PEG chains grafted onto a polymer backbone. PEG can also be linear. The number often included in the name of PEG indicates its average molecular weight (for example, PEG with n=9 has an average molecular weight of approximately 400 daltons and is classified as PEG400).

[0174] As used herein, “PEGylation” is the action of conjugating a PEG structure to a hepcidin analog of the present invention (e.g., by covalent bond), and in certain embodiments, it is referred to as the “PEGylated hepcidin analog.” In certain embodiments, the PEG of the PEGylated side chain is a PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG portion of the conjugated half-life extension portion is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, 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 comprises a hepcidin analog peptide (or its dimer) conjugated with PEG, which is covalently bonded, for example, via an amide, thiol, by click chemistry, or by any other suitable means known in the art. In certain embodiments, PEG is bonded via an amide bond, and therefore, the specific PEG derivative used is appropriately functionalized. For example, in certain embodiments, PEG11, which is O-(2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol, has both an amine and a carboxylic acid for bonding to the peptide of the present invention. In certain embodiments, PEG25 comprises a diacid and 25 glycol moieties.

[0176] Other suitable polymer moieties include poly-amino acids, such as poly-lysine, poly-aspartic acid, and poly-glutamic acid (see, for example, Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57 and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol. 73, : 721-729). The polymer moieties may be linear or branched. In some embodiments, they have molecular weights of 500 to 40,000 Da, for example, 500 to 10,000 Da, 1,000 to 5,000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da.

[0177] In some embodiments, the hepcidine analog of the present invention may comprise two or more such polymeric moieties, in which case the total molecular weight of all such moieties is generally within the range provided above.

[0178] In some embodiments, the polymer moiety can be (covalently) attached to an amino group, carboxyl group, or thiol group of an amino acid side chain. Specific examples are the thiol group of a Cys residue and the epsilon amino group of a Lys residue, and the carboxyl groups of Asp and Glu residues can also be used.

[0179] One of ordinary skill in the art will be familiar with suitable techniques that can be used to perform the conjugation reaction. For example, a PEG moiety having a methoxy group can be attached to a Cys thiol group by a maleimide linkage using reagents commercially available from Nektar Therapeutics AL. See also WO2008 / 101017 and the references listed above for details of suitable chemicals. 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 in a two-step process. As used herein, for a single oxidation step, a trityl protecting group is often used during assembly, which allows for deprotection during cleavage followed by solution oxidation. If a second disulfide bond is required, there are options of native oxidation or selective oxidation. For selective oxidation, which requires orthogonal protecting groups, Acm and trityl are used as protecting groups for cysteine. Cleavage results in removal of one protecting pair of cysteine, allowing oxidation of this pair. A second oxidative deprotection step of the Acm group protected with cysteine is then performed. For native oxidation, a trityl protecting group is used on all cysteines, allowing for the native folding of the peptide.

[0181] One of ordinary skill in the art will be familiar with suitable techniques that can be used to perform the oxidation step.

[0182] In certain embodiments, the hepcidin analogs of the present invention may include, but are not limited to, a half-life extension moiety selected from 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 includes a half-life extension moiety having the structure shown below, where n = 0 to 24 or n = 14 to 24.

Chemical formula

[0184] In certain embodiments, the hepcidin analogs of the present invention include a conjugated half-life extension moiety shown in Table 3.

Table 5-1

Table 5-2

[0187] In certain embodiments, the hepcidin analogs of the invention include any of the linker portions shown in Table 4 and any of the half-life extension portions shown in Table 3, and include 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 analogs include two or more linkers. In certain embodiments, the two or more linkers are concatamerized, i.e., joined to each other. In related embodiments, the invention includes a polynucleotide encoding a polypeptide having a peptide sequence present in any of the hepcidin analogs described herein.

[0189] Furthermore, the invention includes a vector, e.g., an expression vector, comprising the polynucleotide of the invention.

[0190] Treatment Methods Polycythemia vera (PV) is a chronic, progressive tricytic clonal disorder characterized by increased bone marrow, erythrocyte, and megakaryocyte proliferation / accumulation, and is 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 criterion is increased erythrocyte mass, bone marrow biopsy showing tricytic polycythemia, and the presence of a JAK2V617F or JAK2 exon 12 mutation; the second criterion incorporates polycythemia, confirmation by bone marrow biopsy, and subnormal serum erythropoietin levels (Arber et al., Blood, 2016, 127(20):2391-405).

[0191] In normerythrocytes, erythropoiesis is regulated by JAK2-dependent erythropoietin during erythropoiesis; however, when JAK2 is constitutively activated, erythropoietin-independent erythropoiesis leads to polycythemia. Approximately 95% of PV patients have the JAK2V617F mutation (Rampal et a., Blood. 2014, 123(22):e123-33). PV can progress to myelofibrosis or undergo leukemia transformation. Polycythemia in PV men is characterized by Hgb > 16.5 g / dL or Hct > 49%, while in women it is characterized by Hgb > 16.0 g / dL or Hct > 48%. A hematocrit of over 44% in PV patients is associated with a rapid increase in the number of thromboembolic complications (Pearson et al., Lancet. 1978;2:1219-1221). Bone marrow biopsy is performed to confirm PV originating from essential thrombocythemia and should show cytophilia for age with proliferation of the three blood cell lines.

[0192] In the United States, an estimated 148,000 people are living with PV, with a median age of 61 years at diagnosis (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, itching, erythromelalgia, and paresthesia (Tefferi et al., Blood Cancer J. 2018, 8(1):3). Clinical features include splenomegaly, thrombotic and hemorrhagic complications, and an increased risk of leukemia transformation.

[0193] PV is classified into two risk categories that define subsequent treatment regimens: high-risk (age ≥ 60 years and 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 receive 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, and death was due to thrombotic complications (Tefferi et al., 2018). With current treatment, the median survival time from diagnosis is 24 years in patients under 60 years of age compared to 14 years in patients over 60 years of age. The hematocrit targets for therapeutic phlebotomy are less than 45% in men, less than 42% in women, and less than 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 iron-deficient state, thereby reducing erythrocyte production. Furthermore, iron-deficient erythrocytes are less viscous than normocytic blood due to their smaller size. Fortunately, evaluations have confirmed that, after exercise, patients with chronic iron-deficient PV do not experience the iron-deficiency-associated aerobic deficiency expected in their normocytic counterparts (Rector et al., Medicine (Baltimore). 1982 Nov; 61(6):382-9).

[0194] Cytoreductive therapy is not suitable for high-risk patients, or those who cannot tolerate phlebotomy, exhibit progressive splenomegaly, or require 1500x10 9It is used in low-risk patients with platelet counts greater than / L or progressive leukocytosis. Cytodepressants include, but are not limited to, hydroxyurea, interferon alfa, 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 improves myelosuppression and reduces the risk of thrombosis compared to phlebotomy alone. However, concerns about the long-term risks of hydroxyurea in secondary leukemia are justified. After a median follow-up of over 8 years, the polycythemia vera trial group reported that 5.4% of evaluated patients with PV developed leukemia after receiving hydroxyurea, compared to 1.5% of patients treated with phlebotomy alone (Fruchtman et al., Semin Hematol. 1997, 34:17-23). Patients who are intolerant to or resistant to hydroxyurea can be managed with pegylated IFN-α or busulfan. IFN-α is preferred in patients under 65 years of age, while busulfan is preferred in elderly patients.

[0195] Approximately one in four PV patients are considered uncontrollable due to an inadequate or intolerant response to hydroxyurea. Jakafi® (ruxolitinib) is a JAK1 / JAK2 inhibitor approved by the U.S. Food and Drug Administration for PV patients with hydroxyurea resistance or intolerance. Approval is based on a Phase 3 trial called the Efficacy and Safety Response Randomized, Open-Label, Multicenter Phase 3 Trial in Patients with Polycythemia Vera Resistant or Tolerant of Hydroxyurea: JAK Inhibitor INC4 24 Tablets vs. Best Available Treatment (BAT). The trial evaluated patients with phlebotomy-dependent PV and splenomegaly who were intolerant or tolerant of hydroxyurea and were randomized to receive either ruxolitinib (n=110) or the best available treatment (n=112). The composite primary endpoint included hematocrit control (<45%) and splenomegaly reduction (≥35%) at week 32. From week 32 onward, patients randomized for the best available therapy were able to cross over to ruxolitinib. At week 32, 77% of patients randomized for ruxolitinib met at least one component of the primary endpoint, while only 1% of patients who received BAT achieved the primary endpoint. The majority (91%) of ruxolitinib-treated patients who achieved the primary endpoint had a confirmed response at week 48, with a 94% probability of maintaining a primary response over one year. The incidence of thromboembolic events was low in the ruxolitinib group, with only one event (portal vein thrombosis) reported by week 32, compared to six events in patients who received BAT. Researchers in the response trial concluded that in PV patients who had an inadequate response to hydroxyurea or were intolerant of it, ruxolitinib was superior to BAT in controlling hematocrit without phlebotomy, normalizing blood cell counts, reducing spleen volume, and improving PV-related symptoms including itching, fatigue, and nocturnal sweating.

[0196] Patients in the United States undergo bloodletting approximately eight times a year, and the procedure causes pain, nuisance, and inconvenience (Boccia et al., Blood, 2017, 130(Suppl 1), 5271). A considerable amount of time is spent undergoing bloodletting, each procedure involving approximately half a day of work. Furthermore, many redox / metabolic cycles occur (which is where more is needed), and therefore, if the ultimate goal of bloodletting therapy in polycythemia vera is to achieve a state of chronic iron deficiency that limits erythrocyte formation, this can theoretically be achieved with hepcidin / hepcidin mimetic.

