Conjugate hepcidin mimetic

Abstract

The present invention provides hepcidin analogs with improved in vivo half-lives, as well as related pharmaceutical compositions and methods of use. The present invention generally relates to hepcidin analog peptides and methods of making and using them. In certain embodiments, the hepcidin analogs exhibit one or more hepcidin activities. In certain embodiments, the present invention relates to hepcidin peptide analogs comprising one or more peptide subunits, which form a cyclized structure via an intramolecular bond, such as an intramolecular disulfide bond. In certain embodiments, the cyclized structure exhibits increased potency and selectivity compared to non-cyclized hepcidin peptides and analogs thereof. In certain embodiments, the hepcidin analog peptides of the present invention exhibit an extended half-life when delivered orally compared to hepcidin or conventional hepcidin analogs.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 057,582, filed July 28, 2020; U.S. Provisional Patent Application No. 63 / 057,577, filed July 28, 2020; U.S. Provisional Patent Application No. 63 / 169,527, filed April 1, 2021; U.S. Provisional Patent Application No. 63 / 169,533, filed April 1, 2021; U.S. Provisional Patent Application No. 63 / 169,515, filed April 1, 2021; U.S. Provisional Patent Application No. 63 / 057,583, filed July 28, 2020; and U.S. Provisional Patent Application No. 63 / 057,574, filed July 28, 2020, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on July 28, 2021, is named PRTH_054_02WO_ST25.txt and is 154KB in size.

[0003] The present invention relates particularly to specific hepcidin peptide analogs comprising both peptide monomers and peptide dimers, as well as their conjugates and derivatives, and compositions comprising such peptide analogs, and to the use of such peptide analogs in the treatment and / or prevention of various diseases, conditions or disorders, including, for example, polycythemia vera, iron overload diseases, such as hereditary hemochromatosis, iron overload anemia, and other conditions and disorders described herein. [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 transport channel ferroportin, and undergoing internal translocation and degradation. Human hepcidin is a 25-amino acid peptide (Hep25). See Krause et al. (2000) FEBS Lett 480:147-150 and Park et al. (2001) J Biol Chem 276:7806-7810. The structure of the bioactive 25-amino acid form of hepcidin is a simple hairpin structure, as described in Jordan et al. J Biol Chem 284:24155-67, with eight cysteine ​​groups forming four disulfide bonds. The N-terminal region is required for iron regulation, and the deletion of five N-terminal amino acid residues results in loss of iron regulation 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-overload anemia. Hereditary hemochromatosis is a genetic iron-overload disorder caused primarily by hepcidin deficiency, or in some cases, hepcidin resistance. As a result, iron is absorbed excessively from the diet, leading to iron overload. Clinical signs of HH include liver disease (e.g., cirrhosis, NASH, and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is regular phlebotomy, which is very burdensome for patients. Iron-overload anemia is a hereditary anemia accompanied by poor red blood cell production, such as β-thalassemia. Complications due to iron overload are the main cause of morbidity and death in these patients. Hepcidin deficiency is the main cause of iron overload in non-transfusion patients and also contributes to iron overload in transfusion patients. The current treatment for iron overload in these patients is iron chelation, but this is very burdensome. Furthermore, it is ineffective in some cases and frequently accompanied by side effects.

[0006] Hepcidin has several limitations that restrict its use as a drug, including the difficulty of its synthesis process, which is partly due to aggregation and precipitation during protein folding. Aggregation and precipitation during folding also lead to low bioavailability, injection site reactions, immunogenicity, and high commercial costs. What is needed in this field is a compound that possesses hepcidin activity and also has other beneficial physical properties, such as improved solubility, stability, and / or potency. Such hepcidin-like compounds can be manufactured inexpensively and can be used to treat hepcidin-related diseases and disorders, such as those described herein. The present invention addresses such needs and provides novel peptide analogs, including both peptide monomer analogs and peptide dimer analogs, that possess hepcidin activity and other beneficial properties, which make the peptides of the present invention suitable hepcidin substitutes. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Krause et al.(2000)FEBS Lett 480:147-150 [Non-Patent Document 2] Park et al. (2001) J Biol Chem 276:7806-7810 [Non-Patent Document 3] Jordan et al. J Biol Chem 284:24155-67 [Non-Patent Document 4] Nemeth et al.(2006)Blood 107:328-33 [Overview of the project] [Means for solving the problem]

[0008] The present invention generally relates to hepcidin-active peptide analogs, including both monomeric and dimeric forms, and methods for using the same.

[0009] In one embodiment, the present invention relates to formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ia) The hepcidin analog containing the peptide, or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 It is a cycloalkanoyl, R 2 is NH2, substituted amino, OH, or substituted hydroxy, X1 is either nonexistent, or is Asp, isoAsp, Asp(OMe), Glu, GluOMe, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, Lys, substituted Lys, (D)Lys or substituted (D)Lys. X2 is Ala, Thr, Gly, N-substitution Gly, or Ser. X3 is Ala, Gly, N-substituted Gly, His, or substituted His. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal. X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. X6 is either absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either nonexistent, or is Ala, Gly, N-substitution Gly, Ile, Val, Leu, NLeu, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, or 123 triazole. X9 is either nonexistent or is Ala, Ile, Gly, N-substitution Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X10 is either nonexistent or is Ala, Gly, N-substitution Gly, Ile, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X11 is either nonexistent or is Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. and Each of X12 to X14 is either nonexistent or an arbitrary amino acid independently. However, the following conditions apply: i) The peptide may be further conjugated with any amino acid. ii) Any of the amino acids in the peptide may be the corresponding (D)-amino acid, or may be N-substituted, and iii) The peptide is either a linear peptide or a cyclized lactam, and In the formula, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, and 2-Nal is 2-naphthyl Alanine, Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and Phe is substituted for phenyl. F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, ben Phenylalanine is substituted with zyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, and substituted bhPhe is β-homophenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, and substituted Trp is Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid. 123-triazole is [Chemical formula] and Dab is [Chemical formula] is.

[0010] In one aspect, the present invention provides a hepcidin analog comprising a peptide of formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ib) or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 is hydrogen, C1-C6 alkyl, C6-C 12 aryl, C6-C 12 aryl-C1-C6 alkyl, C1-C 20 alkanoyl, or C1-C 20 cycloalkanoyl, R 2 is NH2, substituted amino, OH, or substituted hydroxy, X1 is absent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, Gln, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, X2 is Ala, Thr, Gly, N-substituted Gly, or Ser, X3 is Ala, Gly, N-substituted Gly, His, or substituted His, X4 is Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal, X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), or 2-pyrrolidinebutanoic acid (Pba), X6 is either absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either nonexistent, or is Ala, Gly, N-substitution Gly, Ile, Val, Leu, NLeu, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X8 is either nonexistent or is Ala, (D)Ala, Ile, Gly, N-substitution Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, substitution (D)Lys, or aMeLys. X9 is either nonexistent or is Ala, Ile, Gly, N-substitution Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X10 is either nonexistent or is Ala, Gly, N-substitution Gly, Ile, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X11 is either nonexistent or is Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. and Each of X12 to X14 is either nonexistent or an arbitrary amino acid independently. However, the following conditions apply: i) The peptide does not contain disulfide bonds or thioether bonds; ii) The peptide may be further conjugated with any amino acid; iii) Any of the amino acids in the peptide may be the corresponding (D)-amino acid or N-substituted, where Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, and 2-Nal is 2-naphthylalanine. Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, Substituted Phe is phenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, Substituted bhPhe is phenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,β-homophenylalanine is substituted with 6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH or t-Bu, substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH or t-Bu, Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and, Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid.

[0011] In one embodiment, X1 is Glu, X2 is Thr, X4 is Dpa, or X5 is Pro.

[0012] In another embodiment, the present invention relates to formula (II): R 1 -Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (II) The formula comprises a hepcidin analog containing the peptide, or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2 X3, X6-X14 are as shown in equation (I).

[0013] In another embodiment, the present invention relates to formula (IXa): R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); The formula comprises a hepcidin analog containing the peptide, or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2, and X11-X14 are as shown in equation (I).

[0014] In another embodiment, the present invention relates to formula (XXI): R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (XXI) The formula includes a hepcidin analog containing the peptide, where R 1 , R 2 , and X10~X14 are as described in equation (I), X6 is nonexistent, Ala, or substitute Lys; X7 is nonexistent, Ile, substitute Lys, or substitute (D)Lys; X9 is nonexistent or bhPhe. And X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is the linker and Z is the half-life extension portion.

[0015] In one embodiment, R 1 It is IVA or isovaleric acid.

[0016] In one embodiment, R 2 is NH2. In one embodiment, R 2 It is OH.

[0017] In any particular embodiment of the hepcidin analog of the present invention, the substituted Lys or substituted (D)Lys is a Lys or (D)Lys that is substituted directly or via a linker with an acid selected from C12 (lauric acid), C14 (mysteric acid), C16 (palmitic acid), C18 (stearic acid), C20, C12 diacitate, C14 diacitate, C16 diacitate, C18 diacitate, C20 diacitate, biotin, and isovaleric acid, or a residue thereof. In one embodiment, the linker is Ahx, PEG, or PEG-Ahx.

[0018] In any particular embodiment of the hepcidin analog of the present invention, X8 or X10 is (D)Lys substituted with Lys or L1Z, where L1 is absent, Dapa, D-Dapa or isoGlu, PEG, Ahx, isoGlu-PEG, PEG-isoGlu, PEG-Ahx, isoGlu-Ahx, or isoGlu-PEG-Ahx, where Ahx is an aminohexanoic acid moiety and PEG is -[C(O)-CH2-(Peg) n -N(H)] m -or-[C(O)-CH2-CH2-(Peg) n -N(H)] m - and Peg is -OCH2CH2-, m is 1, 2 or 3, n is an integer between 1 and 100K, and Z is the half-life extension portion. In one embodiment, the half-life extension portion is C 10 -C 21 It is Alkanoyl.

[0019] In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal X1 residue. In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal Asp residue. In certain embodiments, the peptide analog or dimer of the present invention includes an isovaleric acid moiety conjugated to the N-terminal Glu residue.

[0020] In certain embodiments, the peptide analog of the present invention comprises an amidated C-terminal residue.

[0021] In related embodiments, the present invention includes polynucleotides encoding a hepcidin analog or dimer (or monomeric subunit of the dimer) peptide of the present invention.

[0022] In further relevant embodiments, the present invention includes a vector comprising a polynucleotide of the present invention. In certain embodiments, the vector is an expression vector comprising a promoter operably ligated to the polynucleotide, for example, in a manner that promotes the expression of the polynucleotide.

[0023] In another embodiment, the present invention includes a pharmaceutical composition comprising the hepcidin analog, dimer, polynucleotide, or vector of the present invention, and a pharmaceutically acceptable carrier, excipient, or vehicle.

[0024] In another embodiment, the present invention provides a method for binding to ferroportin or for inducing the internal migration and degradation of ferroportin, the method comprising contacting ferroportin with at least one hepcidin analog, dimer, or composition of the present invention.

[0025] In further embodiments, the present invention includes a method for treating an iron metabolism disorder in a subject where such need is provided, the method comprising providing an effective amount of the hepcidin analog or pharmaceutical composition of the present invention to the subject. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject by oral, intravenous, intraperitoneal, 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 iron metabolism disorder is iron overload. In certain embodiments, the hepcidin analog or pharmaceutical composition is provided to the subject at a maximum or approximately twice a day, at a maximum or approximately once a day, at a maximum or approximately once every two days, at a maximum or approximately once a week, or at a maximum or approximately once a month. In certain embodiments, the hepcidin analog is provided to the subject in doses of approximately 1 mg to approximately 100 mg, or approximately 1 mg to approximately 5 mg.

[0026] In another embodiment, the present invention provides a device comprising the hepcidin analog or pharmaceutical composition of the present invention for optionally delivering the hepcidin analog or dimer of the present invention orally or subcutaneously to a target.

[0027] In yet another embodiment, the present invention includes a kit comprising a hepcidin analog or pharmaceutical composition of the present invention, packaged together with a reagent, device, or reference material, or a combination thereof. [Modes for carrying out the invention]

[0028] The present invention generally relates to hepcidin analog peptides, as well as methods for preparing and using the same. In certain embodiments, the hepcidin analog exhibits one or more hepcidin activities. In certain embodiments, the present invention relates to a hepcidin peptide analog comprising one or more peptide subunits, the peptide subunits forming a cyclized structure via intramolecular bonding, such as intramolecular disulfide bonds. In certain embodiments, the cyclized structure exhibits 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 extended half-life compared to hepcidin or conventional hepcidin analogs when delivered orally. In embodiments of the present invention, for example, the following items are provided. (Item 1) Equation Ia: R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ia) A hepcidin analog containing a peptide according to, or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 is hydrogen, C 1 -C 6 Alkyl, C 6 -C 12 Ariel, C 6 -C 12 Aryl-C 1 -C6 Alkyl, C 1 -C 20 Alkanoyl, or C 1 -C 20 It is a cycloalkanoyl, R 2 NH 2 , substituted amino, OH, or substituted hydroxy, X1 is either nonexistent or is Asp, isoAsp, Asp(OMe), Glu, Glu-OMe, bhGlu, bGlu, substituted Glu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, Lys, substituted Lys, (D)Lys or substituted (D)Lys. X2 is Ala, Thr, Gly, N-substitution Gly, or Ser. X3 is Ala, Gly, N-substituted Gly, His, or substituted His. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal. X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. X6 is either absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either nonexistent, or is Ala, Gly, N-substitution Gly, Ile, Val, Leu, NLeu, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, or 123 triazole. X9 is either nonexistent or is Ala, Ile, Gly, N-substitution Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X10 is either nonexistent or is Ala, Gly, N-substitution Gly, Ile, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X11 is either nonexistent or is Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. and Each of X12 to X14 is either nonexistent or an arbitrary amino acid independently. However, the following conditions apply: i) The peptide may be further conjugated with any amino acid, ii) Any of the amino acids of the peptide may be the corresponding (D)-amino acid of the amino acid, or may be N-substituted, and iii) The peptide is a linear peptide or a cyclized lactam, and In the formula, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, and Orn is ornithine. Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting bhPhe is β-homophenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN, or guanidine. Substituting Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH or t-Bu. Substituting bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH or t-Bu. Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid, 123-triazole is

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[0029] Definitions and Terminology Unless otherwise defined herein, scientific and technical terms used herein shall have the same meaning as those commonly understood by those skilled in the art. In general, the terminology and techniques used herein in relation to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein chemistry and nucleic acid chemistry are publicly known and commonly used in the art.