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

[0198] When JAK2 is constitutively activated, it triggers erythropoietin production independent of erythrocytes, leading to polycythemia. An approach to prevent the effects of mutant Jak2 is to induce iron restriction with hepcidin or hepcidin-mimicking peptides. Low iron levels inhibit erythropoietin signaling downstream of Jak2, thus providing an override signal. When hepcidin-mimicking peptides were administered to transgenic mice expressing the human Jak2 gene with a mutation that causes polycythemia, increased erythropoiesis and hematocrit characteristic of polycythemia reverted to the normal range. (Reference: 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). Throughout, references to hepcidin analogs are understood to 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 activity or expression of ferroportin, comprising administering to the subject an amount of the hepcidin analog or composition of the present invention sufficient to bind (partially or completely) to ferroportin in the subject and to agonize ferroportin. In another embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by dysregulation of iron metabolism, comprising administering to the subject the 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 of developing a disease or disorder characterized by dysregulation of iron levels (e.g., a disease or disorder of iron metabolism, a disease or disorder related to iron overload, and a disease or disorder related to 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, polycythemia vera is polycythemia vera requiring phlebotomy. In some embodiments, polycythemia vera is polycythemia vera requiring phlebotomy in low-risk patients. In some embodiments, the subjects are high-risk patients with polycythemia vera. In some embodiments, the subjects are low-risk patients with polycythemia vera. In some embodiments, the subjects are symptomatic patients with polycythemia vera requiring phlebotomy. In some embodiments, the subjects are low-risk patients with polycythemia vera requiring phlebotomy. In some embodiments, the subjects are high-risk patients with polycythemia vera requiring phlebotomy. In some embodiments, the subjects are diagnosed with polycythemia vera and have undergone at least three phlebotomies in the 24 weeks prior to administration of the pharmaceutical composition to the subjects, targeting a hematocrit of 45% or less. In some embodiments, the subjects are mammals, e.g., humans.

[0203] Accordingly, 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 that is effective in treating polycythemia vera. In certain embodiments, polycythemia vera is polycythemia vera requiring phlebotomy. In some embodiments, polycythemia vera is polycythemia vera requiring phlebotomy in low-risk patients. In some embodiments, polycythemia vera is polycythemia vera requiring phlebotomy in high-risk patients. In some embodiments, the subject is a low-risk patient with polycythemia vera. In some embodiments, the subject is a high-risk patient with polycythemia vera. In some embodiments, the subject is a symptomatic patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject is a low-risk patient with polycythemia vera requiring phlebotomy. In some embodiments, the subject is diagnosed with polycythemia vera and has undergone at least three phlebotomies in the 24 weeks prior to administration of the pharmaceutical composition to the subject, with a target hematocrit of 45% or less. In some embodiments, the subject is a mammal, e.g., human.

[0204] In certain embodiments, the Disclosure provides a method for treating polycythemia vera in a human subject requiring treatment for polycythemia vera, comprising administering to the subject an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof disclosed herein, a peptide, or a composition, 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, polycythemia vera is polycythemia vera requiring phlebotomy. In some embodiments, polycythemia vera is polycythemia vera requiring phlebotomy in low-risk or high-risk patients. In some embodiments, the subject is a low-risk patient with polycythemia vera. In some embodiments, the subject is a high-risk patient with polycythemia vera. In some embodiments, the subject is a symptomatic patient with polycythemia vera 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 subjects are diagnosed with polycythemia vera and have undergone at least three phlebotomies in the 24 weeks prior to administration of the pharmaceutical composition to the subjects, with a target hematocrit of 45% or less.

[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, for example, 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 for treating a human subject having polycythemia vera requiring phlebotomy, comprising subcutaneously administering an effective amount of a hepcidin analog disclosed herein, for example, 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 to the subject. In a particular embodiment, the effective amount is about 10 mg to about 100 mg, for example, 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 week, or about once a month. In certain embodiments, subjects are administered approximately once a week in doses of about 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, or 80 mg of hepcidin analog or a pharmaceutically acceptable salt thereof. In certain embodiments, when the hepcidin analog is administered to women, reduced doses, for example, about 10 mg to about 60 mg, for example, about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or 60 mg of the hepcidin analog, may be administered. In some embodiments, the hepcidin analog or a pharmaceutically acceptable salt thereof is administered approximately once every two weeks or approximately once a month. In some embodiments, a 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 1 year or about 1 year, at least 2 years or about 2 years, at least 5 years or about 5 years, or over the lifetime of the subject.

[0207] In certain embodiments of the methods disclosed herein, a 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 about weekly over a period of time, for example, for as long as necessary. In some embodiments, the hepcidin analog or peptide (or composition) is administered about every 3 days, about twice a week, about once every 4 days, about once every 5 days, about weekly, about once every 2 weeks, about once a month, about once every 6 weeks, about once every 8 weeks, about once every 2 months, or about once every 3 months. In certain embodiments, it is administered about once a week or about once every 2 weeks. In certain embodiments, it is administered about once a week. In some embodiments, this is administered approximately once every two weeks, approximately once a month, or approximately once every two months.

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

[0209] In certain embodiments, the Disclosure provides a method for treating PV in a subject, comprising providing an effective amount of hepcidin analog or a pharmaceutically acceptable salt thereof to the subject, wherein the amount of hepcidin analog or a pharmaceutically acceptable salt thereof to the subject is such that the plasma or serum concentration of the hepcidin analog or a pharmaceutically acceptable salt thereof is approximately 2 ng / mL to approximately 3500 ng / mL, approximately 5 ng / mL to approximately 3500 ng / mL, approximately 100 ng / mL to approximately 3000 ng / mL, approximately 5 ng / mL to approximately 900 ng / mL, approximately 5 ng / mL to approximately 250 ng / mL, or approximately 20 ng / mL to approximately 150 ng / mL. In certain embodiments, the plasma or serum concentrations achieved are 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 concentrations achieved are, for example, for compound A, 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. In certain embodiments, the plasma or serum concentration achieved is, for example, 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 for compound A. In certain embodiments, the plasma or serum concentration achieved is, for example, at least about 4 ng / mL or at least about 17 ng / mL for compound A. In certain embodiments, this plasma or serum level is achieved after a single dose and maintained until the next dose of the hepcidin analog, for example, 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 the administration of the hepcidin analog, for example, compound A or compound B.In certain embodiments, the plasma or serum concentrations achieved are, for example, about 20 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1500 ng / mL, 2000 ng / mL, 2500 ng / mL, or 3000 ng / mL for compound A. In certain embodiments, the plasma or serum concentrations achieved are, 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 concentrations achieved are, for example, for compound A, approximately 25 ng / mL to approximately 1000 ng / mL within 20 to 48 hours after administration, approximately 100 ng / mL to approximately 1000 ng / mL within 20 to 48 hours after administration, approximately 200 ng / mL to approximately 1000 ng / mL within 20 to 48 hours after administration, and approximately 400 ng / mL to approximately 850 ng / mL within 20 to 48 hours after administration. In certain embodiments, the plasma or serum concentrations achieved are, for example, for compound B, approximately 25 ng / mL to approximately 125 ng / mL, or approximately 50 ng / mL to approximately 125 ng / mL. In certain embodiments, the plasma or serum concentrations achieved are, for example, for compound B, at least approximately 25 ng / mL, at least approximately 50 ng / mL, or at least approximately 100 ng / mL. In certain embodiments, this plasma or serum concentration is the maximum plasma or serum concentration after administration of the hepcidin analog or a pharmaceutically acceptable salt thereof.In certain embodiments, the plasma or serum concentration is maintained for a certain period following administration of the hepcidin analog or a pharmaceutically acceptable salt thereof, for example, for 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, 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 over about 4 hours, about 8 hours, about 12 hours, about 24 hours, or about 48 hours. In certain embodiments, the plasma or serum concentrations achieved are, for example, for compound A, 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 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 concentrations achieved are, for example, for compound B, at least about 25 ng / mL to about 125 ng / mL, or about 50 ng / mL to about 125 ng / mL over at least 4 hours, about 8 hours, about 12 hours, about 24 hours, or about 48 hours. In certain embodiments, the plasma or serum concentrations achieved are, for example, for compound B, at least about 25 ng / mL, at least about 50 ng / mL, or at least about 100 ng / mL over about 4 hours, about 8 hours, about 12 hours, about 24 hours, or about 48 hours, respectively. 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 approximately 10 mg to approximately 100 mg of a hepcidin analog or a pharmaceutically acceptable salt thereof, for example, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, or approximately 100 mg, and the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject approximately twice a week, approximately once a week, approximately once every week, or approximately once a month. In certain embodiments, a subject is administered approximately 15 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, or approximately 80 mg of a hepcidin analog or a pharmaceutically acceptable salt thereof approximately once a week or approximately once every two weeks. In certain embodiments, subjects are administered approximately once a week a hepcidin analog or a pharmaceutically acceptable salt thereof in an amount of approximately 15 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, or approximately 80 mg.

[0210] In certain embodiments, the subject is treated with a hepcidin analog or a pharmaceutically acceptable salt thereof, such as SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, or a hepcidin analog having the structure of compound A or compound B, in combination with a cytoreductive therapy, such as hydroxyurea, interferon, or ruxolitinib. In certain embodiments, when the hepcidin analog is used in combination with a cytoreductive therapy, a reduced amount of the hepcidin analog may be administered, for example, about 10 mg to about 60 mg, such as 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 a step of determining the hematocrit of a subject after administration of a hepcidin analog. As shown in Figure 7, the hematocrit level 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 the concentration / dose of hepcidin analog that is optimal for maintaining a hematocrit below a target level. In some embodiments, the target level for humans is 45% or less, as measured herein, for example. In certain embodiments, the target level in males is 45% or less (e.g., about 37% to about 45%), in some cases the target threshold level for females (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 method is 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 the hepcidin analog dose and the hematocrit response (i.e., reduction of hematocrit). Therefore, the present disclosure provides a dosing regimen that can maximize the time the 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 during a period of approximately 1 to 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 in 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 above the desired target level for the subject's sex and pregnancy status, a higher dose of the hepcidin analog is administered to the subject in 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, for example, 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 in the next scheduled treatment compared to the dose administered immediately before the hematocrit was determined. In some embodiments, if the subject's hematocrit is determined to be within an acceptable range, for example, 35%–42%, 35%–45%, 37.5%–45%, 40%–45%, or 40%–44%, then in the next scheduled treatment, the subject is administered the same dose of hepcidin analog compared to the dose administered immediately before the hematocrit was determined. The acceptable range may vary depending on the subject's sex and pregnancy status. In certain embodiments, the acceptable range for males is approximately 37%–45%, for non-pregnant women it is approximately 35%–42% or 43%, and for pregnant women it is approximately 30%–36%.

[0213] In certain embodiments, the method includes determining the target hematocrit level multiple times over the course of treatment to monitor the effectiveness of the dosage, and modifying the dosage as necessary to maintain the target 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 target hematocrit level is determined approximately every two weeks, every three weeks, every four weeks, or every eight weeks over the course of treatment. In certain embodiments, it is determined approximately every four weeks over the course of treatment. In certain embodiments, the target range is the acceptable range of the target's sex and pregnancy status. In certain embodiments, any of these methods includes maintaining or adjusting the amount of hepcidin analog or a pharmaceutically acceptable salt thereof administered to a 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 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 disclosure provides a method for treating PV, comprising subcutaneously administering a hepcidin analog, e.g., compound A, in doses of about 10 mg to about 80 mg to a patient diagnosed with PV, about once a week for a period of at least 7 weeks, wherein the subject does not require or has not undergone therapeutic phlebotomy during the 7 weeks. 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 phlebotomies prior to treatment with the hepcidin analog. In certain embodiments, the subject has undergone one or more therapeutic phlebotomies within 8 weeks prior to treatment with the hepcidin analog. In certain embodiments, the subject is male, and his hematocrit level is maintained below 45% for at least 7 weeks. In certain embodiments, the subjects are non-pregnant and their hematocrit levels are maintained at less than 42% or less than 43% for at least 7 weeks. In certain embodiments, the subjects are pregnant and their hematocrit levels are maintained at less than 36% for at least 7 weeks. In certain embodiments, the hematocrit levels are 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 disclosure provides a method for treating PV, comprising subcutaneously administering a hepcidin analog, e.g., compound A, in doses of about 10 mg to about 80 mg to a patient diagnosed with PV, about once a week for a period of at least 7 weeks, wherein the subject does not require or has not required therapeutic phlebotomy during the 7 weeks. In a particular embodiment, the subject received one or more therapeutic phlebotomies prior to treatment with the hepcidin analog. In a particular embodiment, the subject received one or more therapeutic phlebotomies within 8 weeks of treatment with the hepcidin analog. In a particular embodiment, the subject's hematocrit level is measured at least once during the 7 weeks, and the dose for the following week is increased if the subject's hematocrit level is above an acceptable range, or decreased if the subject's hematocrit level is below an acceptable range. In a particular embodiment, the subject is male, and the acceptable range is about 37% to about 45%. In certain embodiments, the increased or decreased dose is also about 10 mg to about 80 mg. In certain embodiments, the dose is increased or decreased by about 5 mg, about 10 mg, about 15 mg, or about 20 mg. 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 one embodiment, the subjects are non-pregnant and the tolerable range is about 35% to about 43%. In one embodiment, the subjects are pregnant and the tolerable range is about 30% to about 36%. In certain embodiments, the subjects do not require or have bloodletting after the first administration of the hepcidin analog and 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 during treatment. In certain embodiments, the treatment is continued 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 method comprises administering an effective amount of a hepcidin analog or a pharmaceutically acceptable salt thereof multiple times over a period of time, for example, the hepcidin analog or a pharmaceutically acceptable salt thereof being administered to a subject about once or twice a week over a period of time. The effective amount may vary or remain 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, where optionally, the hepcidine analog contains a disulfide bond between two Cys amino acids. In certain embodiments, a hepcidin analog or a pharmaceutically acceptable salt thereof is administered to a subject by subcutaneous administration in doses 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 over a period of time. The effective dose may vary or remain the same between doses. In certain embodiments, the dose is adjusted based on the subject's hematocrit level over a period of time. Thus, in certain embodiments, the method further comprises determining the subject's hematocrit after one or more of the multiple doses of the hepcidin analog or a pharmaceutically acceptable salt thereof, and the dose given to the subject during subsequent doses is maintained or adjusted based on the hematocrit value. For example, if the determined hematocrit of a subject exceeds an acceptable range based on the subject's sex and pregnancy status, the next dose may be increased compared to the previously administered dose; if the determined hematocrit of a subject is below an acceptable range, the next dose may be decreased compared to the previously administered dose; or if the determined hematocrit of a subject is within an acceptable range, the next dose may be the same as the previously administered dose. In certain embodiments, the subject's hematocrit is measured approximately 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 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 hematocrit level of 45% or less of the subject. In certain embodiments, the subject's hematocrit is maintained within a range of about 37.5% to about 45% (or within an acceptable range for the subject's sex and pregnancy status) for a period of time, for example, at least one month, at least two months, at least six months, or longer. In certain embodiments, the methods or treatment regimens result in a reduction of at least 3%, at least 5%, or at least 10% of hematocrit (Hct%), and / or a reduction of at least 10%, at least 20%, at least 40%, or at least 50% of phlebotomy (e.g., in patients requiring phlebotomy). As used herein, a 3% reduction in hematocrit means an absolute reduction, for example, a reduction from 46% to 43%.

[0218] In various embodiments of the methods disclosed herein, the methods result in an increase in the serum ferritin level of the subject. 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 one month, at least two months, at least six months, or longer. In certain embodiments, the serum ferritin level of the subject is maintained within the range of about 25 ng / mL to about 150 ng / mL over a period of time, for example, at least one month, at least two months, at least six months, or longer.

[0219] In various embodiments of the methods disclosed herein, the method results in or causes a decrease of at least 60% or at least 80% in the subject's transferrin saturation (TSAT) level and / or serum iron level. In some embodiments, the subject's TSAT level decreases to less than 40%. In some embodiments of the methods disclosed herein, the method results in a slight increase in or no change in the TSAT and / or serum iron level, and in certain embodiments, the TSAT and / or serum iron level remains below normal levels.

[0220] In various embodiments of the methods disclosed herein, the methods result in an increase in the MCV and / or MCH of the subject. 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 one month, at least two months, at least six months, or longer.

[0221] In various embodiments of the methods disclosed herein, the method results in a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 80% of the subject's hematocrit and / or red blood cell count. In some embodiments, the subject's TSAT level is reduced to less than 40%. In some embodiments of the methods disclosed herein, the method results in a slight increase in or no change 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 method results in the subject not requiring or having therapeutic phlebotomy for, for example, 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 may 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 doses, three or more doses, four or more doses, or consecutive doses of a hepcidin analog over a period of time, for example, approximately once a week or approximately once every two weeks for at least one month, at least two months, at least six months, or longer.

[0225] In various embodiments of the methods disclosed herein, the methods do not result in a substantial change in the number of platelets of interest, for example, an increase or decrease of more than 50%. In certain embodiments, the number of platelets of interest 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 number of red blood cells in question, for example, an increase or decrease of more than 50%. In certain embodiments, the number of red blood cells in question 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 number of leukocytes or leukocytes of interest, for example, an increase or decrease of more than 50%. In certain embodiments, the number of leukocytes or leukocytes of interest does not increase or decrease by more than 10%, 20%, 30%, 40%, or 50%.

[0228] In any particular embodiment of the method of this disclosure, the method provides a therapeutic benefit to the subject, which may include the relief or alleviation of one or more symptoms of PV. Such symptoms include, but are not limited to, itching, hair loss, fatigue, headache, visual disturbances, nocturnal sweating, and thrombotic events.

[0229] In some embodiments, the method of the present invention includes providing a hepcidin analog (i.e., a first therapeutic agent) to a subject in need of a second therapeutic agent. In certain embodiments, the second therapeutic agent is provided to the subject before and / or simultaneously with and / or after the administration of the hepcidin analog. In certain embodiments, the second therapeutic agent is an iron chelating agent. In certain embodiments, the second therapeutic agent is selected from deferoxamine and deferasirox (Exjade®), which are iron chelating agents. In other embodiments, the method includes administering a third therapeutic agent to the subject.

[0230] The present invention provides compositions (e.g., pharmaceutical compositions) comprising one or more hepcidin analogs of the present invention and pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically acceptable carriers, diluents, or excipients refer to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation aid.

[0231] The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers. 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 may be used. Suitable pH buffers may be, for example, phosphates, citrates, acetates, tris(hydroxymethyl)aminomethane (Tris), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetates (e.g., sodium acetate), or mixtures thereof. The term further encompasses any carriers listed in the United States Pharmacopeia 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 hepcidin analog peptide monomers shown herein, (ii) any two or more hepcidin analog peptide dimers disclosed herein, (iii) any one or more hepcidin analog peptide monomers disclosed herein and any one or more hepcidin analog peptide dimers disclosed herein.

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

[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, for example, the therapeutic methods herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide monomer or a pharmaceutically acceptable salt or solvate thereof for treating various conditions, diseases, or disorders disclosed elsewhere in this specification (see, for example, the therapeutic methods herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide dimer or a pharmaceutically acceptable salt or solvate thereof for treating various conditions, diseases, or disorders disclosed herein.