[0030] As used herein, unless otherwise specified, the following terms have the meanings associated with them.

[0031] Throughout this specification, the term “comprise,” or variations such as “comprises” or “comprising,” shall be understood to mean that it includes a specified integer (or component) or set of integers (or components), but does not exclude any other integer (or component) or set of integers (or components).

[0032] The singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise.

[0033] The term "including" is used to mean "including but not limited to the following." "Including" and "including but not limited to the following" are interchangeable.

[0034] 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.

[0035] As used herein, the term "peptide" broadly refers to a sequence of two or more amino acids linked together by a peptide bond. It should be understood that this term does not imply an amino acid polymer of a specific length, much less suggest or identify whether a polypeptide was created using recombinant techniques, chemical synthesis, or enzymatic synthesis, or whether it occurred spontaneously.

[0036] As used herein, the terms “peptide analog” or “hepcidin analog” broadly refer to peptide monomers and peptide dimers having one or more structural features and / or functional activity common to hepcidin or its functional domain. In certain embodiments, a peptide analog includes peptides that share substantial amino acid sequence identity with hepcidin, including, for example, peptides that include one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence of wild-type hepcidin, such as human hepcidin. In certain embodiments, a peptide analog includes one or more additional modifications, such as, for example, conjugation into another compound. The term “peptide analog” encompasses any peptide monomer or peptide dimer of the present invention. In certain examples herein, “peptide analog” may also be referred to as “hepcidin analog,” “hepcidin peptide analog,” or “hepcidin analog peptide.”

[0037] When used herein, “sequence identity,” “identity percentage,” “homology percentage,” or, for example, “a sequence that is 50% identical to ~,” refers to the degree to which sequences are identical on a comparison window, either on a nucleotide basis or on an amino acid basis. Thus, “sequence identity percentage” can be calculated by comparing two optimally aligned sequences on a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in both sequences to calculate 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.

[0038] The calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) may be carried out as follows: To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences may be aligned for the purpose of optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for the purpose of optimal alignment, and non-homologous sequences may be ignored for the purpose of comparison). In certain embodiments, the length of the reference sequence to be aligned for the purpose of comparison is at least 30%, for example, 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, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. The molecules are identical at the position when the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence.

[0039] The percentage of identity between two sequences is a function of the number of identical positions shared by those sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences, and the length of each gap.

[0040] The comparison of sequences and the determination of the percentage of identity between two sequences may be performed using mathematical algorithms. In some embodiments, the percentage of identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453), incorporated into the GAP program of the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program of the GCG software package, using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. Another exemplary parameter setting is the Blossum 62 scoring matrix, which uses a gap penalty of 12, a gap elongation penalty of 4, and a frameshift gap penalty of 5. The identity percentage between two amino acid or nucleotide sequences may be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4:11-17), incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0041] The peptide sequences described herein may be used as "query sequences" to perform searches against public databases to identify, for example, other family species or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215:403-10). A BLAST nucleotide search may be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. A BLAST protein search may be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of the present invention. To obtain gapped alignment for comparative purposes, Gapped BLAST may be used as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and Gapped BLAST programs, you may use the default parameters for each program (e.g., XBLAST and NBLAST).

[0042] 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 having similar properties, 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). Nle is a biological equivalent of Met, but in contrast to Met, it 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. On the other hand, 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 using residues not normally present in endogenous mammalian peptides or proteins is, for example, a 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 details on phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et al., Science 247, 1306-1310, 1990. In the following scheme, conserved amino acid substitutions are grouped by their physicochemical properties: I: neutral, hydrophilic; II: acidic and amide; III: basic; IV: hydrophobic; V: aromatic, giant amino acids. [Table 1]

[0043] In the following scheme, conserved amino acid substitutions are grouped according to their physicochemical properties: VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. [Table 2]

[0044] As used herein, the terms “amino acid” or “any amino acid” refer to all amino acids, including natural amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. This includes both D-amino acids and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that are integrated into peptide chains to form the building blocks of countless proteins. These are primarily L-stereoisomers, although some D-amino acids exist in bacterial envelopes and some antibiotics. Twenty “standard” natural amino acids are listed in the table above. “Non-standard” natural amino acids are pyrrolicin (found in methanogenic organisms and other eukaryotes), selenocysteine ​​(found in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non-proteinogenic amino acids (i.e., not naturally encoded or present in the genetic code) that do not exist in nature or are chemically synthesized. Over 140 naturally occurring amino acids are known, and thousands of combinations are possible. An example of a "non-natural" amino acid is the β-amino acid (β 3 and β 2 Examples include homo-amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methylamino acids. Non-natural or non-natural amino acids also include modified amino acids. "Modified" amino acids include amino acids that have been chemically modified to contain groups, multiple groups, or chemical moieties that do not exist on an amino acid in nature (e.g., natural amino acids).

[0045] As will be apparent to those skilled in the art, the peptide sequences disclosed herein are presented in progression from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end being the C-terminus of the peptide. Some of the sequences disclosed herein incorporate a "Hy-" moiety at the amino terminus (N-terminus) and either an "-OH" moiety or an "-NH2" moiety at the carboxyl terminus (C-terminus). In such cases, and unless otherwise indicated, the "Hy-" moiety at the N-terminus of the sequence in question represents a hydrogen atom, which corresponds 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, which corresponds to the presence of an amide (CONH2) group at the C-terminus. In each of the sequences of the present invention, the C-terminus "-OH" moiety may be substituted for the C-terminus "-NH2" moiety, and vice versa. It is further understood that the amino-terminus or carboxy-terminus can be bonded in a covalent or other manner, especially when the amino-terminus or carboxy-terminus is bonded to another chemical part, such as a linker or PEG moiety.

[0046] 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 via acylation of the C-terminus or N-terminus of a polypeptide.

[0047] As used herein, the term "carboxy" refers to -CO2H.

[0048] In most cases, the names of natural and non-natural aminoacyl residues used herein follow the nomenclature rules proposed by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature, as described in “Nomenclature of α-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). Wherever the names and abbreviations of amino acids and aminoacyl residues used herein and in the appended claims differ from those proposed, they are made clear to the reader. Some abbreviations useful for describing the present invention are defined below in Tables 1A and 1B. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 4]

[0049] Throughout this specification, unless natural amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they will be designated by conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). For less common or non-natural amino acids, unless they are referred to by their full names (e.g., sarcosine, ornithine, etc.), the three-letter or four-letter codes commonly used for those residues will be used. Examples include Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Daba (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), pGlu (pyroglutamic acid), Gaba (γ-aminobutanoic acid), β-Pro (pyrrolidine-3-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).

[0050] Furthermore, R 1 In all sequences, isovaleric acid or its equivalent can be substituted. In some embodiments, if the peptide of the present invention is conjugated with an acidic compound such as isovaleric acid, isobutyric acid, or valeric acid, the presence of such a conjugate will be referred to as the acidic form. Therefore, in some embodiments, the present application may refer to such a conjugate as isovaleric acid, instead of indicating the conjugation of isovaleric acid to the peptide by referring to isovaleroyl, for example, but not limited to.

[0051] It should be understood that for each of the hepcidine analog formulas provided herein, bonds may be indicated by "-" or implied based on the formula and its components. For example, "B7(L1Z)" is understood to include a bond between B7 and L1 if L1 is present, and a bond between B7 and Z if L1 is absent. Similarly, "B5(L1Z)" is understood to include a bond between B5 and L1 if L1 is present, and a bond between B5 and Z if L1 is absent. Furthermore, if both L1 and Z are present, it is understood that there is also a bond between L1 and Z. Thus, the definitions of certain substituents, such as B7, L1, and J, may include "-" before and / or after the defined substituent. However, it should be understood that in each case, the substituent is bonded to the other substituent via a single bond. For example, if "J" is defined as Lys, D-Lys, Arg, Pro, -Pro-Arg-, etc., it is understood that J is bonded to Xaa2 and Y1 via a single bond. Therefore, while the definition of a substituent may or may not include a "-", in either case, it should be understood that it is bonded to an adjacent substituent.

[0052] As used herein, the term "L-amino acid" refers to the "L" isomer of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomer of a peptide. In certain embodiments, the amino acid residues described herein are in the L isomer, but residues in the "D" isomer can be substituted with any L amino acid residue, provided that the desired functionality is retained in the peptide.

[0053] Unless otherwise indicated, references are made to the L-isomers of the native and unnatural amino acids in question that possess a chiral center. Where appropriate, the D-isomers of amino acids are indicated in the conventional style by prefixing them with "D" before their conventional three-letter code (e.g., Dasp, (D)Asp or D-Asp; Dphe, (D)Phe or D-Phe).

[0054] 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.

[0055] As used herein, "higher homolog 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.

[0056] As used herein, the term "DRP" refers to a disulfide-rich peptide.

[0057] 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).

[0058] The terms “isostere replacement” or “isostere substitution” are used herein to refer to any amino acid or other analog moiety that is mutually interchangeable and has chemical and / or structural properties similar to a given amino acid. In certain embodiments, isostere replacement is a conservation substitution with a native or non-native amino acid.

[0059] As used herein, the term “cyclization” refers to a reaction in which one portion of a polypeptide molecule is linked to another portion of a polypeptide molecule to form a disulfide crosslink or other similar bond, thereby forming a closed ring.

[0060] As used herein, the term “subunit” refers to one of a pair of polypeptide monomers that are linked together to form a dimeric peptide composition.

[0061] 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.

[0062] In the context of the present invention, the term “solvate” refers to a complex formed stoichiometrically between a solute (e.g., a hepcidin analog or a pharmaceutically acceptable salt thereof according to the present invention) and a solvent. In this context, the solvent may be, for example, water, ethanol, or another pharmaceutically acceptable, typically low-molecular-weight organic substance, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is usually referred to as a hydrate.

[0063] As used herein, “iron metabolism disorders” include diseases in which abnormal iron metabolism is the direct cause of the disease, or diseases resulting from dysregulation of blood iron levels, or diseases in which iron dysregulation is a consequence of another disease, or diseases that can be treated by regulating iron levels. More specifically, iron metabolism disorders as defined herein include iron overload, iron deficiency, impaired biodistribution of iron, other disorders of iron metabolism, and other disorders that may be related to iron metabolism. Iron metabolism disorders include hemochromatosis, HFE variant hemochromatosis, ferroportin variant hemochromatosis, transferrin receptor 2 variant hemochromatosis, hemomodoverin variant hemochromatosis, hepcidin variant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, fluid-induced iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sideroblastic anemia, porphyria, late-onset cutaneous porphyria, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital erythrodysplasia anemia, hypochromic microcytic anemia, sickle cell anemia, and polycythemia vera (primary). These include idiopathic and secondary polycythemia, secondary polycythemia, such as chronic obstructive pulmonary disease (COPD), post-renal transplantation, Chuvash, HIF and PHD mutations, as well as idiopathic myelodysplasia, pyruvate kinase deficiency, iron deficiency in obesity, other anemias, benign or malignant tumors that overproduce or induce overproduction of hepcidin, hepcidin overdose, ataxia of Friedreich, Gracil syndrome, Haller-Vorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer, hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes mellitus, atherosclerosis, neurodegenerative disorders, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0064] In some embodiments, the diseases and disorders are related to iron overload disorders, such as iron hemochromatosis, HFE variant hemochromatosis, ferroportin variant hemochromatosis, transferrin receptor 2 variant hemochromatosis, hemomodoverin variant hemochromatosis, hepcidin variant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, fluid-induced iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sickle cell disease, myelodysplasia, sideroblastic infections, diabetic retinopathy, and pyruvate kinase deficiency.

[0065] In some embodiments, the hepcidin analogs of the present invention are used to treat diseases and disorders not typically identified as iron-related. For example, since hepcidin is highly expressed in the pancreas of mice, it is suggested that diabetes mellitus (type I or II), insulin resistance, impaired glucose tolerance, and other disorders may be ameliorated by treating underlying iron metabolic disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26. This document is incorporated herein by reference. Thus, the peptides of the present invention may be used to treat these diseases and conditions. Those skilled in the art can easily determine, using methods known in the art, whether a given disease can be treated with the peptides of the present invention, such as the assay of WO 2004092405. This patent application is incorporated herein by reference, and the assay monitors the levels and expression of hepcidin, hemoduverin, or iron, such as the assay described in U.S. Patent No. 7,534,764, which is known in the art. The said patent is incorporated herein by reference.

[0066] In certain embodiments of the present invention, iron metabolism disorders are iron overload disorders, which include hereditary hemochromatosis, iron-overload anemia, alcoholic liver disease, and chronic hepatitis C.

[0067] As used herein, the term “pharmaceutically acceptable salt” refers to a salt or amphoteric form of the peptide or compound of the present invention that is soluble in water or oil, or dispersible, suitable for the treatment of a disease without excessive toxicity, irritation, and allergic reactions, commensurate with a reasonable benefit / risk ratio, and effective for its intended use. Salts can be prepared during the final isolation and purification of the compound, or separately by reacting the amino group with a suitable acid. Typical acid addition salts include acetate, adipine, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, and mesitylenesulfonate. Examples include methanesulfonates, naphthylenesulfonates, nicotinates, 2-naphthalenesulfonates, oxalates, pamoates, pectinates, persulfates, 3-phenylproprionate, picrates, pivalates, propions, succinates, tartrates, trichloroacetates, trifluoroacetates, phosphates, glutamates, bicarbonates, para-toluenesulfonates, and undecanoates. Furthermore, the amino groups in the compounds of the present invention may be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. A pharmaceutically acceptable salt may preferably be a salt selected from among 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, in which case R1, R2, R3, and R4 independently represent typically hydrogen, optionally substituted C1-6-alkyl, or optionally substituted C2-6-alkenyl. Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl, and 2-propyl groups. Examples of potentially relevant C2-6-alkenyl groups include ethenyl, 1-propenyl, and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its latest edition), “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66:2 (1977). For an overview of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Other suitable basic 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. Hemi salts of acids and bases may also be formed, such as hemisulfates and hemicalcium salts.

[0068] As used herein, the term "N(alpha)methylation" describes the methylation of an alpha-amine of an amino acid, and is also commonly referred to as N-methylation.

[0069] As used herein, the terms "symmethylation" or "Arg-Me-sym" describe the symmetric methylation of two nitrogen atoms of the guanidine group of arginine. Furthermore, the terms "asymmethylation" or "Arg-Me-asym" describe the methylation of one nitrogen atom of the guanidine group of arginine.

[0070] As used herein, the term “acylated organic compound” refers to a variety of compounds having a carboxylic acid functional group used to acylate the N-terminus of an amino acid subunit before forming a C-terminal dimer. Non-limiting examples of acylated organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3,3,3-trifluoropropionic acid, 3-fluoromethylbutyric acid, and tetrahedro-2H-pyran-4-carboxylic acid.

[0071] The term "alkyl" includes linear or branched, acyclic or cyclic saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Representative saturated linear alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Representative saturated branched alkyls include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl and cyclohexenyl.

[0072] As used herein, the “therapeutic effective dose” of the peptide agonist of the present invention is intended to describe a sufficient amount of the peptide agonist to treat hepcidin-related diseases, including, but not limited to, any of the diseases and disorders described herein (e.g., diseases of iron metabolism). In particular embodiments, the therapeutic effective dose achieves a desirable benefit / risk ratio applicable to any medical treatment.

[0073] Hepcidin peptide analog The present invention provides a peptide analog of hepcidin, which may be a monomer or a dimer (collectively, "hepcidin analog").

[0074] In some embodiments, the hepcidin analog of the present invention binds to ferroportin, for example, human ferroportin. In certain embodiments, the hepcidin analog of the present invention binds specifically to human ferroportin. As used herein, “specifically binds” means that a specific binding agent preferentially interacts with a given ligand over other substances in a sample. For example, a specific binding agent that specifically binds to a given ligand binds to the given ligand in an amount or degree observed to the extent that it exceeds any nonspecific interactions with other components in the sample under suitable conditions. Suitable conditions are those that enable interaction between a given specific binding agent and a given ligand. These conditions include pH, temperature, concentration, solvent, incubation time, etc., and may vary depending on the pair of specific binding agent and ligand, but are readily determined by those skilled in the art. In some embodiments, the hepcidin analog of the present invention binds 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 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 of the hepcidin reference compounds provided herein).

[0075] 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, such as the in vitro or in vivo activity described herein. In some embodiments, the hepcidin analogs of the present invention exhibit at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99% of the activity exhibited by a hepcidin reference compound (for example, any one of the hepcidin reference compounds provided herein).

[0076] In some embodiments, the hepcidin analog of the present invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99% of the ferroportin binding capacity exhibited by the hepcidin reference compound. In some embodiments, the hepcidin analog of the present invention exhibits a lower EC50 or IC50 with respect to binding to ferroportin (e.g., human ferroportin) compared to the hepcidin reference compound. 50 (i.e., high binding affinity). In some embodiments, the hepcidin analog of the present invention exhibits an EC50 or IC50 that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than that of the hepcidin reference compound in a ferroportin competitive binding assay. 50 It has.

[0077] In certain embodiments, the hepcidin analogs of the present invention exhibit increased hepcidin activity compared to a hepcidin reference compound. In some embodiments, the activity is in vitro or in vivo activity, such as the in vitro or in vivo activity described herein. In certain embodiments, the hepcidin analogs of the present invention exhibit hepcidin activity that is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times higher than that of a hepcidin reference compound. In certain embodiments, the hepcidin analogs of the present invention exhibit activity at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more than 99%, more than 100%, more than 200%, more than 300%, more than 400%, more than 500%, more than 700%, or more than 1000% higher than the hepcidin reference compound.

[0078] In some embodiments, the peptide analogs of the present invention exhibit in vitro activity with respect to the induction of degradation of human ferroportin protein that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 700%, or greater than 1000% higher than the activity of the hepcidin reference compound, in which case such activity is measured according to the method described herein.

[0079] In some embodiments, the peptides or peptide dimers of the present invention exhibit in vivo activity with respect to inducing a decrease in free plasma iron that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 700%, or greater than 1000% higher than the activity of the hepcidin reference compound, in which case such activity is measured according to the method described herein.

[0080] In some embodiments, the activity is in vitro or in vivo activity, for example, the in vitro or in vivo activity described herein. In certain embodiments, the hepcidin analog of the present invention is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 times higher than the hepcidin reference compound, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% The activity is 700% or 1000% higher, in which case the activity is either an in vitro activity that induces ferroportin degradation as measured, for example, according to the examples herein, or an in vivo activity that reduces free plasma iron as measured, for example, according to the examples herein.

[0081] In some embodiments, the hepcidin analogs of the present invention mimic the hepcidin activity of Hep25, a bioactive human 25-amino acid type, and are referred to herein as “mini-hepcidin.” 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 a subject (e.g., parenteral injection or oral administration). See, for example, Rivera et al. (2005), Blood 106:2196-9. In some embodiments, the peptides of the present invention reduce the plasma iron concentration in a subject by at least about 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

[0082] In some embodiments, the hepcidin analogs of the present invention possess in vitro activity and are analyzed by their ability to induce internal translocation and degradation of ferroportin in ferroportin-expressing cell lines, as taught in Nemeth et al. (2006) Blood 107:328-33. In some embodiments, in vitro activity is measured by dose-dependent fluorescence loss in cells engineered to display ferroportin fused to green fluorescent protein, as presented by Nemeth et al. (2006) Blood 107:328-33. Cell aliquots are incubated for 24 hours with reference preparations or mini-hepcidin of stepwise concentrations of Hep25. As provided herein, EC 50 The value is provided as the concentration of a given compound (e.g., the hepcidin analog peptide or peptide dimer of the present invention) at which 50% of the maximum fluorescence produced by the reference compound disappears. The EC of the Hep25 preparation in the assay. 50 The EC is in the range of 5 to 15 nM, and in certain embodiments, the preferred hepcidin analog of the present invention has an EC of about 1,000 nM or less in in vitro activity analysis. 50 In certain embodiments, the hepcidin analog of the present invention has an EC of less than approximately one of the following values ​​in in vitro activity analysis (as described in Nemeth et al. (2006) Blood 107:328-33 or the examples herein): 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 50 It has. In some embodiments, the hepcidin analog or biopharmaceutical composition (for example, any one of the pharmaceutical compositions described herein) has an EC of about 1 nM or less. 50 Value or IC 50 It has a value.

[0083] Other methods known in the art may be used to calculate the hepcidin activity and in vitro activity of the hepcidin analog according to the present invention. For example, in certain embodiments, the in vitro activity of the hepcidin analog or reference peptide is measured by its ability to internalize ferroportin into cells, which is determined by immunohistochemistry or flow cytometry using an antibody that recognizes the extracellular epitope of ferroportin. Alternatively, in certain implementations, the in vitro activity of the hepcidin analog or reference peptide is determined by its ability to dose-dependently inhibit iron efflux from ferroportin-expressing cells pre-loaded with a radioisotope or stable isotope, as described in Nemeth et al. (2006) Blood 107:328-33.

[0084] In some embodiments, the hepcidin analogs of the present invention exhibit increased stability (e.g., measured by half-life, proteolytic rate) compared to the hepcidin reference compound. In certain embodiments, the stability of the hepcidin analogs of the present invention is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times higher than the hepcidin reference compound, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% higher. In some embodiments, the stability is the stability described herein. In some embodiments, stability is plasma stability, which is optionally measured according to a method described herein, for example. In some embodiments, stability is stability when delivered orally.

[0085] In certain embodiments, the hepcidin analog of the present invention exhibits a longer half-life than the hepcidin reference compound. 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 1 The half-life is 0 days, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months or more, or any half-life or range in between, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 4 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months or more, or any half-life or range in between. 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 extension moieties disclosed herein. In certain embodiments, the hepcidin analog of the present invention has the above-mentioned 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 pH about 7.4.

[0086] In certain embodiments, the hepcidin analog of the present invention, which includes a conjugated half-life extension portion, has an increased serum half-life after oral, intravenous, or subcutaneous administration compared to the analog without 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, the serum half-life is 12–168 hours, 24–168 hours, 36–168 hours, or 48–168 hours.

[0087] In certain embodiments, the hepcidin analog of the present invention, for example, a hepcidin analog comprising a conjugated half-life extension portion, lowers the serum iron concentration after oral, intravenous, or subcutaneous administration to a subject. In certain embodiments, the subject's serum iron concentration is reduced 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 that the subject would have had without administration of the hepcidin analog. In certain embodiments, the reduction in serum iron concentration is maintained for at least 1 hour, at least 4 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours after administration to the subject. In certain embodiments, the reduction in serum iron concentration is maintained for 12–168 hours, 24–168 hours, 36–168 hours, or 48–168 hours. In one embodiment, the serum iron concentration of the subject decreases to less than 20% approximately 4 hours or 10 hours after administration, for example, intravenous, oral, or subcutaneous. In another embodiment, the serum iron concentration of the subject decreases to less than 50% or 60% approximately 24 to 30 hours after administration, for example, intravenous, oral, or subcutaneous.

[0088] In some embodiments, the half-life is measured in vitro using any suitable method known in the art. For example, in some embodiments, the stability of the hepcidin analog of the present invention is determined by incubating the hepcidin analog with human serum (Sigma) pre-warmed at 37°C. Samples are typically taken at various time points up to 24 hours, and the stability of the samples is analyzed by separating the hepcidin analog from the serum proteins and then analyzing the presence of the hepcidin analog of interest using LC-MS.

[0089] In some embodiments, the stability of the hepcidin analog is measured in vivo using any suitable method known in the art. For example, in some embodiments, the stability of the hepcidin analog is determined in vivo by administering a peptide or peptide dimer to a subject such as a human or any mammal (e.g., a mouse), and then taking samples from the subject by blood collection at various time points, typically up to 24 hours. The samples are then analyzed as described above with respect to in vitro methods for measuring the half-life. In some embodiments, the in vivo stability of the hepcidin analog of the present invention is determined by the methods disclosed in the examples herein.

[0090] In some embodiments, the present invention provides hepcidin analogs described herein, which exhibit improved solubility or improved aggregation properties compared to hepcidin reference compounds. Solubility can be determined by any suitable method known in the art. In some embodiments, suitable methods known in the art for determining solubility include incubating the peptide (e.g., the hepcidin analog of the present invention) in various buffers (acetate pH 4.0, acetate pH 5.0, Phos / citrate pH 5.0, Phos citrate pH 6.0, Phos pH 6.0, Phos pH 7.0, Phos 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 other buffers known in the art), and testing for aggregation or solubility using standard methods. These methods, but are not limited to, include, for example, precipitation visualization, dynamic light scattering, circular dichroism, and fluorescent dyes to measure surface hydrophobicity and detect aggregation or fibrillation. In some embodiments, improved solubility means that the peptide (e.g., the hepcidin analog of the present invention) dissolves more in a given liquid than the hepcidin reference compound.

[0091] In certain embodiments, the present invention provides a hepcidin analog described herein that exhibits increased solubility in certain solutions or buffers, such as water or buffers known in the art or disclosed herein, by at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 times, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%.

[0092] In certain embodiments, the present invention provides a hepcidin analog described herein, which exhibits reduced aggregation, and 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 that of a hepcidin reference compound in certain solutions or buffers, such as water or buffers known in the art or disclosed herein.

[0093] In some embodiments, the present invention provides hepcidin analogs described herein that exhibit a degradation reduction (i.e., high degradation stability) of, for example, about 10% or lower, about 20% or lower, about 30% or lower, about 40% or lower, or about 50% higher or lower than that of a hepcidin reference compound. In some embodiments, degradation stability is determined by any suitable method known in the art. In some embodiments, a suitable method known in the art for determining degradation stability is cited as Hawe et al. J Pharm Sci, VOL.101, NO.3, 2012, pp. 895-913, which is incorporated herein by reference in its entirety. In some embodiments, this method is used to select a viable sequence with a long shelf life.

[0094] In some embodiments, the hepcidine analog of the present invention is manufactured synthetically. In other embodiments, the hepcidine analog of the present invention is manufactured recombinantly.

[0095] The monomeric and dimeric peptides of the various hepcidin analogs of the present invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may include non-natural or non-natural amino acids, including, but not limited to, modified amino acids. In certain embodiments, modified amino acids include natural amino acids that have been chemically modified to include groups, multiple groups, or chemical moieties on the amino acid that do not exist on the amino acid in nature. The hepcidin analogs of the present invention may additionally contain D-amino acids. Furthermore, the hepcidin analog peptide monomers and dimers of the present invention may contain amino acid analogs. In certain embodiments, the peptide analogs of the present invention include any of those described herein, wherein 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.

[0096] In certain embodiments, the hepcidin analog of the present invention comprises one or more modified or unnatural amino acids. For example, in certain embodiments, the hepcidin analog comprises one or more of the following: Daba, Dapa, Pen, Sar, Cit, Pba, Cav, HLeu, 2-Nal, 1-Nal, d-1-Nal, d-2-Nal, Bip, Phe(4-OMe), Tyr(4-OMe), βhTrp, βhPhe, Phe(4-CF3), 2-2-indan, 1-1-indan, cyclobutyl, βhPhe, hLeu, Gla, Phe(4-NH2), hPhe, 1-Nal, Nle, 3-3-diPhe, cyclobutyl-Ala, Cha, Bip, β-Glu, Phe(4-Guan), homoamino acids, D-amino acids, and various N-methylated amino acids. Those skilled in the art will recognize that, in order to achieve similar desirable results, modified or non-natural amino acids may be used with other modified or non-natural amino acids and various other substitutions of natural amino acids, and such substitutions are within the scope of the teachings and spirit of the present invention.