[0235] The compounds described herein include isotope-labeled compounds, which are identical to the compounds described in the various formulas and structures presented herein, except that one or more atoms are substituted with atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that may be incorporated into these compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, for example, respectively. 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Examples include Cl. Specific isotope-labeled compounds described herein, for example, 3 H and 14 Compounds incorporating radioactive isotopes such as 13C are useful in tissue distribution assays of drugs and / or substrates. Furthermore, deuterium, i.e. 2 Substitution with isotopes such as 1H can offer certain therapeutic benefits resulting from higher metabolic stability, for example, an increased in vivo half-life or reduced dosage requirements.

[0236] The hepcidin analogs of the present invention are suitable for administration with or without storage and can be formulated as pharmaceutical compositions typically containing 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 compositions of the present invention are unit dosage forms. In such forms, the composition is divided into unit doses containing an appropriate amount(s) of the active ingredient. The unit dosage form may be provided as a packaged preparation, the package containing a distinct amount of the preparation, e.g., packaged tablets, capsules, or powders in a vial or ampoule. The unit dosage form may also be, for example, a capsule, cachet, or tablet itself, or any appropriate number of these packaging forms. The unit dosage form may also be provided in an injectable form for single-dose administration, e.g., in the form of a pen device containing a liquid-phase (typically aqueous) composition. The composition may be formulated for any preferred route and means of administration, e.g., any one of the routes and means of administration disclosed herein.

[0238] In certain embodiments, a hepcidin analog or a pharmaceutical composition containing a hepcidin analog is suspended in a sustained-release matrix. The sustained-release matrix used herein is a matrix made of a material (usually a polymer) that is degradable by enzymatic hydrolysis, acid-base hydrolysis, or dissolution. Once inserted into the body, enzymes and body fluids act on this matrix. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolides (polymers of glycolic acid), polylactidecoglycolides (copolymers of lactic acid and glycolic acid), polyacid anhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. The preferred biodegradable matrix is ​​one of the following: 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, intracisionally, vaginally, intraperitoneally, rectally, topically (including delivery by intravitreal, intranasal, and inhalation, such as by powders, ointments, drops, suppositories, or transdermal patches), or orally. As used herein, the term “parenteral” refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intra-articular injections and infusions. Accordingly, in certain embodiments, the composition is formulated for delivery by any of these routes of administration.

[0240] In certain embodiments, pharmaceutical compositions for parenteral injection comprise a pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, or sterile powder for reconstitution into a sterile injectable solution or dispersion immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), carboxymethylcellulose and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (olive oil), and injectable organic esters such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Long-term absorption of injectable pharmaceutical dosage forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0241] Injectable depot formulations include those prepared by forming a microcapsule matrix of hepcidine analogs in one or more biodegradable polymers such as polylactide-polyglycolide, poly(orthoester), poly(anhydrous), and (poly)glycols such as PEG. The release rate of the hepcidine analog can be controlled depending on the peptide-to-polymer ratio and the properties of the specific polymer used. Depot-injectable formulations are also prepared by capturing the hepcidine analogs in liposomes or microemulsions that are compatible with body tissues.

[0242] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed immediately before use in sterile water or other sterile injectable media.

[0243] The hepcidin analog of the present invention may also be administered in liposomes or other lipid-based carriers. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by monolayers or multilayers of hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipids capable of forming liposomes may be used. The composition of the present invention in liposomal form may include stabilizers, preservatives, excipients, etc., in addition to the hepcidin analog of the present invention. In certain embodiments, the lipids include phospholipids containing both natural and synthetic phosphatidylcholine (lecithin) and serine. Methods for forming liposomes are known in the art.

[0244] Pharmaceutical compositions used in the present invention, which are suitable for parenteral administration, may include sterile aqueous solutions and / or suspensions of peptide inhibitors that are isotonic with the recipient's blood, typically using sodium chloride, glycerin, glucose, mannitol, sorbitol, etc.

[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, although not disclosed herein, are known in the art and can be modified or integrated into systems or delivery vehicles suitable for use in the oral delivery of peptides.

[0246] In certain embodiments, formulations for oral administration may include adjuvants to artificially increase permeability of the intestinal wall (e.g., nonionic surfactants such as resorcinol and / or polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether), and / or enzyme inhibitors to inhibit enzymatic degradation (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFF), or tracylol). In certain embodiments, hepcidin analogs in solid dosage forms 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, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants, such as magnesium stearate, parabens, preservatives, such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants, such as cysteine, disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavorings, or fragrances.

[0247] In certain embodiments, an oral dosage form or unit dose suitable for use with the hepcidin analog of the present invention may comprise a mixture of the hepcidin analog with a non-pharmacological component or excipient, and other non-reusable materials that may be considered components or packaging. The oral composition may comprise at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, an oral dosage form comprising an effective amount of the hepcidin analog is provided, and the dosage form comprises at least one of pills, tablets, capsules, gels, pastes, beverages, syrups, ointments, and suppositories. In some cases, an oral dosage form is provided that is designed and configured to achieve delayed release of the hepcidin analog in the small intestine and / or colon of the subject.

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

[0249] In one embodiment, the pharmaceutical composition comprising the hepcidin analog of the present invention is provided with an enteric coating, which is designed to protect and release the pharmaceutical composition in a controlled manner within the lower digestive system of the target, thereby avoiding systemic adverse reactions. In addition to the enteric coating, the hepcidin analog of the present invention may be encapsulated, coated, engaged, or otherwise associated within any suitable oral drug delivery system or component. For example, in some embodiments, the hepcidin analog of the present invention is provided in a lipid carrier system comprising 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, thereby protecting the hepcidin analog from protein degradation in the small intestine and / or colon. The hepcidin analog of the present invention may be further formulated for use in conjunction with carrier systems designed to increase solubility and enhance intestinal absorption of peptides. These methods include the use of liposomes, micelles, and nanoparticles to increase the gastrointestinal penetration of peptides.

[0251] Various bioreactive 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 bioreactive systems such as hydrogels and mucosal adhesive polymers having hydrogen bonding groups (e.g., PEG, poly(methacrylic acid) [PMAA], cellulose, Eudragit®, chitosan, and alginates) 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, wherein the surface of the hepcidin analog is modified to include mucosal adhesive properties via hydrogen bonding, polymers having linked mucins, and / or hydrophobic interactions. These modified peptide molecules can demonstrate increased drug residence time in a subject according to the desired features of the present invention. Furthermore, targeted mucosal adhesion systems can specifically bind to receptors on the surface of intestinal cells and M cells, thereby further increasing the uptake of particles containing the hepcidin analog.

[0252] Other embodiments include methods for oral delivery of the hepcidin analog of the present invention, in which the hepcidin analog is provided to subjects in combination with a permeabiliser that facilitates the transport of the peptide across the intestinal mucosa by increasing paracellular or transcellular permeation. For example, in one embodiment, the permeabiliser is combined with the hepcidin analog, and the permeabiliser comprises at least one of long-chain fatty acids, bile salts, amphiphilic surfactants, and chelating agents. In one embodiment, a permeabiliser comprising sodium N-[hydroxybenzoyl)amino]caprylate is used to form a weak non-covalent association with the hepcidin analog of the present invention, and the permeabiliser acts favorably for membrane transport and further dissociation once it reaches the bloodstream. In another embodiment, the hepcidin analog of the present invention is conjugated to oligoarginine, thereby increasing the cellular permeability of the peptide into various cell types. Furthermore, in at least one embodiment, a non-covalent bond is provided between the peptide inhibitor of the present invention and a penetration enhancer selected from the group consisting of cyclodextrin (CD) and dendrimers, the penetration enhancer reducing peptide aggregation and increasing the stability and solubility of the hepcidin analog molecule.

[0253] Other embodiments of the present invention provide methods for treating a subject with a hepcidin analog of the present invention having an increased half-life. In one embodiment, the present invention provides a hepcidin analog having a half-life of at least several hours to one day in vitro or in vivo (e.g., when administered to a human subject) sufficient for a therapeutically effective dose administered once daily (qd) or twice daily (bid). In another embodiment, the hepcidin analog has a half-life of three days or more, sufficient for a therapeutically effective dose administered once weekly (qw). Furthermore, in yet another embodiment, the hepcidin analog has a half-life of eight days or more, sufficient for a therapeutically effective dose administered once every two weeks (biw) or once a month. In yet another embodiment, the hepcidin analog is derivatized or modified so that it has a longer half-life compared to a non-derivativeated or unmodified hepcidin analog. In yet another embodiment, the hepcidin analog includes one or more chemical modifications to increase the serum half-life.

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

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

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

[0257] In certain embodiments, the total dose is, for example, about 10 mg to about 100 mg, 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 for a human patient. In certain embodiments, the hepcidin analog is provided to the subject once a week. In another particular embodiment, the hepcidin analog is provided to the subject twice a week, for example for a human patient.

[0258] In a more specific embodiment, the total dose for a human patient is approximately 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, or 80 mg once or twice a week. In a more specific embodiment, the total dose for a human patient is approximately 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, or 80 mg every other week or once a month.

[0259] In various embodiments, the hepcidin analogs of the present invention may be administered continuously (e.g., by intravenous administration or another continuous drug administration method), or at intervals, typically at regular intervals, depending on the desired dosage and pharmaceutical composition selected by those skilled in the art for a particular subject. Regular dosing intervals include, for example, once daily, twice daily, once every two days, once every three days, once every four days, once every five or six days, once or twice per week, once or twice per month, and so on.

[0260] Such regular hepcidin analog administration regimens of the present invention may be advantageously interrupted for a period of time to reduce the level of the drug or discontinue drug administration, often referred to as taking a “drug break,” in certain situations, such as during chronic long-term administration. Drug breaks are useful, for example, to maintain or restore sensitivity to the drug, particularly during long-term chronic treatment, or to reduce undesirable side effects of long-term chronic treatment of the subject with the drug. The timing of the drug break depends on the timing of the regular administration regimen and the purpose of taking the drug break (e.g., to restore drug sensitivity and / or to reduce undesirable side effects of continuous long-term administration). In some embodiments, the drug break may be a reduction in the dose of the drug (e.g., below the therapeutically effective dose over a specific interval). In other embodiments, the administration of the drug is stopped at a certain interval before administration is restarted using the same or a different administration regimen (e.g., a lower or higher dose and / or administration frequency). Thus, drug breaks of the present invention can be selected from a wide range of periods and administration regimens. Exemplary drug-free periods include those lasting two days or more, one week or more, or one month or more, up to approximately 24 months. Therefore, for example, a regular daily dosing regimen using the peptide, peptide analogue, or dimer of the present invention may be interrupted by a drug-free period of one week, two weeks, or four weeks, after which the previous regular dosing regimen (e.g., a daily or once-weekly dosing regimen) is resumed. Various other drug-free regimens are intended to be useful for administering the hepcidin analogue of the present invention.

[0261] Therefore, hepcidin analogs can be delivered by a dosing regime that includes two or more dosing periods separated by their respective drug-free periods.

[0262] During each administration period, the hepcidin analog is administered to the recipient in a therapeutically effective dose according to a predetermined administration pattern. The administration pattern may include continuous administration of the drug to the recipient over the duration of the administration period. Alternatively, the administration pattern may include administration of multiple doses of the hepcidin analog to the recipient, with intervals set by the administration interval.

[0263] The dosing pattern may include at least two doses per administration period, at least five doses per administration period, at least ten doses per administration period, at least twenty doses per administration period, at least thirty doses per administration period, or more.

[0264] The above-mentioned dosing intervals may be regular intervals set out above, including once daily, twice daily, once every two days, once every three days, once every four days, once every five days or once every six days, once or twice a week, once or twice a month, or regular and even less frequent dosing intervals, depending on the specific dosage form, bioavailability and pharmacokinetic profile of the hepcidin analog of the present invention.

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

[0266] If the dosing pattern involves multiple doses, the duration of the following drug-free period will be longer than the dosing interval used in that dosing pattern. If the dosing intervals are irregular, the duration of the drug-free period may be longer than the average interval between doses throughout the dosing period. Alternatively, the duration of the drug-free period may be longer than the longest interval between consecutive doses during the dosing period.

[0267] The drug-free period may be at least twice the relevant dosing interval (or its average), at least three times, at least four times, at least five times, at least ten times, or at least twenty times the relevant dosing interval or its average.

[0268] Within these constraints, the drug-free 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 dosing pattern during the previous administration period.

[0269] A dosing regime consists of at least two dosing periods. Consecutive dosing periods are separated by their respective rest periods. Thus, a dosing regime may consist of at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty or more dosing periods, each separated by a rest period.

[0270] While the same dosing pattern may be used during consecutive dosing periods, this is not always desirable or necessary. However, when other drugs or activators are administered in combination with the hepcidin analog of the present invention, typically the same combination of drugs or activators is given during consecutive dosing periods. In certain embodiments, the recipient is human.

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

[0272] In some embodiments, the present invention provides a process for producing hepcidin analogs or hepcidin analog compositions (e.g., pharmaceutical compositions) as disclosed herein.

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

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

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

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

[0277] In some embodiments, the hepcidin analogs of the present invention have a measured value of less than 500 nM (e.g., EC50) in an Fpn internalization assay. Those skilled in the art will understand that the function of the hepcidin analog depends on the tertiary structure and binding surface of the presented hepcidin analog. Therefore, it is possible to make slight modifications to the sequence encoding a hepcidin analog that does not affect folding or is not on the binding surface, and yet maintains function. In other embodiments, the present invention provides a hepcidin analog having 85% or more (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity or homology to the amino acid sequence of any hepcidin analog described herein, exhibiting activity (e.g., hepcidin activity) or alleviating symptoms of a disease or indication in which hepcidin is involved.

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

[0279] In some embodiments, the hepcidin analogs of the present invention may comprise 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 herein in the examples, the hepcidin analogs of the present invention may be prepared by methods known in the art, including chemical synthesis, biosynthesis, or in vitro synthesis using recombinant DNA methods, and solid-phase synthesis. See, for example, Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol. 12, JK Setlow ed., Plenum Press, NY, pp. 1-19, incorporated herein by reference; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce, Rockford, IL. The hepcidin analogs of the present invention may be purified using protein purification techniques known in the art, such as reverse-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 may be prepared by recombinant DNA techniques known in the art. Thus, polynucleotides encoding the polypeptides of the present invention are contemplated herein. In certain preferred embodiments, the polynucleotides are isolated. As used herein, “isolated polynucleotides” refers to polynucleotides in an environment different from the environment in which they naturally occur. [Examples]

[0281] The following examples illustrate specific embodiments of the present invention. Unless otherwise described in detail, the following examples were carried out using standard techniques that are well known and routine to those skilled in the art. It should be understood that these examples are for illustrative purposes only and are not intended to limit the conditions or scope of the present invention in any way. Therefore, they should never be construed as limiting the scope of the present invention. Abbreviation: DCM: Dichlormethane 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 following a lysine residue, the compound or chemical group in parentheses should be understood as a side chain conjugated to the lysine residue. For example, K-[(PEG8)]- indicates, but is not limited to, that the PEG8 portion is conjugated to this lysine side chain.

[0283] Palm: Shows the conjugation of palmitic acid (palmitoyl).

[0284] As used herein, "C()" refers to a cysteine ​​residue involved in a particular disulfide crosslink. For example, in hepcidin, there are four disulfide crosslinks: a first between two C(1) residues, a second between two C(2) residues, a third between two C(3) residues, and a fourth between two C(4) residues. Therefore, in some embodiments, the sequence of hepcidin is as follows: It is described as Hy-DTHFPIC(1)IFC(2)C(3)GC(2)C(4)HRSKC(3)GMC(4)C(1)KT-OH, and other peptide sequences can be described in the same format at will.

[0285] Example 1 Synthesis of peptide analogs Unless otherwise specified, the reagents and solvents used below are commercially available 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 the C-terminal amide, rink amide resin was used, but wang and trityl resins were also used to generate the C-terminal acid. The side chain protecting groups were as follows: Glu, Thr, and Tyr: Ot-butyl; Trp and Lys: t-Boc (t-butyloxycarbonyl); Arg: N-gamma-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; His, Gln, Asn, Cys: trityl. For selective disulfide crosslinking, Acm (acetamidomethyl) was also used as a Cys protecting group. For bonding, a 4-10-fold excess 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 bonding efficiency in difficult areas, 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 cleaving peptides from resin Side-chain deprotection and cleavage of the peptide analogs of the present invention (e.g., compound number 2) were achieved by stirring the dry 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 to decant the ether, followed by a second diethyl ether wash. The peptide was dissolved in an aqueous solution of acetonitrile containing 0.1% TFA (trifluoroacetic acid) (1:1), and the resulting solution was filtered. The quality of the linear peptide was evaluated using electrospray ionization mass spectrometry (ESI-MS).

[0288] Peptide purification procedure The purification of the peptide of the present invention (e.g., compound number 2) was achieved using reversed-phase high-performance liquid chromatography (RP-HPLC). Analysis was performed using a C18 column (3 μm, 50 × 2 mm) at a flow rate of 1 mL / min. Purification of linear peptides was achieved using preparative RP-HPLC with a C18 column (5 μm, 250 × 21.2 mm) at a flow rate of 20 mL / min. Separation was achieved using a linear gradient of buffer B in A (buffer A: 0.05% TFA aqueous solution, buffer B: 0.043% TFA, 90% acetonitrile in water).

[0289] Procedure for peptide oxidation Method A (Single Disulfide Oxidation). Oxidation of the unprotected peptide of the present invention was achieved by dropwise adding iodine in MeOH (1 mL per 1 mg) to the peptide in a solution (ACN:H2O, 7:3, 0.5% TFA). After stirring for 2 minutes, ascorbic acid was added in small amounts until the solution became clear, and the sample was immediately loaded into 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 free cysteine ​​was achieved with a pH 7.6 NH4CO3 solution of 1 mg / 10 mL of peptide. After stirring for 24 hours and before purification, the solution was acidified to pH 3 with TFA and subsequently lyophilized. The resulting single oxidized peptide (containing 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 reactant at room temperature (final concentration: 5 mg / mL). After stirring the solution for 7 minutes, ascorbic acid was added in small amounts until the solution became clear. The solution was then directly loaded into the HPLC.

[0291] Method C (Natural Oxidation). When two or more disulfides were present and selective oxidation was not performed, natural oxidation was carried out. Natural oxidation was achieved using a 100 mM NH4CO3 (pH 7.4) solution in the presence of oxidized and reduced glutathione (peptide / GSH / GSSG, 1:100:10 molar ratio) (peptide:GSSG:GSH, 1:10, 100). After stirring for 24 hours and before RP-HPLC purification, the solution was acidified to pH 3 with TFA and subsequently freeze-dried.

[0292] Procedure for cysteine ​​oxidation to produce dimers. Oxidation of the unprotected peptide of the present invention was achieved by dropwise adding iodine in MeOH (1 mL per 1 mg) to the peptide in a solution (ACN:H2O, 7:3, 0.5% TFA). After stirring for 2 minutes, ascorbic acid was added in small amounts until the solution became clear, and the sample was immediately loaded into an HPLC for purification.