[0097] The present invention comprises any of the hepcidin analogs described herein, either in free form or in salt form.

[0098] The compounds described herein include isotope-labeled compounds that are identical to those enumerated in the various formulas and structures presented herein, but differ in that one or more atoms are substituted by atoms having atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, for example, 2 H, 3 H, 1 3C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Examples include Cl. 3 H and 14 Certain isotope-labeled compounds described herein, incorporating radioactive isotopes such as 13C, are useful in drug-tissue distribution assays and / or substrate-tissue distribution assays. Furthermore, for example, deuterium, i.e. 2 Substitution using isotopes such as H can yield specific therapeutic benefits derived from increased metabolic stability, such as an extended in vivo half-life or a reduced required dose. In certain embodiments, the compound isotope-substituted with deuterium. In even more specific embodiments, the most unstable hydrogen atom is substituted with deuterium.

[0099] The hepcidin analog of the present invention comprises either a peptide monomer or dimer described herein, linked to one of the specific linker moieties described herein, which includes one of the specific linker moieties described herein.

[0100] The hepcidin analogs of the present invention include, but are not limited to, a monomeric or dimeric peptide 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 comprises any of the amino acid sequences shown in Tables 2 and 3.

[0101] In certain embodiments, the peptide analog of the present invention, or the monomeric subunit of the dimeric peptide analog of the present invention, comprises or consists of 7-35 amino acid residues, 8-35 amino acid residues, 9-35 amino acid residues, 10-35 amino acid residues, 7-25 amino acid residues, 8-25 amino acid residues, 9-25 amino acid residues, 10-25 amino acid residues, 7-18 amino acid residues, 8-18 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues, and optionally one or more additional non-amino acid moieties, such as a half-life extension moiety, a PEG or linker moiety. 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 conjugate 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 any of the various formulas described herein, X comprises or consists of 7-35 amino acid residues, 8-35 amino acid residues, 9-35 amino acid residues, 10-35 amino acid residues, 7-25 amino acid residues, 8-25 amino acid residues, 9-25 amino acid residues, 10-25 amino acid residues, 7-18 amino acid residues, 8-18 amino acid residues, 9-18 amino acid residues, or 10-18 amino acid residues.

[0102] In certain embodiments, the hepcidin analog or dimer of the present invention does not contain any of the compounds described in PCT / US2014 / 030352 or PCT / US2015 / 038370.

[0103] Peptide hepcidin analog In certain embodiments, the hepcidin analog of the present invention comprises a single peptide subunit, which is optionally conjugated to an acidic moiety. In certain embodiments, the acidic moiety is conjugated directly or via a linker.

[0104] In one embodiment, the present invention relates to formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ia) The hepcidin analog containing the peptide, or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 It is a cycloalkanoyl, R 2 is NH2, substituted amino, OH, or substituted hydroxy, X1 is either nonexistent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Dab, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2, Lys, substituted Lys, (D)Lys or substituted (D)Lys. X2 is Ala, Thr, Gly, N-substitution Gly, or Ser. X3 is Ala, Gly, N-substituted Gly, His, or substituted His. X4 is Ala, Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal. X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), 2-pyrrolidinebutanoic acid (Pba), Glu, Lys, substituted Lys, (D)Lys, or substituted (D)Lys. X6 is either absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either nonexistent, or is Ala, Gly, N-substitution Gly, Ile, Val, Leu, NLeu, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X8 is either absent or is Ala, (D)Ala, Ile, Gly, N-substituted Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substituted Lys, (D)Lys, substituted (D)Lys, aMeLys, or 123 triazole. X9 is either nonexistent or is Ala, Ile, Gly, N-substitution Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X10 is either nonexistent or is Ala, Gly, N-substitution Gly, Ile, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X11 is either nonexistent or is Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. and Each of X12 to X14 is either nonexistent or an arbitrary amino acid independently. However, the following conditions apply: i) The peptide may be further conjugated with any amino acid. ii) Any of the amino acids in the peptide may be the corresponding (D)-amino acid, or may be N-substituted, and iii) The peptide is either a linear peptide or a cyclized lactam, and In the formula, Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, and bhTrp is β-homo Liptophan is, 1-Nal is 1-naphthylalanine, 2-Nal is 2-naphthylalanine, Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Ppa is 2-(R)-pyrrolidinepropanoic acid, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenyl with F, Cl, Br, I, OH, methoxy, dimethyl Phenylalanine is substituted with c, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, substituted bhPhe is β-homophenylalanine in which phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, substituted Trp is N-methyl-L-tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH or t-Bu. Substituting bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH or t-Bu, Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid. 123-triazole is [ka] and Dab is [ka] That is the case.

[0105] In one embodiment, the present invention relates to formula (I): R 1 -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (Ib) The hepcidin analog containing the peptide, or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, R 1 C1-C6 alkyl, C6-C 12 Ariel, C6-C 12 Aryl-C1-C6 alkyl, C1-C 20 Alkanoyl, or C1-C 20 It is a cycloalkanoyl, R 2 is -NH2 or -OH, X1 is either nonexistent or is Asp, isoAsp, Asp(OMe), Glu, bhGlu, bGlu, Gly, N-substituted Gly, Gla, Glp, Ala, Arg, Leu, Lys, Dap, Orn, (D)Asp, (D)Arg, Tet1 or Tet2. X2 is Ala, Thr, Gly, N-substitution Gly, or Ser. X3 is Ala, His, or substitution His. X4 is Phe, Dpa, Gly, N-substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal. X5 is Pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, Gaba, 2-pyrrolidinepropanoic acid (Ppa), or 2-pyrrolidinebutanoic acid (Pba). X6 is either absent or any amino acid other than Cys, (D)Cys, aMeCys, hCys, or Pen. X7 is either nonexistent, or is Ala, Gly, N-substitution Gly, Ile, Val, Leu, NLeu, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X8 is either nonexistent or is Ala, (D)Ala, Ile, Gly, N-substitution Gly, Glu, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, substitution (D)Lys, or aMeLys. X9 is either nonexistent or is Ala, Ile, Gly, N-substitution Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X10 is either nonexistent or is Ala, Gly, N-substitution Gly, Ile, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X11 is either nonexistent or is Ala, Pro, bhPhe, Lys, substitute Lys, or (D)Lys. and Each of X12 to X14 is either nonexistent or an arbitrary amino acid independently. However, the following conditions apply: i) the peptide does not consist of a disulfide or thioether bond, ii) the peptide may be further conjugated with any amino acid, and iii) any of the amino acids of the peptide may be the corresponding (D)-amino acid of that amino acid, or may be further N-substituted. Dapa is diaminopropanoic acid, Dpa or DIP is 3,3-diphenylalanine or β,β-diphenylalanine, bhPhe is β-homophenylalanine, Bip is biphenylalanine, bhPro is β-homoproline, Tic is L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, NPC is L-nipecotinic acid, bhTrp is β-homotryptophan, 1-Nal is 1-naphthylalanine, 2-Nal is 2- Naphthylalanine, Orn is orinithine, Nleu is norleucine, 2Pal is 2-pyridylalanine, Pba is 2-(R)-pyrrolidinebutanoic acid, and substituted Phe is phenyl, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t - is a phenylalanine substituted with Bu, carboxyl, CN or guanidine, where substituted bhPhe is a β-homophenylalanine where phenyl is substituted with F, Cl, Br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azide, nitro, 4-carbamoyl-2,6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine, where substituted Trp is an N-methyl-L -Tryptophan, α-methyltryptophan, or tryptophan substituted with F, Cl, OH or t-Bu, substituted bhTrp is N-methyl-Lb-homotryptophan, α-methyl-β-homotryptophan, or β-homotryptophan substituted with F, Cl, OH or t-Bu, Tet1 is (S)-(2-amino)-3-(2H-tetrazole-5-yl)propanoic acid, and Tet2 is (S)-(2-amino)-4-(1H-tetrazole-5-yl)butanoic acid.

[0106] In any particular embodiment of the hepcidin analog of the present invention, X8 or X10 is (D)Lys substituted with Lys or L1Z, where L1 is absent, Dapa, D-Dapa or isoGlu, PEG, Ahx, isoGlu-PEG, PEG-isoGlu, PEG-Ahx, isoGlu-Ahx, or isoGlu-PEG-Ahx, where Ahx is an aminohexanoic acid moiety and PEG is -[C(O)-CH2-(Peg) n -N(H)] m -or-[C(O)-CH2-CH2-(Peg) n -N(H)] m - and Peg is -OCH2CH2-, m is 1, 2 or 3, n is an integer between 1 and 100K, and Z is the half-life extension portion. In one embodiment, the half-life extension portion is C 10 -C 21 It is Alkanoyl.

[0107] In any one embodiment of a peptide including, but not limited to, formula (Ia) or (Ib), X1 is Asp, Glu, (D)Asp, Tet1, or Tet2. X2 is either Thr or Ser. X3 is His or substitute His, X7 is nonexistent, Ile, Val, Leu, NLeu, Lys, substitute Lys, (D)Lys, or substitute (D)Lys. X8 is either nonexistent or is Ile, Val, Leu, NLeu, Phe, bhPhe, Lys, substitute Lys, (D)Lys, substitute (D)Lys, or aMeLys. X9 is either nonexistent or is Ala, Ile, Gly, N-substitution Gly, Val, Leu, NLeu, Phe, bhPhe, Lys, substitution Lys, (D)Lys, or substitution (D)Lys. X10 is either nonexistent, or is Ala, Ile, Phe, bhPhe, Lys, substitute Lys, (D)Lys or substitute (D)Lys, and X11 is either non-existent or is Pro, bhPhe, Lys, substitute Lys, or (D)Lys.

[0108] In any one embodiment of a peptide including, but not limited to, formula (Ia) or (Ib), X1 is Glu, Dab, Dap, Orn, Lys, or Tet1. X2 is Thr, X3 is His or 1MeHis. X4 is Dpa, The X5 is a Pro model, X6 is either nonexistent, Ala, Glu, or a substitute Lys; X7 is either not present, or is Ile, Lys, substitute Lys, (D)Lys, or substitute (D)Lys. X8 is either absent or is Ile, Glu, Asp, 123-triazole, Lys, substituted Lys, (D)Lys, substituted (D)Lys, or aMeLys. X9 either does not exist or is bhPhe. X10 is either nonexistent, or is Ala, Ile, Phe, bhPhe, Lys, substitute Lys, (D)Lys or substitute (D)Lys, and X11 is either non-existent or is Pro, bhPhe, Lys, substitute Lys, or (D)Lys.

[0109] In one embodiment, X1 is Glu.

[0110] In one embodiment, X2 is Thr.

[0111] In one embodiment, X4 is Dpa.

[0112] In one embodiment, X5 is Pro.

[0113] In one embodiment, the peptide is formula II: R 1-Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (II) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 X3, X6-X14 are as described in formula (Ia) or formula (Ib).

[0114] In one embodiment, X9 is nonexistent, bhPhe, Lys, substitute Lys, (D)Lys, or substitute (D)Lys.

[0115] In one embodiment, X9 does not exist.

[0116] In one embodiment, X9 is bhPhe.

[0117] In one embodiment, the peptide is formula III: R 1 -Glu-Thr-X3-[Dpa]-Pro-X6-X7-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (III) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 X3, X6-X8, and X10-X14 are as described in formula (Ia) or formula (Ib).

[0118] In one embodiment, X6 is Ala, Lys, or substitute Lys.

[0119] In one embodiment, X6 is Ala.

[0120] In one embodiment, the peptide is formula IV: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-X7-X8-[bhPhe]-X10-X11-X12-X13-X14-R2 (IV) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 X3, X7-X8, and X10-X14 are as described in formula (Ia) or formula (Ib).

[0121] In one embodiment, X7 is nonexistent, Ile, Lys, or a substitute Lys.

[0122] In one embodiment, X7 does not exist.

[0123] In one embodiment, X7 is Ile.

[0124] In one embodiment, the peptide is of formula V: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-X8-[bhPhe]-X10-X11-X12-X13-X14-R 2 (V) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 X3, X8, and X10-X14 are as described in formula (Ia) or formula (Ib).

[0125] In one embodiment, X8 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0126] In one embodiment, X8 is (D)Lys, or substitute (D)Lys.

[0127] In one embodiment, X8 is Lys or Lys(Ac).

[0128] In one embodiment, X8 is (D)Lys or (D)Lys(Ac).

[0129] In one embodiment, X8 is a conjugated amino acid.

[0130] In one embodiment, X8 is a conjugated Lys or (D)Lys.

[0131] In one embodiment, X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is the linker and Z is the half-life extension portion.

[0132] In one embodiment, the peptide is of formula VIa or VIb: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIa); or R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIb) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 X3 and X10-X14 are as described in formula (Ia) or formula (Ib).

[0133] In one embodiment, the peptide is of formula VIc: R 1 -Glu-Thr-X3-[Dpa]-Pro-Ala-Ile-[Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIc); In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 X3 and X10-X14 are as described in formula (Ia) or formula (Ib).

[0134] In one embodiment, X3 is His.

[0135] In one embodiment, the peptide is of formula VIIa or VIIb: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIa); or R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIb) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , and X10-X14 are as described in formula (Ia) or formula (Ib).

[0136] In one embodiment, the peptide is of formula VIIc: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIc); In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , and X10-X14 are as described in formula (Ia) or formula (Ib).

[0137] In one embodiment, X3 is (1-Me)His.

[0138] In one embodiment, the peptide is of formula VIIIa or VIIIb: R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-X10-X11-X12-X13-X14-R 2(VIIIa); or R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (VIIIb) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , and X10-X14 are as described in formula (Ia) or formula (Ib).

[0139] In one embodiment, X10 is Lys, substituted Lys, (D)Lys, or substituted (D)Lys.

[0140] In one embodiment, X10 is (D)Lys, or substitute (D)Lys.

[0141] In one embodiment, X10 is (D)Lys or (D)Lys(Ac).

[0142] In one embodiment, X10 is Lys(Ahx_Palm).

[0143] In one embodiment, X10 is a conjugated amino acid.

[0144] In one embodiment, X10 is a conjugated Lys or (D)Lys.

[0145] In one embodiment, X10 is Lys(L1Z) or (D)Lys(L1Z), where L1 is the linker and Z is the half-life extension portion.

[0146] In one embodiment, PEG is -[C(O)-CH2-(Peg) n -N(H)] m -, or -[C(O)-CH2-CH2-(Peg) n -N(H)] m-, Peg is -OCH2CH2-, m is 1, 2 or 3, and n is an integer between 1 and 100, or between 10K, 20K or 30K.

[0147] In one embodiment, m is 1. In another embodiment, m is 2.

[0148] In one embodiment, n is 2. In another embodiment, n is 4. In another embodiment, n is 8. In another embodiment, n is 11. In another embodiment, n is 12. In another embodiment, n is 20K.

[0149] In one embodiment, PEG is 1Peg2, and 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0150] In another embodiment, PEG is 2Peg2, and 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0151] In another embodiment, PEG is 1Peg2-1Peg2, where each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0152] In another embodiment, PEG is 1Peg2-1Peg2, and 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-.

[0153] In another embodiment, PEG is 2Peg4, where 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-.

[0154] In another embodiment, PEG is 1Peg8, where 1Peg8 is -C(O)-CH2-(Peg)8-N(H)- or -[C(O)-CH2-(OCH2CH2)8-NH]-.

[0155] In another embodiment, PEG is 2Peg8, where 2Peg8 is -C(O)-CH2-CH2-(Peg)8-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)8-NH]-.

[0156] In another embodiment, PEG is 1Peg11, and 1Peg11 is -C(O)-CH2-(Peg) 11 -N(H)-, or -[C(O)-CH2-(OCH2CH2)] 11 -NH]- is

[0157] In another embodiment, PEG is 2Peg11, and 2Peg11 is -C(O)-CH2-CH2-(Peg) 11 -N(H)-, or -[C(O)-CH2-CH2-(OCH2CH2)] 11 -NH]- is

[0158] In another embodiment, PEG is 2Peg11' or 2Peg12, where 2Peg11' or 2Peg12 is -C(O)-CH2-CH2-(Peg) 12 -N(H)-, or -[C(O)-CH2-CH2-(OCH2CH2)] 12 -NH]- is

[0159] In one embodiment, when PEG binds to Lys, the -C(O)- of PEG becomes the N of Lys. ε Combine.

[0160] In one embodiment, when PEG binds to isoGlu, the -N(H)- of PEG binds to the -C(O)- of isoGlu.

[0161] In one embodiment, when PEG binds to Ahx, the -N(H)- of PEG binds to the -C(O)- of Ahx.

[0162] In one embodiment, when PEG is coupled to Palm, the -N(H)- of PEG is coupled to the -C(O)- of Palm.

[0163] In one embodiment, the peptide is formula IX: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); According to the formula, or a pharmaceutically acceptable salt or solvate thereof, where R 1 , R 2 X6, X7 and X11-X14 are as described in formula (Ia) or formula (Ib).

[0164] In one embodiment, the peptide is of formula IXa or IXb: R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXa); or R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (IXb) In accordance with, or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , and X11-X14 are as described in formula (Ia) or formula (Ib).

[0165] In one embodiment, the peptide is of formula Xa or Xb: R 1 -Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (Xa); or R 1-Glu-Thr-[(1-Me)His]-[Dpa]-Pro-Ala-Ile-[Lys(Ac)]-[bhPhe]-[Lys(Ahx_Palm)]-X11-X12-X13-X14-R 2 (Xb) A peptide that conforms to [the specified formula], or a pharmaceutically acceptable salt or solvate thereof, In the formula, R 1 , R 2 , and X11-X14 are as described in formula (Ia) or formula (Ib).

[0166] In one embodiment, the peptide is a linear peptide.

[0167] In one embodiment, the peptide is a lactam.

[0168] In one embodiment, the peptide is a lactam, in which case any free -NH2 is cyclized with any free -C(O)2H.

[0169] In one embodiment, the peptide is of formula XXI: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-X8-X9-X10-X11-X12-X13-X14-R 2 (XXI) Accordingly, in the formula, R 1 , R 2 , and X10~X14 are as described in formula (Ia) or formula (Ib), X6 is nonexistent, Ala, or substitute Lys; X7 is nonexistent, Ile, substitute Lys, or substitute (D)Lys; X9 is nonexistent or bhPhe. And X8 is Lys(L1Z) or (D)Lys(L1Z), where L1 is the linker and Z is the half-life extension portion.

[0170] In one embodiment, X8 is Lys(L1Z).

[0171] In one embodiment, X8 is (D)Lys(L1Z).

[0172] In one embodiment, the peptide is of formula XXII: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXII) Accordingly, in the formula, R 1 , R 2 , and X10~X14 are as described in formula (Ia) or formula (Ib), X6 is nonexistent, Ala, or substitute Lys; X7 is nonexistent, Ile, substitute Lys, or substitute (D)Lys; and X9 is nonexistent or bhPhe.

[0173] In one embodiment, X6 does not exist.

[0174] In one embodiment, X6 is a substitution Lys.

[0175] In one embodiment, X6 is Ala.

[0176] In one embodiment, the peptide is of formula XXIIIa or XXIIIb: R 1 -Glu-Thr-His-[Dpa]-Pro-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIIIa) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIIIb) Accordingly, in the formula, R 1 , R 2 , and X10~X14 are as described in formula (Ia) or formula (Ib), X7 is nonexistent, Ile, substitution Lys, or substitution (D)Lys, and X9 is nonexistent or bhPhe.

[0177] In one embodiment, X7 does not exist.

[0178] In one embodiment, X7 is the substitution (D)Lys.

[0179] In one embodiment, X7 is a substitution Lys.

[0180] In one embodiment, X7 is Ile.

[0181] In one embodiment, the peptide is of formula XXIVa, XXIVb, XXIVc, or XXIVd: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVa) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVb) R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVc) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXIVd) Accordingly, in the formula, R 1 , R 2 , and X10~X14 are as described in formula (Ia) or formula (Ib), X9 either does not exist or is bhPhe.

[0182] In one embodiment, X9 does not exist.

[0183] In one embodiment, the peptide has the formula XXVa, XXVb, XXVc, or XXVd: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVa) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVb) R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVc) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-X10-X11-X12-X13-X14-R 2 (XXVd) Accordingly, in the formula, R 1 , R 2 , and X10-X14 are as described in formula (Ia) or formula (Ib).

[0184] In one embodiment, X9 is bhPhe.

[0185] In one embodiment, the peptide has the formula XXVIa, XXVIb, XXVIc, or XXVId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIa) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIb) R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVIc) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-X10-X11-X12-X13-X14-R 2 (XXVId) Accordingly, in the formula, R 1 , R 2 , and X10-X14 are as described in formula (Ia) or formula (Ib).

[0186] In one embodiment, X10 is Lys or (D)Lys.

[0187] In one embodiment, X10 is (D)Lys.

[0188] In one embodiment, the peptide is of formula XXVIIa, XXVIIb, XXVIIc, or XXVIId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-[(D)Lys]-X11-X12-X13-X14-R 2 (XXVIIa) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIIb) R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2 (XXVIIc) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-[(D)LYS]-X11-X12-X13-X14-R 2(XXVIId) Accordingly, in the formula, R 1 , R 2 , and X11-X14 are as described in formula (Ia) or formula (Ib).

[0189] In one embodiment, X10 does not exist.

[0190] In one embodiment, the peptide is of formula XXVIIIa, XXVIIIb, XXVIIIc, or XXVIIId: R 1 -Glu-Thr-His-[Dpa]-Pro-Ile-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIa) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-Ile-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIb) R 1 -Glu-Thr-His-[Dpa]-Pro-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIIc) R 1 -Glu-Thr-His-[Dpa]-Pro-Ala-[Lys(L1Z)]-[bhPhe]-X11-X12-X13-X14-R 2 (XXVIIId) Accordingly, in the formula, R 1 , R 2 , and X11-X14 are as described in formula (Ia) or formula (Ib).

[0191] In one embodiment, L1 is a single bond.

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

[0193] In one embodiment, L1 is Ahx.

[0194] In one embodiment, L1 is iso-Glu-Ahx.

[0195] In one embodiment, L1 is PEG.

[0196] A hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 78 to 96, wherein L1 is PEG-Ahx.

[0197] In one embodiment, L1 is iso-Glu-PEG-Ahx.

[0198] In one embodiment, PEG is -[C(O)-CH2-(Peg)nN(H)]m- or -[C(O)-CH2-CH2-(Peg)nN(H)]m-, where Peg is -OCH2CH2-, m is 1, 2, or 3, and n is an integer between 1 and 100, or between 10K, 20K, or 30K.

[0199] In one embodiment, m is 1.

[0200] In one embodiment, m is 2.

[0201] In one embodiment, n is 2.

[0202] In one embodiment, n is 4.

[0203] In one embodiment, n is 8.

[0204] In one embodiment, n is 11.

[0205] In one embodiment, n is 12.

[0206] In one embodiment, n is 20K.

[0207] In one embodiment, PEG is 1Peg2, and 1Peg2 is -C(O)-CH2-(Peg)2-N(H)-.

[0208] In one embodiment, PEG is 2Peg2, and 2Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0209] In one embodiment, PEG is 1Peg2-1Peg2, and each 1Peg2 is -C(O)-CH2-CH2-(Peg)2-N(H)-.

[0210] In one embodiment, PEG is 1Peg2-1Peg2, and 1Peg2-1Peg2 is -[(C(O)-CH2-(OCH2CH2)2-NH-C(O)-CH2-(OCH2CH2)2-NH-]-.

[0211] In one embodiment, PEG is 2Peg4, where 2Peg4 is -C(O)-CH2-CH2-(Peg)4-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)4-NH]-.

[0212] In one embodiment, PEG is 1Peg8, and 1Peg8 is -C(O)-CH2-(Peg)8-N(H)- or -[C(O)-CH2-(OCH2CH2)8-NH]-.

[0213] In one embodiment, PEG is 2Peg8, where 2Peg8 is -C(O)-CH2-CH2-(Peg)8-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)8-NH]-.

[0214] In one embodiment, PEG is 1Peg11, where 1Peg11 is -C(O)-CH2-(Peg)11-N(H)- or -[C(O)-CH2-(OCH2CH2)11-NH]-.

[0215] In one embodiment, PEG is 2Peg11, where 2Peg11 is -C(O)-CH2-CH2-(Peg)11-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)11-NH]-.

[0216] In one embodiment, PEG is 2Peg11' or 2Peg12, where 2Peg11' or 2Peg12 is -C(O)-CH2-CH2-(Peg)12-N(H)- or -[C(O)-CH2-CH2-(OCH2CH2)12-NH]-.

[0217] In one embodiment, when PEG binds to Lys, the -C(O)- of PEG binds to the Ne of Lys.

[0218] In one embodiment, when PEG binds to isoGlu, the -N(H)- of PEG binds to the -C(O)- of isoGlu.

[0219] In one embodiment, when PEG binds to Ahx, the -N(H)- of PEG binds to the -C(O)- of Ahx.

[0220] In one embodiment, when PEG is coupled to Palm, the -N(H)- of PEG is coupled to the -C(O)- of Palm.

[0221] In one embodiment, Z is a Palm.

[0222] In one embodiment, L1Z is -Ahx_Palm.

[0223] In one embodiment, L1Z is -bAla_Palm.

[0224] In one embodiment, L1Z is -IsoGlu_Palm.

[0225] In one embodiment, L1Z is PEG12_Palm.

[0226] In one embodiment, L1Z is -1PEG2_1PEG2_Ahx_C18_diacid.

[0227] In one embodiment, X11, X12, X13, and X14 are not present.

[0228] In one embodiment, the peptide is of formula XXI: R 1 -Glu-Thr-His-[Dpa]-Pro-X6-X7-[Lys(L1Z)]-X9-X10-X11-X12-X13-X14-R 2 (XXI) Accordingly, in the formula, R 1 , R 2 , and X10~X14 are as described in formula (Ia) or formula (Ib), X6 is either nonexistent or substituted with Lys, X7 is either nonexistent or substituted with Lys, and X9 is either nonexistent or bhPhe.

[0229] In one embodiment, each of -L1Z is independently: PEG11_OMe; PEG12 C18 acid; 1PEG2_1PEG2_Ahx_Palm; 1PEG2_Ahx_Palm; Ado_Palm; Ahx_Palm; Ahx_PEG20K; PEG12_Ahx_IsoGlu_Behenic; PEG12_Ahx_Palm; PEG12_DEKHKS_Palm; PEG12 IsoGlu C18 acid; PEG12_Ahx_C18 acid; PEG12_IsoGlu_Palm; PEG12_KKK_Palm; PEG12_KKKG_Palm; PEG12_DEKHKS_Palm; PEG12_Palm; PEG12_PEG12_Palm; PEG20K; PEG4_Ahx_Palm; PEG4_Palm; PEG8_Ahx_Palm; or IsoGlu_Palm; -1PEG2_1PEG2_Dap_C18_Diacid; -1PEG2_1PEG2_IsoGlu_C10_Diacid; -1PEG2_1PEG2_IsoGlu_C12_Diacid; -1PEG2_1PEG2_IsoGlu_C14_Diacid; -1PEG2_1PEG2_IsoGlu_C16_diacid; -1PEG2_1PEG2_IsoGlu_C18_Diacid; -1PEG2_1PEG2_IsoGlu_C22_Diacid; -1PEG2_1PEG2_Ahx_C18_Diacid; -1PEG2_1PEG2_C18_Diacid; -1PEG8_IsoGlu_C18_Diacid; -IsoGlu_C18_Diacid; -PEG12_Ahx_C18_Diacid; -PEG12_C16_diacid; -PEG12_C18_diacid; -1PEG2_1PEG2_1PEG2_C18_Diacid; -1PEG2_1PEG2_1PEG2_IsoGlu_C18_Diacid; -PEG12_IsoGlu_C18_Diacid; -PEG4_IsoGlu_C18_Diacid; or -PEG4_PEG4_IsoGlu_C18_diacid, During the ceremony, PEG11_OMe is -[C(O)-CH2-CH2-(OCH2CH2) 11 -OMe] 1PEG2 is -C(O)-CH2-(OCH2CH2)2-NH-, PEG4 is -C(O)-CH2-CH2-(OCH2CH2)4-NH-, PEG8 is -[C(O)-CH2-CH2-(OCH2CH2)8-NH-, 1PEG8 is -[C(O)-CH2-(OCH2CH2)8-NH-, PEG12 is -[C(O)-CH2-CH2-(OCH2CH2) 12 -NH-, Ado is -[C(O)-(CH2) 11 -NH]- Cn acids are -C(O)(CH2) n-2 -CH3, and C18 acids are -C(O)-(CH2) 16 -Me, Palm is -C(O)-(CH2) 14 -Me, isoGlu is isoglutamic acid, isoGlu_Palm is [ka] And, Ahx is -[C(O)-(CH2)5-NH]-, Cn diacids are -C(O)-(CH2) n-2 -COOH, where n is 10, 12, 14, 16, 18, or 22.