[0293] The procedure for dimerization. Glyoxylic acid (DIG), IDA, or Fmoc-β-Ala-IDA were pre-activated as N-hydroxysuccinimide esters by treating them with 1 equivalent (abbreviated as "eq") of the acid in both N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) in 2.2 equivalents of 0.1 M NMP (N-methylpyrrolidone) at a final concentration. For PEG13 and PEG25 linkers, these chemicals were purchased and pre-formed as activated succinimide esters. Approximately 0.4 equivalents of the activated ester were slowly added in small amounts to the peptide (1 mg / mL) in NMP. After stirring the solution for 10 minutes, 2-3 additional aliquots of approximately 0.05 equivalents of the linker were slowly added. After stirring the solution for a further 3 hours, the solvent was removed under vacuum, and the residue was purified by reverse-phase HPLC. Additional reverse-phase HPLC purification was performed after an additional step (2 × 10 mins) of stirring the peptide in 20% piperidine in DMF.

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

[0295] Linker activation and dimerization As described below, peptide monomer subunits were linked to form a hepcidin analog peptide dimer.

[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 equivalents, 2.2 mmol), and a stirring rod. This mixture was stirred at room temperature to completely dissolve the solid starting materials. Then, N,N'-dicyclohexylcarbodiimide (DCC, 453.9 mg, 2.2 equivalents, 2.2 mmol) was added to this mixture. A precipitate appeared within 10 minutes, and the reaction mixture was stirred further at room temperature overnight. The reaction mixture was then filtered to remove the precipitated dicyclohexylurea (DCU). The activated linker was kept in a sealed vial before use for dimerization. The nominal concentration of the activated linker was approximately 0.20 M.

[0297] The dimerization using PEG linkers did not involve a prior 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 the 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, 0.048 mmol relative to the monomer) was added to the monomer solution. The reaction mixture was stirred at room temperature for 1 hour. The completion of the dimerization reaction was monitored using analytical HPLC. The time required for the completion of the dimerization reaction varied depending on the linker. After the 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. Next, the crude dimer was purified using reversed-phase HPLC (Luna C18 support, 10u, 100A; mobile phase A: water containing 0.1% TFA; mobile phase B: acetonitrile (ACN) containing 0.1% TFA, gradient from 15% B to 45% B, over 60 minutes, flow rate 15 ml / min). The fraction containing the pure product was then freeze-dried in a freeze-dryer.

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

[0300] Example 2 Activity of peptide analogs The induction of human ferroportin protein internalization was tested in vitro using peptide analogs. After internalization, the peptide is degraded. This assay measures the decrease in receptor fluorescence.

[0301] cDNA encoding human ferroportin (SLC40A1) was cloned from a cDNA clone of Origene (NM_014585). The ferroportin-encoding DNA was amplified by PCR using primers encoding terminal restriction sites for subcloning, but without using terminal codons. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker so that the ferroportin reading frame fused with the GFP protein within the frame. The fidelity of the protein-encoding DNA was confirmed by DNA sequencing. HEK293 cells were used for transfection with the ferroportin GFP receptor expression plasmid. Cells were grown in growth medium according to a standard protocol and transfected with the plasmid using lipofectamine (manufacturer's protocol, Invitrogen). Cells stably expressing ferroportin-GFP were selected using G418 in the growth medium (in which case only cells incorporating the cDNA expression plasmid survive), and sorted several times using a Cytomation MoFlo® cell sorter to obtain GFP-positive cells (488nm / 530nm). The cells were grown 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. The compound was added in an incubator for at least 18 hours to reach the desired final concentration. After incubation, residual GFP fluorescence was determined by either the GFP fluorescence of all cells (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). The compound was added in an incubator for at least 18 hours but less than 24 hours to reach the desired final concentration.

[0303] In specific experiments, the reference compounds included native hepcidin, minihepcidin, and R1-minihepcidin, an analog of minihepcidin. The "RI" in RI-minihepcidin stands for Retro Inverse. A retroinverse peptide is a peptide that has the reverse sequence at all D amino acids. One example is Hy-Glu-Thr-His-NH2 becoming Hy-DHis-DThr-DGlu-NH2. The EC of these reference compounds for ferroportin degradation... 50 The activity assays described above determined the peptides. These peptides served as control standards. [Table 8]

[0304] The determined efficacy EC for various peptide analogues of the present invention 50 Values ​​(nM) and other activity data are provided in patents US9,822,157 and US10,030,061. These patents are incorporated herein by reference in their entirety.

[0305] Example 3 Hepcidin inhibition in polycythemia vera The hepcidin analog of the present invention was tested for activity in polycythemia vera as described in Casuetal.,Blood. 2016;128(2):265-276.

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

[0307] When JAK2 is constitutively activated, it triggers erythropoietin production independent of erythrocytes, leading to polycythemia. An approach to prevent the effects of mutant Jak2 is to induce iron restriction with hepcidin or hepcidin-mimicking peptides. Low iron levels inhibit erythropoietin signaling downstream of Jak2, thus providing an override signal. When hepcidin-mimicking peptides were administered to transgenic mice expressing the human Jak2 gene with a mutation that causes polycythemia, increased erythropoiesis and hematocrit characteristic of polycythemia reverted to the normal range. (Casu et al, Blood. 2016;128(2):265-276).

[0308] Example 4 In vivo verification of hepcidin peptide analogs The hepcidin analog of the present invention was tested for in vivo activity to determine its ability to reduce free Fe2+ in serum.

[0309] In the PK-PD experiment, hepcidin analogs (compound A (sequence number 45) or compound B (sequence number 55)) or vehicle controls were subcutaneously administered to cynomolgus monkeys (n=3 / group) at a dose of 2.44 mg / kg of compound A or 2.93 mg / kg of compound B. Serum samples were collected from the monkey groups administered with the hepcidin analogs at 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, and 144 hours after dose administration. Iron content in plasma / serum was measured using a colorimetric assay with Cobas c 111 according to the manufacturer's instructions for the assay (assay: IRON2: ACN661). Data obtained from Cobas Iron2 analysis are shown in Figure 1A for compound A and Figure 1B for compound B (as mean values ​​with standard deviation). Compound A induced a reduction in serum iron levels of approximately five times eight hours after administration. The reduction in serum iron levels from compound B was greatest approximately 12 hours after administration, reaching 12 times lower than pre-administration levels.

[0310] These tests demonstrate that the hepcidin analogs of the present invention reduce serum iron levels in cynomolgus monkeys for at least 60 hours after administration. Both compound A and compound B showed a concentration-dependent effect on serum iron reduction. There was also a delay in the effect between the serum concentration of the hepcidin analog and its corresponding effect; that is, the lowest point of the effect occurred after a delay from the peak serum concentration of the compound. For compound A, serum concentrations in the range of 200–3200 ng / mL were effective, with 1000–3200 ng / mL showing the maximum effect. For compound B, serum concentrations of 25–125 ng / mL were effective, with 50–125 ng / mL showing the maximum effect.

[0311] Example 5 Effectiveness of peptides that limit erythrocyte formation The hepcidin analog of the present invention was tested for its efficacy in restricting erythropoiesis when administered to healthy cynomolgus monkeys. In repeated-dose studies, cynomolgus monkeys received subcutaneous (SC) administration of compound A or vehicle control once a week for four doses at three different dose levels: 0.6 mg / kg / dose, 2 mg / kg / dose, or 6 mg / kg / dose (n=6 monkeys / sex / high-dose or control group and n=3 monkeys / sex / low-dose or medium-dose group).

[0312] Compound A at doses of 3 mg / kg / dose or higher induced pharmacologically mediated anemia, specifically a dose- and time-dependent decrease in hematocrit (Hct) and hemoglobin (Hgb) (Table 7 and Figure 2). On day 29, Hgb levels in males treated with 1, 3, and 10 mg / kg of compound A were reduced by 0.1, 2.7, and 7.2 g / dL, respectively, compared to the concurrent control. Absolute Hct levels decreased by 6% and 22% in the 3 mg / kg / dose and 10 mg / kg / dose male groups, respectively. Male animals showed similar pharmacological responses to females. After a prolonged reduction in red blood cells (RBCs), reticulocyte plaque developed, with a statistically significant increase observed on day 29. After discontinuation of administration, RBC parameters returned to concurrent control levels. [Table 9]

[0313] Figure 2 shows secondary hematological indicators, including hematocrit values ​​and mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin per cell (MCH), which reflect RBC size and Hgb content. RBCs generated after compound A-induced iron-restricted erythropoiesis were similar in size (MCV) to those of the concurrent control, but showed a dose-dependent decrease in Hgb concentration per cell (MCHC and MCH) compared to the concurrent control. At recovery, both the vehicle control and the 10 mg / kg compound A dose group showed similar reversibility of hematological changes. The observed hematological findings are expected after excessive and persistent iron-restricted anemia in animals that were originally iron-rich.

[0314] Example 6 Efficacy of peptides in limiting erythrocyte formation In another repeated-dose study, cynomolgus monkeys were given subcutaneously for 13 weeks either compound A or a vehicle control at three different doses, followed by a 5-week recovery period.

[0315] Cynomolgus monkeys (6 animals / sex / group) were subcutaneously administered compound A at doses of 0 (0.9% saline), 0.6, 2, or 6 mg / kg / dose once weekly (QW) for 3 months, for 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 animals / sex / group) was sacrificially killed on day 92 (main group), and the remaining animals (2 animals / sex / group) were sacrificially killed on day 120 after a 35-day recovery period (recovery). Hematological samples were obtained twice before the start of treatment (-7 and -3 days), and on days 27, 55, and 90 (corresponding to 5 days after the 4th, 8th, and 13th doses for the main and recovery animals), and on day 119 for the recovery animals.

[0316] Hematological changes were consistent with the expected pharmacology of hepcidin mimetic administered to iron-rich NHP, including dose-dependent decreases in RBC parameters (RBC count, Hgb, and Hct), an increase in reticulocytes, and changes in RBC indices (decreased MCHC and MCH) and RBC morphology (e.g., microcytosis and hypochromia) (data not shown). Consistent with compound A-induced anemia, hematopoietic hyperplegia occurred in the bone marrow (femur, sternum), and extramedullary hematopoiesis and hemosiderin deposition were observed in the liver and spleen at doses of 2 mg / kg / dose and above (correlating with increased organ weight at 6 mg / kg / dose) (data not shown).

[0317] Hematological changes consistent with the known erythropoiesis pharmacological effects of the hepcidin mimetic in inducing iron deficiency anemia occurred (Table 8). On day 90, the Hgb level was 0.6. Females treated with compound A at 2 and 6 mg / kg showed reductions of 1.3, 3.0, and 5.6 g / dL, respectively, compared to the concurrent control. Male animals showed a similar pharmacological response to females. At all administration time points, retinal cells increased in a dose-dependent manner in response to anemia. After discontinuation of administration, the RBC parameter returned to the values ​​of the concurrent control. [Table 10]

[0318] In the same study, compound A induced significant changes in secondary hematological indicators, 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 erythrocyte formation. The hematological effects were similar to those of the concurrent control 35 days after discontinuation of administration.

[0319] An increase in total bilirubin (tBili) at doses of ≥2 mg / kg / day was observed even during the treatment period associated with the destruction of red blood cells (RBCs) related to 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, a significant increase in platelet levels was observed in both male and female animals at the highest evaluation dose of 6 mg / kg / dose (Figure 5). After discontinuation of administration at the recovery point, bilirubin levels returned to normal levels, but platelet levels were in the process of returning to within the normal limit (compared to the concurrent control).

[0320] Example 7 Phase 2 trial of compound A in patients with polycythemia vera requiring bloodletting. Background: Polycythemia vera (PV) patients are typically treated with regular therapeutic phlebotomy (with or without concurrent cytopenic therapy) to maintain a hematocrit of less than 45%. As a result, PV patients with a high need for phlebotomy are likely to have a hematocrit greater than 45% between appointments. Meanwhile, the majority of PV patients are iron deficient at diagnosis and worsen after repeated phlebotomy. PV patients can be symptomatic from iron deficiency (accompanied by cognitive impairment and fatigue even without anemia), and iron supplementation typically leads to an increased rate of phlebotomy. Recent studies have shown that available therapies partially improve PV-related symptoms by reversing iron deficiency. Therefore, symptomatic PV patients requiring phlebotomy have an unmet therapeutic need. We hypothesize that hepcidin mimetic promotes iron sequestration in splenic macrophages, reducing the availability of iron for malignant erythropoiesis and thus reducing the need for phlebotomy, while reversing iron deficiency-related symptoms.

[0321] Compound A is a hepcidin mimetic used in clinical trials for multiple hematological 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 trial of Compound A as a single dose in 62 healthy subjects showed a 65% reduction in serum iron concentration and a 70% reduction in transferrin saturation from baseline without significant adverse events.

[0322] Objective: The primary objective of this three-part Phase II clinical trial (outlined in Figure 6) was to demonstrate the efficacy (reduction of phlebotomy requirements) and safety of compound A in PV patients requiring phlebotomy. A secondary objective was to determine the effects of compound A on patient-reported outcomes and iron metabolism markers.

[0323] Methods: Eligibility criteria included a PV diagnosis (according to WHO criteria 2016) and at least three phlebotomies targeting a hematocrit of ≤45% in the 6 months prior to enrollment, regardless of whether stable doses of cytoreductive therapy were administered. Eligible patients were enrolled in the 28-week dose-finding part of the Phase 2 clinical trial. As outlined in Figure 7, patients were given Compound A doses of 10, 20, 40, 60, and 80 mg subcutaneously once weekly in individualized adjustments to maintain a hematocrit of <45%. Iron status in the body was quantified by monitoring serum ferritin, serum iron, transferrin saturation (TSAT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH).

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

[0325] All subjects maintained a hematocrit of less than 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 storage (Figure 8). TSAT (Figure 9) and serum iron levels fluctuated transiently but remained below the normal range, reflecting the pharmacodynamic effect of compound A in 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 suggesting normalization of iron distribution.

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

[0327] These studies demonstrate that compound A is well-tolerated. Treatment adherence subjects showed a significant decrease in hematocrit and an absolute level of less than 45% compared to pre-enrollment levels, as well as increased ferritin levels at the end of the evaluable treatment period, suggesting improvement in iron deficiency symptoms. Platelet counts remained generally stable throughout the course of treatment (Figure 14). Reticulocyte percentages showed an increasing trend throughout the course of treatment (Figure 15), but no similar increase in mature red blood cells was observed (data not shown). White blood cell counts remained generally stable throughout the course of treatment, suggesting that the treatment did not induce an inflammatory response (Figure 16). There appeared to be no progression of PV disease, as evidenced by the absence of increases in platelets and white blood cells.

[0328] Plasma concentrations of compound A were measured at various time points after subcutaneous administration of 10 mg to 80 mg in PV patients. Concentrations increased in a dose-dependent manner and varied based on the sampling dose and time. The concentrations of compound A ranged from less than undetectable (<2 ng / mL) to 866 ng / mL (Figures 17A and 17B).

[0329] Conclusion: These results support the use of hepcidin mimetic compounds, such as Compound A, in the treatment of PV patients, including low-risk PV patients requiring high levels of therapeutic phlebotomy. Compound A and other hepcidin analogs are hypothesized to reverse iron deficiency-related symptoms while reducing the need for phlebotomy by promoting iron sequestration in splenic macrophages and decreasing the availability of iron for malignant erythrocyte formation. 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 PV patients. The elimination of the need for TP over 7 months in TP-dependent PV patients is significant and unexpected. These results indicate that Compound A is an effective agent for hematocrit control, iron deficiency reversal, and elimination of therapeutic phlebotomy in both low-risk and high-risk patients. These results also establish methods for monitoring and adjusting dosages throughout treatment, as well as administration regimens, routes of administration, and overall treatment.