[0230] In one embodiment, X8 or X10 is Lys(1PEG2_1PEG2_IsoGlu_C n (Diacid) and (1PEG2_1PEG2_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0231] In one embodiment, X8 or X10 is (D)Lys(1PEG2_1PEG2_IsoGlu_C n(Diacid) and (D)Lys(1PEG2_1PEG2_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0232] In one embodiment, X8 or X10 is Lys(1PEG8_IsoGlu_C n _Diacid) and Lys(1PEG8_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0233] In one embodiment, X8 or X10 is (D)Lys(1PEG8_IsoGlu_C n (Diacid) and (D)Lys(1PEG8_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0234] In one embodiment, X8 or X10 is Lys(1PEG2_1PEG2_Dap_C n _Diacid) and Lys(1PEG2_1PEG2_Dap_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0235] In one embodiment, X8 or X10 is Lys(IsoGlu_C n _Diacid) and Lys(IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0236] In one embodiment, X8 or X10 is (D)Lys(IsoGlu_C n (Diacid) and (D)Lys(IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0237] In one embodiment, X8 or X10 is Lys(PEG12_IsoGlu_C n It is a diacid, and Lys(PEG12_IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0238] In one embodiment, X8 or X10 is (D)Lys(PEG12_IsoGlu_C n (Diacid) and (D)Lys(PEG12_IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0239] In one embodiment, X8 or X10 is (PEG4_IsoGlu_C n _Diacid) and Lys(PEG4_IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0240] In one embodiment, X8 or X10 is (D)Lys(PEG4_IsoGlu_C n (Diacid) and (D)Lys(PEG4_IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0241] In one embodiment, X8 or X10 is Lys(PEG4_PEG4_IsoGlu_C n It is a diacid, and Lys(PEG4_PEG4_IsoGlu_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0242] In one embodiment, X8 or X10 is (D)Lys(PEG4_PEG4_IsoGlu_C n (D)Lys(PEG4_PEG4_IsoGlu_C) n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0243] In one embodiment, X8 or X10 is Lys(IsoGlu_C n _Diacid) and Lys(IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0244] In one embodiment, X8 or X10 is (D)Lys(IsoGlu_C n (Diacid) and (D)Lys(IsoGlu_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0245] In one embodiment, X8 or X10 is Lys(PEG12_Ahx_C n _Diacid) and Lys(PEG12_Ahx_C n (Diacid) is, [ka] And n is 10, 12, 14, 16, or 18.

[0246] In one embodiment, X8 or X10 is Lys(PEG12_Ahx_C n _Diacid) and Lys(PEG12_Ahx_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0247] In one embodiment, X8 or X10 is (D)Lys(PEG12_Ahx_C n (Diacid) and (D)Lys(PEG12_Ahx_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0248] In one embodiment, X8 or X10 is Lys(PEG12_C n _Diacid) and Lys(PEG12_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0249] In one embodiment, X8 or X10 is (D)Lys(PEG12_C n (Diacid) and (D)Lys(PEG12_C n (Diacid) is, [ka] ; And n is 10, 12, 14, 16, or 18.

[0250] In one embodiment, X8 or X10 is 123-triazole.

[0251] In one embodiment, X11 is nonexistent, Ala, (D)Lys, or substitute Lys.

[0252] In one embodiment, X11 does not exist.

[0253] A hepcidin analog or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 156, wherein X11 is Ala.

[0254] In one embodiment, X11 is (D)Lys.

[0255] In one embodiment, X11 is Lys(Ahx_Palm).

[0256] In one embodiment, X12 is either absent or Ala.

[0257] In one embodiment, X12 does not exist.

[0258] In one embodiment, X12 is Ala.

[0259] In one embodiment, X13 does not exist.

[0260] In one embodiment, X14 does not exist.

[0261] In one embodiment, R 2 It is NH2.

[0262] In one embodiment, R 2 It is a substituted amino acid.

[0263] In one embodiment, R 2 It is an N-alkylamino compound.

[0264] In one embodiment, R 2 This is an N-alkylamino compound, in which case the alkyl group is either further substituted or unsubstituted.

[0265] In one embodiment, R 2 This is an N-alkylamino, in which case the alkyl is further substituted with an aryl or heteroaryl.

[0266] In one embodiment, R 2 is an alkylamino, in which case the alkyl is either unsubstituted or substituted with an aryl, and the alkyl is ethyl, propyl, butyl, or pentyl.

[0267] In one embodiment, R 2is an alkylamino, in which case the alkyl is either unsubstituted or substituted with phenyl, and the alkyl is ethyl, propyl, butyl, or pentyl.

[0268] In one embodiment, R 2 It is OH.

[0269] In one embodiment, R 1 C1-C 20 It is Alkanoyl.

[0270] In one embodiment, R 1 It is IVA or isovaleric acid.

[0271] In one embodiment, the peptide is a linear peptide.

[0272] In one embodiment, the peptide is a lactam.

[0273] In one embodiment, the peptide is a lactam, in which case any free -NH2 is cyclized with any free -C(O)2H.

[0274] In one embodiment, X11 is nonexistent, Ala, (D)Lys, or substitute Lys.

[0275] In one embodiment, X11 does not exist.

[0276] In one embodiment, X11 is Ala.

[0277] In one embodiment, X11 is (D)Lys.

[0278] In one embodiment, X11 is Lys(Ahx_Palm).

[0279] In one embodiment, X12 is either absent or Ala.

[0280] In one embodiment, X12 does not exist.

[0281] In one embodiment, X12 is Ala.

[0282] In one embodiment, X13 does not exist.

[0283] In one embodiment, X14 does not exist.

[0284] In one embodiment, R 2 In another embodiment, R 2 is a substituted amino acid. In another embodiment, R 2 is an alkylamino or (substituted alkyl)amino. In another embodiment, R 2 These are methylamino, ethylamino, propylamino, benzylamino, or phenethylamino.

[0285] In one embodiment, R 2 It is OH.

[0286] In one embodiment, R 1 C1-C 20 It is Alkanoyl.

[0287] In one embodiment, R 1 It is IVA or isovaleric acid.

[0288] In any particular embodiment of a peptide analog having any of the various formulas described herein, R 1 The selected amides are methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octanyl, and conjugated amides of lauric acid, hexadecanoic acid, and γ-Glu-hexadecanoic acid.

[0289] In certain embodiments, the substitution Lys is Ac, PEG, Ahx, isoGlu, C 10 -C 20Alkanoyl, PEG-Ahx, PEG-isoGlu, Ahx-C 10 -C 20 Alkanoyl, isoGlu-C 10 -C 20 Alkanoyl, PEG-Ahx-C 10 -C 20 Alkanoyl, PEG-isoGlu-C 10 -C 20 Lys is substituted with an alkanoyl or any other as described herein. In one embodiment, Lys is N ε It is replaced by this.

[0290] In certain embodiments, the substitution (D)Lys is Ac, PEG, Ahx, isoGlu, C 10 -C 20 Alkanoyl, PEG-Ahx, PEG-isoGlu, Ahx-C 10 -C 20 Alkanoyl, isoGlu-C 10 -C 20 Alkanoyl, PEG-Ahx-C 10 -C 20 Alkanoyl, PEG-isoGlu-C 10 -C 20 (D)Lys is substituted with an alkanoyl or any other as described herein. In one embodiment, (D)Lys is N of (D)Lys ε It is replaced by this.

[0291] In a particular embodiment, C 10 -C 20 The alkanoyl is Palm.

[0292] In certain embodiments, the present invention includes polypeptides comprising amino acid sequences listed in Tables 6A to C, or polypeptides having any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity to any of these amino acid sequences.

[0293] In certain embodiments, the present invention includes a hepcidin analog having the structure described below, or comprising the amino acid sequence described below: Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PA-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-P-[Lys(Ahx_Palm)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ahx_Palm)]-[bhPhe]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[(D)Lys]-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-PA-[Lys(Ac)]-NH2; Isovaleric acid-ETH-[Dpa]-P-[Lys(Ac)]-NH2; Iso-glycine-ETH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-NH2; Iso-glycine-ETH-[Dpa]-PLI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-L-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid)]-[bhPhe]-[(D)Lys]-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid)]-NH2; Iso-glycine-ETH-[Dpa]-PI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PSI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PII-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PFI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-PEI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-P-[(D)Lys]-I-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Iso-glycine-ETH-[Dpa]-P-[Lys(Ahx_Palm)]-I-[(D)Lys]-[bhPhe]-[Lys(Ac)]-NH2; Iso-glycine-ATH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid-[(D)Lys]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(1Peg2_1Peg2_Ahx_C18_dioic acid)]-[(D)Lys]-[bhPhe]-NH2; Iso-glycine-ETH-[Dpa]-PAI-[(D)Lys]-A-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ETH-[Dpa]-PAI-[(D)Ala]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ETH-[Dpa]-PAA-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ATH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-EAH-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Iso-glycine-ETA-[Dpa]-PAI-[(D)Lys]-[bhPhe]-[Lys(Ahx_Palm)]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-ETH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Iso-glycine-ET-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-[Tet1]-TH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-[Tet2]-TH-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Yoshiki oxalic acid-[Tet1]-T-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Isovaleric acid-[Tet2]-T-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-A-NH2; Isovaleric acid-[Tet2]-T-[(1-Me)His]-[Dpa]-PAI-[Lys(Ac)]-[bhPhe]-[(D)Lys]-NH2; Isovaleric acid-ETH-[Dpa]-PAI-[bhPhe]-[(D)Lys]-A-NH2; Isovaleric acid-[Tet1]-T-[(1-Me)His]-[Dpa]-PAI-[bhPhe]-[(D)Lys]-A-NH2, or Isovaleric acid-[Tet1]-T-[(1-Me)His]-[Dpa]-PAI-[bhPhe]-[(D)Lys]-NH2.

[0294] In certain embodiments, the present invention includes a hepcidin analog having the structure described below, or comprising the amino acid sequence described below: ID number 321 [ka] ; ID number 319 [ka] ; ID number 322 [ka] ; ID number 318 [ka] ; ID number 320 [ka] ; ID number 56 [ka] ; ID number 286 [ka] ; ID number 58 [ka] ; ID number 287 [ka] ; ID number 156 [ka] ,or ID number 292 [ka] ;

[0295] In certain embodiments, the peptide is any one of the peptides having an FPN activity less than 100 nM. In another specific embodiment, the peptide is any one of the peptides having an FPN activity less than 50 nM. In yet another specific embodiment, the peptide is any one of the peptides having an FPN activity less than 20 nM. In yet another specific embodiment, the peptide is any one of the peptides having an FPN activity less than 10 nM. In yet another specific embodiment, the peptide is any one of the peptides having an FPN activity less than 5 nM.

[0296] Peptide analog conjugate In certain embodiments, the hepcidin analogs of the present invention, including both monomers and dimers, comprise one or more conjugated chemical substituents, such as lipophilic substituents and polymeric moieties, collectively referred to herein as half-life extension moieties. While we do not wish to be bound by any particular theory, it is thought that lipophilic substituents bind to albumin in the bloodstream, thereby concealing the hepcidin analog from enzymatic degradation and extending its half-life. Furthermore, it is thought that polymeric moieties extend the half-life, reducing clearance in the bloodstream, and in some cases increasing epithelial permeability and enhancing retention in the lamina propria. It is also speculated that in some cases these substituents may further increase epithelial permeability and enhance retention in the lamina propria. Those skilled in the art will be well aware of appropriate compound preparation methods employed under the circumstances of the present invention. For examples of non-restrictive appropriate chemical methods, 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).

[0297] 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 extender. 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 moieties, if present, can provide a gap between the hepcidin analog and the lipophilic substituent.

[0298] In certain embodiments, the lipophilic substituent or half-life extension portion 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 a bond to an 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, which may then form part of an alkanoyl group, for example, palmitoyl, caproyl, lauroyl, myristoyl, or stearoyl.

[0299] Lipophilic substituents can be conjugated to any amino acid side chain in the hepcidine analog of the present invention. In certain embodiments, the amino acid side chain includes a carboxyl group, hydroxyl group, thiol group, amide group, or amine group to form an ester, sulfonyl ester, thioester, amide, or sulfonamide with a spacer or lipophilic substituent. For example, the lipophilic substituent may be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys, or Dbu, Dpr, or Orn. ​​In certain embodiments, the lipophilic substituent is conjugated to Lys. The amino acid represented as Lys in any of the formulas provided herein may be replaced, for example, with Dbu, Dpr, or Orn to which the lipophilic substituent is added.

[0300] Alternatively, in further embodiments of the present invention, the side chains of one or more amino acid residues in the hepcidin analog of the present invention may be conjugated to a polymer moiety or other half-life extension moiety, for example, to increase solubility and / or to extend the in vivo half-life (e.g., plasma half-life) and / or to increase bioavailability. Such modifications are also known to reduce the clearance (e.g., renal clearance) of therapeutic proteins and therapeutic peptides.

[0301] As used herein, "polyethylene glycol" or "PEG" refers to the general formula H-(O-CH2-CH2) nPEG is a polyether compound of -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), and as used herein, depending on its molecular weight, PEO, PEE, or POG refers to an oligomer or polymer of ethylene oxide. The three names are chemically synonymous, but PEG tends to refer to oligomers or polymers with a molecular weight of less than 20,000 g / mol, PEO tends to refer to polymers with a molecular weight greater than 20,000 g / mol, and POE tends to refer to polymers of 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 interchangeably. PEG is prepared by polymerization of ethylene oxide and is commercially available over a wide range of molecular weights from 300 g / mol to 10,000,000 g / mol. PEG and PEO of different molecular weights are 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 portion is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymer portions include polyethylene glycol (PEG), homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995), and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992). PEGs prepared for the purpose of extending half-lives are also included, for example, monoactivated alkoxy-terminated polyalkylene oxides (POAs), such as mono-methoxy-terminated polyethylene glycol (mPEG); bisactivated polyethylene glycol oxide (glycol), or other PEG derivatives. Suitable polymers vary considerably in weight from about 200 to about 40,000, which are typically selected for the purposes of the present invention. In certain embodiments, PEG having a molecular weight of 200 to 2,000 daltons, or 200 to 500 daltons, is used.Depending on the initiator used in the polymerization process, various forms of PEG may also be used. For example, common initiators include monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), or the abbreviated form mPEG. Other suitable initiators are also known in the art and are suitable for use in the present invention.

[0302] Low molecular weight PEGs are also available as pure oligomers and are referred to as monodispersible, homogeneous, or discontinuous. These are used in specific embodiments of the present invention.

[0303] PEG is available in different structures. Branched PEG has 3 to 10 PEG chains extending from a central core group. Star-shaped PEG has 10 to 100 PEG chains extending from a central core group. Comb-shaped PEG typically has multiple PEG chains bonded to a polymer backbone. PEG can also be linear. The number often included in the name PEG indicates its average molecular weight (for example, PEG with n=9 has an average molecular weight of approximately 400 daltons and is labeled PEG400).

[0304] As used herein, “PEGylation” is the action of conjugating (e.g., covalently) a PEG structure to the hepcidin analog of the present invention, 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 spacer to be PEGylated is PEG3 or PEG8. In some embodiments, the spacer is PEGylated. In certain embodiments, the PEG of the spacer to be PEGylated is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the spacer to be PEGylated is PEG3 or PEG8.

[0305] In some embodiments, the present invention comprises a hepcidin analog peptide (or its dimer) conjugated with PEG, wherein the PEG is covalently bonded, for example, via amide, thiol, click chemistry, or any other suitable means known in the art. In certain embodiments, the PEG is bonded via an amide bond. Thus, 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 and is bonded to the peptide of the present invention. In certain embodiments, PEG25 contains a diacid moiety and 25 glycol moieties.

[0306] Other suitable polymer moieties include, for example, poly-lysine, poly-aspartic acid, and poly-glutamic acid, which are polyamino acids (see, e.g., Gombotz, et al. (1995), Bioconjugate Chem., vol.6:332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol.3, 49-57 and Tsukada, et al. (1984), J. Natl. Cancer Inst., vol.73, :721-729). The polymer moieties may be linear or branched. In some embodiments, the molecular weights are 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).

[0307] In some embodiments, the hepcidin analog of the present invention may contain two or more polymer moieties, in which case the total molecular weight of all such moieties is generally within the range described above.

[0308] In some embodiments, the polymer portion may be covalently bonded to the amino, carboxyl, or thiol groups of the amino acid side chains. Specific examples include the thiol groups of Cys residues and the epsilon-amino groups of Lys residues, and may also include the carboxyl groups of Asp and Glu residues.

[0309] Those skilled in the art will recognize appropriate techniques that can be used to carry out the conjugation reaction. For example, the methoxy-supported PEG moiety can be conjugated to the Cys thiol group via a maleimide bond using reagents commercially available from Nektar Therapeutics AL. For details of preferred chemical methods, see WO 2008 / 101017 and the references cited above. Maleimide-functionalized PEG can also be conjugated to the sulfhydryl side chain group of the Cys residue.

[0310] Where used herein, the oxidation of disulfide bonds can occur in a single step or as a two-step process. Where used herein, in a single oxidation step, a trityl protecting group is often employed during assembly, allowing for deprotection during cleavage, followed by solution oxidation. If a second disulfide bond is required, there are options for spontaneous oxidation or selective oxidation. For selective oxidation requiring an orthogonal protecting group, Acm and trityl are used as protecting groups for cysteine. Cleavage removes the protecting group for the cysteine ​​pair, allowing for oxidation of that pair. A second oxidation-deprotection step is then performed on the Acm-protected cysteine. For spontaneous oxidation, a trityl protecting group is used for all cysteine, and the peptide's original folding occurs.

[0311] Those skilled in the art will recognize appropriate techniques that can be used to carry out the oxidation process.

[0312] In certain embodiments, the hepcidin analog of the present invention includes, but is not limited to, a half-life extension portion which may be selected from: Ahx-Palm, PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-lauric acid, isoGlu-mysteric acid, and isoGlu-isovaleric acid.

[0313] In certain embodiments, the hepcidin analog comprises a half-life extension portion having the structure shown below, where n=0 to 24 or n=14 to 24. [ka]

[0314] In certain embodiments, the hepcidin analog of the present invention includes the conjugate half-life extension portion shown in Table 2. [Table 5-1] [Table 5-2]

[0315] In one embodiment, the half-life extension portion is directly conjugated to the hepcidine analog, while in another embodiment, the half-life extension portion is conjugated to the hepcidine analog peptide via a linker portion, such as one of the linkers shown in Table 3. [Table 6-1] [Table 6-2]

[0316] Regarding the linker structures shown in Table 3, the descriptions for n=1 to 24 or n=1 to 25 (e.g., L4 or L5) indicate that n can be any integer within the enumerated range. Additional linker parts that may be used are shown in the "Abbreviations" table.

[0317] In certain embodiments, the hepcidin analog of the present invention comprises one of the linker portions shown in Table 3 and one of the half-life extension portions shown in Table 2, and comprises one of the following combinations shown in Table 4. [Table 7] [Table 8] [Table 9] [Table 10]

[0318] In certain embodiments, the hepcidin analog comprises two or more linkers. In certain embodiments, the two or more linkers are concatenated, that is, they are bound to each other.

[0319] In related embodiments, the present invention includes a polynucleotide encoding a polypeptide having a peptide sequence present in any of the hepcidin analogs described herein.

[0320] Furthermore, the present invention includes vectors containing the polynucleotides of the present invention, such as expression vectors.

[0321] Treatment method In some embodiments, the present invention provides a method for treating a subject suffering from a disease or disorder related to dysregulation of hepcidin signaling, the method comprising administering the hepcidin analog of the present invention to the subject. In some embodiments, the hepcidin analog administered to the subject is present in a composition (e.g., a pharmaceutical composition). In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by increased activity or expression of ferroportin, the method comprising administering the hepcidin analog or composition of the present invention to the subject in an amount sufficient to bind (partially or completely) to ferroportin, agonize it, or mimic hepcidin. In one embodiment, a method is provided for treating a subject suffering from a disease or disorder characterized by dysregulation of iron metabolism, the method comprising administering the hepcidin analog or composition of the present invention to the subject.

[0322] In some embodiments, the method of the present invention includes providing the hepcidin analog or composition of the present invention to a subject in need. In certain embodiments, the subject in need is diagnosed with, or is determined to be at risk of developing, a disease or disorder characterized by dysregulation of iron levels (e.g., diseases or disorders of iron metabolism, diseases or disorders related to iron overload, and diseases or disorders related to abnormal hepcidin activity or expression). In certain embodiments, the subject is a mammal (e.g., human).

[0323] In certain embodiments, the disease or disorder is a disorder of iron metabolism, such as, for example, iron overload disease, iron deficiency disorder, disorder of iron biodistribution, or another disorder of iron metabolism, and other disorders potentially related to iron metabolism. In certain embodiments, diseases of iron metabolism include hemochromatosis, HFE variant hemochromatosis, ferroportin variant hemochromatosis, transferrin receptor 2 variant hemochromatosis, hemomodoverin variant hemochromatosis, hepcidin variant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, fluid-induced iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, beta-thalassemia, sideroblastic anemia, porphyria, late-onset cutaneous porphyria, African-type iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital erythrodysplasia, hypochromic microcytic anemia, sickle cell anemia, polycythemia vera (primary and secondary), secondary These include polycythemia, such as chronic obstructive pulmonary disease (COPD), post-kidney transplantation, Chuvash, HIF, and PHD mutations, as well as idiopathic myelodysplasia, pyruvate kinase deficiency, hypochromic microcytic anemia, fluid-dependent anemia, hemolytic anemia, iron deficiency in obesity, other anemias, benign or malignant tumors that overproduce or induce overproduction of hepcidin, hepcidin overdose, ataxia of Friedreich, Gracil syndrome, Haller-Vorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer (e.g., liver cancer), hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes mellitus, atherosclerosis, neurodegenerative disorders, dementia, multiple sclerosis, Parkinson's disease, Huntington's disease, or Alzheimer's disease.

[0324] In certain embodiments, the disease or disorder is related to iron overload disorders, such as iron hemochromatosis, HFE variant hemochromatosis, ferroportin variant hemochromatosis, transferrin receptor 2 variant hemochromatosis, hemomodoverin variant hemochromatosis, hepcidin variant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, fluid-induced iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sickle cell disease, myelodysplasia, sideroblastic infection, diabetic retinopathy, and pyruvate kinase deficiency.

[0325] In certain embodiments, the disease or disorder is one that is not typically identified as iron-related. For example, since hepcidin is highly expressed in the pancreas of mice, it is suggested that diabetes mellitus (type I or II), insulin resistance, impaired glucose tolerance, and other disorders may be ameliorated by treating underlying iron metabolic disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26. That document is incorporated herein by reference. Thus, the peptides of the present invention may be used to treat these diseases and conditions. Those skilled in the art can easily determine, using methods known in the art, whether a given disease can be treated with the peptides of the present invention, such as the assay of WO2004092405. That patent application is incorporated herein by reference, and the assay monitors the levels and expression of hepcidin, hemoduberin, or iron, and assays such as those described in U.S. Patent No. 7,534,764 are known in the art. The said patent is incorporated herein by reference.

[0326] In certain embodiments, the disease or disorder is postmenopausal osteoporosis.

[0327] In certain embodiments of the present invention, iron metabolism disorders are iron overload disorders, which include hereditary hemochromatosis, iron-overload anemia, alcoholic liver disease, heart disease and / or heart failure, cardiac myopathy, and chronic hepatitis C.

[0328] In certain embodiments, any of these diseases, disorders, or indications are caused by or related to hepcidin deficiency or iron overload.

[0329] In some embodiments, the method of the present invention includes providing a hepcidin analog of the present invention (i.e., a first therapeutic agent) to a subject in combination with a second therapeutic agent. In certain embodiments, the second therapeutic agent is provided to the subject before and / or simultaneously with and / or after the administration of the pharmaceutical composition to the subject. In certain embodiments, the second therapeutic agent is an iron chelating agent. In certain embodiments, the second therapeutic agent is selected from iron chelating agents, deferoxamine, and deferasirox (Exjade®). In other embodiments, the method includes administering a third therapeutic agent to the subject.

[0330] The present invention provides compositions (e.g., pharmaceutical compositions) comprising one or more hepcidin analogs of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent. A pharmaceutically acceptable carrier, diluent, or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Prevention of microbial influence may be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars or sodium chloride, for example.

[0331] The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers. Pharmacochemically acceptable carriers for therapeutic use are publicly known in the pharmaceutical field and are described, for example, in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, sterile saline and phosphate-buffered saline of weak acid 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., as sodium acetate), or mixtures thereof. The term further includes any carriers listed in the United States Pharmacopeia with respect to use in animals, including humans.

[0332] 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.

[0333] To include 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 should be understood to also include the inclusion of pharmaceutically acceptable salts or solvates of the hepcidin analog of the present invention. In certain embodiments, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers, excipients, or vehicles.

[0334] 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 in other literature (see, for example, the treatment methods herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide monomer, or a pharmaceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders otherwise disclosed herein (see, for example, the treatment methods herein). In certain embodiments, the present invention provides pharmaceutical compositions comprising a hepcidin analog peptide dimer, or a pharmaceutically acceptable salt or solvate thereof, for treating various conditions, diseases, or disorders disclosed herein.

[0335] The hepcidin analogs of the present invention may be formulated as pharmaceutical compositions suitable for administration with or without storage, and typically comprise a therapeutically effective amount of at least one hepcidin analog of the present invention, together with a pharmaceutically acceptable carrier, excipient, or vehicle.

[0336] 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 of the active ingredient. The unit dosage form may be presented as a packaged preparation, which is a package containing individual amounts of the preparation, such as packaged tablets, capsules, or powder in vials or ampoules. The unit dosage form may be, for example, a capsule, a cachet, or a tablet itself, or any appropriate number of these packaged forms. The unit dosage form may be provided in a single-dose injectable form, for example, in the form of a pen-type device containing a liquid-phase (typically aqueous) composition. The composition may be formulated for any suitable route and means of administration, for example, any one of the routes and means of administration disclosed herein.

[0337] 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, as used herein, is a matrix made from a material that is typically a polymer, which is enzymatically soluble, or soluble by acid-base hydrolysis, or soluble. Once inserted into the body, the matrix is ​​activated by enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from, for example, liposomes, polylactides (polylactic acid), polyglycolides (polymers of glycolic acid), polylactidecoglycolides (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and biodegradable materials such as silicon. One embodiment of the biodegradable matrix is ​​a matrix of either polylactide, polyglycolide, or polylactide co-glycolide (a copolymer of lactic acid and glycolic acid).

[0338] 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 intravitreal, intranasal, and inhalation delivery as a powder, ointment, infusion, suppository, or transdermal patch). As used herein, the term “parenteral” refers to modes of administration including intravenous, intramuscular, intraperitoneal, substernal, subcutaneous, intradermal, and intra-articular injections and infusions. Thus, in certain embodiments, the composition is formulated for delivery by any of these routes of administration.