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

[0331] From the above, it will be understood that while specific embodiments of the present invention are described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the invention. The present invention includes the following embodiments. [Section 1] A method for treating polycythemia vera in a subject requiring treatment for polycythemia vera, comprising administering an effective amount of a 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-XY-R2(I)(Sequence ID 1) During the ceremony, R1 is hydrogen, C1-C6 alkyl, C6-C12 aryl, C1-C20 alkanoyl, or pGlu. R2 is NH 2 or OH, X is 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. If Y does not exist, then 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)(Sequence 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 either 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 either 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 containing or consisting of formula I is optionally PEGylated at R1, X, or Y. The method wherein the side chains of the amino acids of the peptide are optionally conjugated to lipophilic substituents or polymer portions. [Section 2] The method according to 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 either Phe or Dpa. X5 is either 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. The method according to paragraph 1, wherein X10 is Lys or absent. [Section 4] 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 Ile, Lys, Glu, Phe, Gln, or Arg. The method according to item 1 or 2, wherein X10 is Lys or absent. [Section 5] The peptide follows formula VI, R1 -XYR 2 (VI)(Sequence ID 6) or a pharmaceutically acceptable salt thereof, in the formula, R 1 However, these are hydrogen, isovaleric acid, isobutyric acid, or acetyl, R 2 However, -NH 2 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 either Phe or Dpa. X5 is either Pro or bhPro. X8 is Ile, Lys, Glu, Phe, Gln, or Arg. X10 is either Lys or absent. Y is a peptide sequence having formula VIII, Y1-Pro-Y3-Ser-Y5-Y6-Y7-Y8-Cys-Y10(VIII)(Sequence No. 8) During the ceremony, Y1 is Gly, Glu, Val, or Lys. Y3 is either Arg or Lys. Y5 is either Arg or Lys. Y6 is Gly, Ser, Lys, Ile, or Arg. Y7 is either Trp or absent, Y8 is Val, Thr, Asp, Glu, or absent. Y10 is either Lys or absent. The peptide contains a disulfide bond between two Cys, The peptide of formula I can be optionally selected as R 1 PEGylated in X or Y, The amino acid side chains of the peptide are optionally conjugated to lipophilic substituents or polymer moieties. 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 (Sequence ID 9), DTHFPCIIFGPRSKGWVCK (Sequence ID 10), DTHFPCIIFEPRSKGWVCK (Sequence ID 11), DTHFPCIIFGPRSKGWACK (Sequence ID 12), DTHFPCIIFGPRSKGWVCKK (Sequence ID 13), DTHFPCIIFVCHRPKGCYRRVCR (Sequence ID 14), DTHFPCIKFGPRSKGWVCK (SEQ ID NO: 15) DTHFPCIKFKPRSKGWVCK (Sequence ID 16), DTHFPCIIFGPRSRGWVCK (Sequence ID 17), DTHFPCIKFGPKSKGWVCK (Sequence ID 18), DTHFPCIKFEPRSKGCK (Sequence ID 19), DTHFPCIKFEPKSKGWECK (Sequence No. 20), DTHFPCIKFEPRSKKCK (Sequence ID 21), DTHFPCIKFEPRSKGCKK (Sequence ID 22), DTHFPCIKFKPRSKGCK (Sequence ID 23), DTHFPCIKFEPKSKGCK (Sequence No. 24), DTHFPCIKF (Sequence ID 25), DTHFPCIIF (Sequence ID 26) or Includes one of the DTKFPCIIF(SEQ ID NO: 27), The method according to claim 1 or 2, wherein the peptide is optionally PEGylated at R1, X, or Y, and the amino acid side chains of the peptide are optionally conjugated to a lipophilic substituent or polymer moiety. [Section 7] The aforementioned hepcidin analog has the following sequence: Isovaleric acid-DTHFPICIFGPRSKGWVC-NH 2 (Sequence ID 9) Isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH 2 (Sequence ID 10) Isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH 2 (Sequence ID 11) Isovaleric acid-DTHFPCIIFGPRSKGWACK-NH 2 (Sequence ID 12) Isovaleric acid-DTHFPCIIFGPRSKGWVCKK-NH 2 (Sequence ID 13) Isovaleric acid-DTHFPCIIFVCHRPKGCYRRVCR-NH 2 (Sequence ID 14) Isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH 2 (Sequence No. 28) Isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH 2 (Sequence ID 16) Isovaleric acid-DTHFPICIFGPRS(K(PEG8))GWVC-NH 2 (Sequence ID 29) Isovaleric acid-DTHFPICIFGPRS(K(PEG4))GWVC-NH 2 (Sequence ID 30) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH 2 (Sequence ID 31) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG4))-NH 2 (Sequence ID 32) Isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG2))-NH 2 (Sequence ID 33) Isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH 2 (Sequence ID 34) Isovaleric acid-DTHFPCIKF)K(Palm))PRSKGWVCK-NH 2 (Sequence ID 35) Isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH 2 (Sequence ID 36) Isovaleric acid-DTHFPCIKFGPRS(K(Palm))GWVCK-NH 2 (Sequence ID 37) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))NH 2 (Sequence No. 38) Isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH 2 (Sequence ID 39) Isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH 2 (Sequence ID 40) Isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH 2 (Sequence ID 41) Isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH 2 (Sequence ID 42) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH 2 (Sequence No. 43) Isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH 2 (Sequence ID 44) Isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH 2 (Sequence ID 45) Isovaleric acid-DTHFPCIKF-K(isoGlu-Palm)-PRSKGCK-NH 2 (Sequence No. 46) Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH 2 (Sequence ID 47) Isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGWECK-NH 2 (Sequence ID 20) Isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH 2 (Sequence No. 48) Isovaleric acid-DTHFPCIKFEPRSK(K(isoGlu-Palm))CK-NH 2 (Sequence ID 21) Isovaleric acid-DTHFPCIKFEPRSKGCK(K(isoGlu-Palm))-NH 2 (Sequence No. 49) Isovaleric acid-DTHFPCI-K(Dapa-Palm)-FEPRSKGCK-NH 2 (Sequence ID 50) Isovaleric acid-DTHFPCIK(F(Dapa-Palm))PRSKGCK-NH 2 (Sequence ID 23) Isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH 2 (Sequence ID 24) Isovaleric acid-DTHFPCIKFEPRS(K(Dapa-Palm))GCK-NH 2 (Sequence ID 51) Isovaleric acid-DTHFPCIKFEPRSK(K(Dapa-Palm))CK-NH 2 (Sequence ID 52) Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))K-NH 2 (Sequence ID 53) Isovaleric acid-DTHFPCIKFEPRSKGC(K(Dapa-Palm))-NH 2 (Sequence ID 54) Isovaleric acid-DTHFPCIKF(K(PEG11-Palm))PRSK[Sar]CK-NH 2 (Sequence ID 55) Isovaleric acid-DTHFPCIKF-NH 2 (Sequence ID 25) Hy-DTHFPCIKF-NH 2 (Sequence ID 25) Isovaleric acid-DTHFPCIIF-NH 2 (Sequence ID 26) Hy-DTHFPCIIKF-NH 2 (Sequence ID 26) Isovaleric acid-DTKFPCIIF-NH 2 (Sequence ID 27) or Hy-DTKFPCIIF-NH 2 (Sequence ID 27) The method described in item 1 or 2, including one of the following. [Section 8] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIIFGPRSKGWVCK-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof. [Section 9] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIIFEPRSKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 11) or a pharmaceutically acceptable salt thereof. [Section 10] The aforementioned hepcidine analog is isovaleric acid-DTHFPCI(K(PEG8))FGPRSKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 28) or a pharmaceutically acceptable salt thereof. [Section 11] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKF(K(PEG8))PRSKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 16) or a pharmaceutically acceptable salt thereof. [Section 12] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIIFGPRSRGWVC(K(PEG8))-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof. [Section 13] The aforementioned hepcidine analog is isovaleric acid-DTHFPCI(K(Palm))FGPRSKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 34) or a pharmaceutically acceptable salt thereof. [Section 14] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKF(K(Palm))PRSKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof. [Section 15] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFGP(K(Palm))SKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 36) or a pharmaceutically acceptable salt thereof. [Section 16] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(Palm))-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 38) or a pharmaceutically acceptable salt thereof. [Section 17] The aforementioned hepcidine analog is isovaleric acid-DTHFPCI(K(PEG3-Palm))FGPRSKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 39) or a pharmaceutically acceptable salt thereof. [Section 18] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKF(K(PEG3-Palm))PRSKGWVCK-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 40) or a pharmaceutically acceptable salt thereof. [Section 19] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFGP(K(PEG3-Palm))SKGWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 41) or a pharmaceutically acceptable salt thereof. [Section 20] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFGPRS(K(PEG3-Palm))GWVCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 42) or a pharmaceutically acceptable salt thereof. [Section 21] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG3-Palm))-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 43) or a pharmaceutically acceptable salt thereof. [Section 22] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFGPRSKGWVC(K(PEG8))-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 44) or a pharmaceutically acceptable salt thereof. [Section 23] The aforementioned hepcidine analog is isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 45) or a pharmaceutically acceptable salt thereof. [Section 24] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKF(K(isoGlu-Palm))PRSKGCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 46) or a pharmaceutically acceptable salt thereof. [Section 25] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFEP(K(isoGlu-Palm))SKGCK-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 47) or a pharmaceutically acceptable salt thereof. [Section 26] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFEPRS(K(isoGlu-Palm))GCK-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 48) or a pharmaceutically acceptable salt thereof. [Section 27] The aforementioned hepcidine analog is isovaleric acid-DTHFPCI(K(Dapa-Palm))FEPRSKGCK-NH 2 The method according to item 1 or 2, wherein (SEQ ID NO: 50) or a pharmaceutically acceptable salt thereof. [Section 28] The aforementioned hepcidine analog is isovaleric acid-DTHFPCIKFEP(K(Dapa-Palm))SKGCK-NH 2 The method according to item 1 or 2, wherein the method is (SEQ ID NO: 24) or a pharmaceutically acceptable salt thereof. [Section 29] The aforementioned hepcidin analog is

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Claims

1. A pharmaceutical composition 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, the method comprising administering an effective amount of the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof to the subject, wherein the hepcidin analog is isovaleric acid-DTHFPCI(K(isoGlu-Palm))FEPRSKGCK-NH ２ It includes the sequence (SEQ ID NO: 45), and 【Chemistry 1】 Having a structure, The effective amount of the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof is 10 mg to 100 mg. The hematocrit of the subject is determined at one or more time points after administration of the hepcidin analog or a pharmaceutically acceptable salt or solvate thereof. A pharmaceutical composition in which, if the determined hematocrit of the subject is greater than 45%, the amount of hepcidin analog or a pharmaceutically acceptable salt or solvate administered is increased; if the determined hematocrit of the subject is less than 37.5% or less than 40%, the amount administered is decreased; and if the measured hematocrit of the subject is between 37.5% and 45% or between 40% and 45%, the amount of hepcidin analog or a pharmaceutically acceptable salt or solvate is maintained.

2. The pharmaceutical composition according to claim 1, further comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is provided to the subject by oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spraying, sublingual, oral, parenteral, rectal, vaginal, or local administration route.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is provided to the subject by an oral or subcutaneous administration route.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is provided to the subject up to twice a week or up to once a week.

6. (a) The hepcidin analog is provided to the subject in doses of 10 mg to 100 mg, 10 mg to 70 mg, 10 mg to 60 mg, 20 mg to 50 mg, 20 mg to 40 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, or 10 mg. (b) The hepcidin analog is provided to the subject in doses of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, or 40 mg. (c) The hepcidin analog is provided to the subject once a week in a dose of 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg. (d) The hepcidin analog is provided to the subject twice a week in doses of 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg, (e) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 10 mg, (f) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 15 mg, (g) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 20 mg, (h) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 25 mg, (i) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 30 mg, (j) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 40 mg, (k) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 50 mg, (l) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 60 mg, (m) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 70 mg, or (n) The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject in a dose of 80 mg. A pharmaceutical composition according to any one of claims 1 to 5.

7. The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject once a week in doses of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, or 80 mg. The hepcidin analog or a pharmaceutically acceptable salt thereof is provided to the subject via a subcutaneous administration route, and The pharmaceutical composition according to claim 6, wherein the subject is a human.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the polycythemia vera is polycythemia vera requiring phlebotomy.

9. (a) Whether the polycythemia vera is polycythemia vera requiring phlebotomy in a low-risk patient, (b) Whether the subject is a low-risk patient with polycythemia vera or a high-risk patient with polycythemia vera, (c) The pharmaceutical composition according to any one of claims 1 to 8, wherein the subject is a low-risk patient having polycythemia vera requiring bloodletting, or a high-risk patient having polycythemia vera requiring bloodletting.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject has been diagnosed with polycythemia vera and has undergone at least three phlebotomies in the 24 weeks prior to administration of the pharmaceutical composition to the subject in order to target a hematocrit of 45% or less.

11. (a) The subject is administered 10 mg to 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof, (b) The subject is administered 20 mg to 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof, (c) The subject is administered 20 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof, (d) The subject is administered 40 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof, (e) The subject is administered 80 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof, or (f) The subject is administered 100 mg of the hepcidin analog or a pharmaceutically acceptable salt thereof. A pharmaceutical composition according to any one of claims 1 to 7, 9, or 10.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition is administered by subcutaneous injection.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is administered weekly over a certain period of time.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the dose of the hepcidin analog or a pharmaceutically acceptable salt thereof is increased over a certain period of time.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the subject is a human and / or the subject is treated by cytoreductive therapy.

16. The pharmaceutical composition according to any one of claims 1 to 15, 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, wherein the hepcidin analog or a pharmaceutically acceptable salt thereof is administered to the subject once a week over the certain period of time.

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