[0339] In certain embodiments, the pharmaceutical composition for parenteral injection comprises a pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension, or emulsion, or sterile powder, which is reconstituted 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 (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose, and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained by using a coating substance such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. These compositions may contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Sustained absorption of the injectable pharmaceutical may be achieved by including an absorption-delaying agent such as aluminum monostearate or gelatin.

[0340] Depot formulations for injection are prepared by forming a microencapsulation matrix of hepcidin analogs in one or more biodegradable polymers, such as polyactide-polyglycolide, poly(orthoester), poly(anhydride), and (poly)glycol, such as PEG. The release rate of the hepcidin analog can be controlled depending on the ratio of peptide to polymer and the properties of the specific polymer used. Depot formulations for injection are also prepared by encapsulating the hepcidin analog in liposomes or microemulsions that are compatible with body tissues.

[0341] Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0342] The hepcidin analog of the present invention may be administered in liposomes or other lipid 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 can be used. The liposomal composition of the present invention may contain stabilizers, preservatives, excipients, etc., in addition to the hepcidin analog of the present invention. In certain embodiments, the lipids include phospholipids containing both natural and synthetic phosphatidylcholine (lecithin) and serine. Methods for forming liposomes are known in the art.

[0343] The pharmaceutical compositions used in the present invention, which are suitable for parenteral administration, may generally include sterile aqueous solutions and / or sterile suspensions of peptide inhibitors that have been made isotonic with the recipient's blood using sodium chloride, glycerin, glucose, mannitol, sorbitol, etc.

[0344] In some embodiments, the present invention provides pharmaceutical compositions for oral delivery. The compositions and hepcidin analogs of the present invention may be prepared for oral administration according to any of the methods, techniques, and / or delivery vehicles described herein. Furthermore, those skilled in the art will recognize that the hepcidin analogs of the present invention may be modified or integrated into systems or delivery vehicles that are not disclosed herein but are known in the art and are suitable for use in the oral delivery of peptides.

[0345] In certain embodiments, the oral formulation may include an adjuvant to artificially increase intestinal permeability (e.g., resorcinol and / or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether), and / or an enzyme inhibitor to inhibit enzymatic degradation (e.g., a pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF), or trazilol). In certain embodiments, the hepcidin analog in solid dosage form for oral administration may be mixed with at least one additive such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, or glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens and sorbic acid, antioxidants such as ascorbic acid and alpha-tocopherol and cysteine, disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavorings, or fragrances.

[0346] 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 and non-pharmaceutical components or excipients, as well as other non-reusable materials that may be considered either components or packaging materials. 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, in which case the dosage form comprises at least one of pills, tablets, capsules, gels, pastes, beverages, syrups, ointments, and suppositories. In some examples, an oral dosage form is provided that is designed and configured to delay the release of the hepcidin analog in the small intestine and / or colon of the subject.

[0347] In one embodiment, an oral pharmaceutical composition comprising the hepcidin analog of the present invention comprises an enteric coating designed to delay the release of the hepcidin analog in the small intestine. In at least some embodiments, a pharmaceutical composition is provided comprising the hepcidin analog of the present invention and a protease inhibitor such as aprotinin in a delayed-release pharmaceutical formulation. In some examples, the pharmaceutical composition of the present invention comprises an enteric coating that is soluble in gastric juice at a pH of about 5.0 or higher. In at least one embodiment, a pharmaceutical composition is provided comprising an enteric coating comprising a polymer having a dissociable carboxylic acid group, such as hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate, as well as cellulose derivatives including cellulose and other carbohydrate polymer analogs.

[0348] In one embodiment, a pharmaceutical composition comprising the hepcidin analog of the present invention is provided in an enteric coating, which is designed to protect and release the pharmaceutical composition in a controlled manner within the lower gastrointestinal tract of the target, thereby avoiding systemic side effects. 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 oral drug delivery 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.

[0349] 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 further be formulated to be compatible with use in carrier systems designed to increase solubility and enhance intestinal absorption of peptides. These methods include the use of liposomes, micelles, and nanoparticles to increase peptide permeability through the GI tube.

[0350] Various biological response systems may be combined with one or more hepcidin analogs of the present invention to provide orally delivered agents. In some embodiments, the hepcidin analogs of the present invention are used in combination with biological response 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 orally administered therapeutic agents. Other embodiments include methods for optimizing or extending the drug residence time of the hepcidin analogs disclosed herein, in which case the surface of the hepcidin analog is modified to possess mucosal adhesion via hydrogen bonding, polymers having linked mucins, and / or hydrophobic interactions. These modified peptide molecules may exhibit extended drug residence time in a subject according to the desired features of the present invention. Furthermore, the targeted mucosal adhesion system may specifically bind to receptors on the surface of intestinal cells and M cells, thereby further increasing the uptake of particles containing the hepcidin analog.

[0351] Other embodiments include methods for the oral delivery of the hepcidin analog of the present invention, in which case the hepcidin analog is provided to the subject in combination with a permeabilis enhancer that promotes peptide transport across the intestinal mucosa by increasing paracellular or intercellular permeability. For example, in one embodiment, the permeabilis enhancer is combined with the hepcidin analog, in which case the permeabilis enhancer comprises at least one of long-chain fatty acids, bile salts, amphiphilic surfactants, and chelating agents. In one embodiment, a permeabilis enhancer containing sodium N-[hydroxybenzoyl)amino]caprylate is used to form a weak covalent bond with the hepcidin analog of the present invention, in which case the permeabilis enhancer acts conveniently for membrane transport and further dissociation upon reaching the bloodstream. In another embodiment, the hepcidin analog of the present invention is conjugated with oligoarginine, thereby increasing the cell penetration of the peptide into various cell types. Furthermore, in at least one embodiment, a non-covalent bond is formed between the peptide inhibitor of the present invention and a permeabilis enhancer selected from the group consisting of cyclodextrin (CD) and dendrimers, in which case the permeabilis enhancer reduces peptide aggregation and increases the stability and solubility of the hepcidine analog molecule.

[0352] Other embodiments of the present invention provide methods for treating a subject with a hepcidin analog of the present invention having an extended half-life. In one embodiment, the present invention provides a hepcidin analog having a half-life of at least several hours to one day, which is sufficient for a therapeutically effective dose administered once daily (qd) or twice daily (bid) in vitro or in vivo (e.g., when administered to a human subject). In another embodiment, the hepcidin analog has a half-life of three days or more, which is sufficient for a therapeutically effective dose administered once weekly (qw). In yet another embodiment, the hepcidin analog has a half-life of eight days or more, which is sufficient for a therapeutically effective dose administered every other week (biw) or once a month. In yet another embodiment, the hepcidin analog is derivatized or modified to have a longer half-life compared to an underivated or unmodified hepcidin analog. In yet another embodiment, the hepcidin analog contains one or more chemical modifications that extend the serum half-life.

[0353] When used in at least one of the therapeutic or delivery systems described herein, the hepcidin analog of the present invention may be employed in pure form, or, if such form exists, in the form of a pharmaceutically acceptable salt.

[0354] dose 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 level of a specific therapeutically effective dose 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 composition being employed, c) the patient's age, weight, overall health, sex, and diet, d) the time of administration, route of administration, excretion rate of the particular hepcidin analog being employed, e) duration of treatment, f) drugs used in combination with or concurrently with the particular hepcidin analog being employed, and similar factors known in the pharmaceutical field.

[0355] 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 per day, or 1 to 300 mg / kg body weight per day. In certain embodiments, the dose of the hepcidin analog of the present invention is administered in one or more doses, for example, 1 to 3 doses, 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, in the range of about 0.01 to about 1 mg / kg body weight per day. In certain embodiments, the total dose is, for example, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg to a human patient approximately once or twice a week. In certain embodiments, the total dose is, for example, once a week, in the range of about 1 mg to about 5 mg, or about 1 mg to about 3 mg, or about 2 mg to about 3 mg per human patient.

[0356] In various embodiments, the hepcidin analog of the present invention may be administered continuously (for example, by intravenous administration or another continuous drug administration method), or it may be administered to a subject periodically, typically at regular intervals, depending on the desired dose and pharmaceutical composition selected by those skilled in the art for a particular subject. Examples of regular administration intervals include once a day, twice a day, once every two days, once every three days, once every four days, once every five days, or once every six days, once or twice a week, once or twice a month, and so on.

[0357] Such a regular hepcidin analog administration regimen of the present invention may be beneficial to interrupt for a period of time under certain circumstances, such as during chronic long-term administration, during which the subject receiving the drug treatment reduces the level of the drug or stops taking the drug. This is often referred to as taking a “drug-free period.” Drug-free periods are useful for maintaining or restoring sensitivity to the drug, for example, particularly during long-term chronic treatment, or for reducing undesirable side effects from long-term chronic treatment of the subject with the drug. The timing of the drug-free period depends on the timing of the regular administration regimen and the purpose of taking the drug-free period (e.g., to restore drug sensitivity and / or to reduce undesirable side effects from continuous long-term administration). In some embodiments, the drug-free period may involve reducing the dose of the drug (e.g., reducing the dose to below the therapeutically effective dose over a specific period). In other embodiments, drug administration is suspended for a specific period of time, after which administration is resumed using the same or a different drug regimen (lower or higher dose, and / or higher or lower dosing frequency). Therefore, the drug-free intervals of the present invention can be selected from a wide range of durations and dosing regimens. Exemplary drug-free intervals range from two days or more, one week or more, or one month or more, up to approximately 24 months. Thus, for example, a regular daily dosing regimen using the peptide, peptide analog, or dimer of the present invention may be interrupted by a drug-free interval of, for example, one week, two weeks, or four weeks, after which the previous regular dosing regimen (e.g., a daily or weekly dosing regimen) is resumed. Various other drug-free interval regimens are anticipated to be useful for the administration of the hepcidin analog of the present invention.

[0358] Therefore, the hepcidin analog may be delivered by a dosing regimen, which comprises two or more dosing phases, each separated by a drug-free period phase.

[0359] During each administration phase, the hepcidin analog is administered to the recipient in a therapeutically effective dose according to a predetermined dosing pattern. The dosing pattern may include continuous administration of the drug to the recipient throughout the administration phase. Alternatively, the dosing pattern may include administering multiple doses of the hepcidin analog to the recipient, in which case the doses are separated by dosing intervals.

[0360] The administration pattern may include at least two doses per administration phase, at least five doses per administration phase, at least 10 doses per administration phase, at least 20 doses per administration phase, at least 30 doses per administration phase, or more.

[0361] The administration interval may be a regular administration interval that can be set as described above, and depending on the specific dosage formulation of the hepcidin analog of the present invention, bioavailability, and pharmacokinetic profile, it may be a regular administration interval that includes once a day, twice a day, once every two days, once every three days, once every five days, or once every six days, once or twice a week, once or twice a month, or a regular but less frequent administration interval.

[0362] The administration phase may be at least two days, at least one week, at least two weeks, at least four weeks, at least one month, at least two months, at least three months, at least six months, or longer.

[0363] If the dosing pattern includes multiple doses, the length of the next rest period phase shall be longer than the dosing interval used in that dosing pattern. If the dosing interval is irregular, the length of the rest period phase may be longer than the average interval between dosing during the dosing phase. Alternatively, the length of the rest period may be longer than the longest interval between consecutive dosing during the dosing phase.

[0364] The length of the drug-free period may be at least twice, 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.

[0365] Within these constraints, the drug-free period phase 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, depending on the dosing pattern during the previous dosing phase.

[0366] The administration regimen includes at least two administration phases. Each consecutive administration phase is separated by a rest period phase. Thus, the administration regimen may include at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 administration phases, or more, each phase being separated by a rest period phase.

[0367] The successive administration phases may utilize the same administration pattern, but this is neither necessarily desirable nor required. 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 administered in successive administration phases. In certain embodiments, the recipient is human.

[0368] In some embodiments, the present invention provides compositions and pharmaceuticals comprising at least one hepcidin analog disclosed herein. In some embodiments, the present invention provides a method for producing pharmaceuticals 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 pharmaceuticals comprising at least one hepcidin analog disclosed herein for the treatment of diabetes mellitus (type I or type II), insulin resistance, or glucose intolerance. Methods for treating iron metabolic disorders in subjects, such as mammalian subjects, and preferably human subjects, are also provided, the method 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.

[0369] In some embodiments, the present invention provides a process for producing hepcidin analogs or hepcidin analog compositions (e.g., pharmaceutical compositions) disclosed herein.

[0370] In some embodiments, the present invention provides a device comprising at least one hepcidin analog of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for delivering a hepcidin analog to a target.

[0371] In some embodiments, the present invention provides a method for binding to ferroportin or for inducing the internal migration and degradation of ferroportin, the method comprising contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0372] In some embodiments, the present invention provides a method for binding to ferroportin to block its pores and its external transport function without causing internal migration of ferroportin. The method involves contacting ferroportin with at least one hepcidin analog or hepcidin analog composition disclosed herein.

[0373] 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.

[0374] 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 via an implant or osmotic pump, by a cartridge or micropump, or by other means recognized by those skilled in the art, as known to those skilled in the art. In some embodiments, the present invention provides a complex comprising at least one hepcidin analog disclosed herein, conjugated to ferroportin, preferably human ferroportin, or conjugated to an antibody, such as a hepcidin analog disclosed herein, Hep25, or a combination thereof.

[0375] In some embodiments, the hepcidin analog of the present invention is measured at a value of less than 500 nM (e.g., EC) in an FPN internal transfer assay. 50) has. As will be recognized by those skilled in the art, the function of a hepcidin analog depends on the tertiary structure of the hepcidin analog and the binding site presented. Therefore, it is possible to maintain the function by making minor changes to the sequence encoding the hepcidin analog that do not affect folding or are not on the binding site. 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 that exhibits activity (e.g., hepcidin activity) or alleviates the symptoms of a disease or indication in which hepcidin is involved.

[0376] 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 any hepcidin analog presented herein, or to the amino acid sequence of a peptide conforming to any one of the formulas or hepcidin analogs described herein.

[0377] In some embodiments, the hepcidin analog of the present invention may include a functional fragment or a variant thereof having up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to one or more of the specific peptide analog sequences listed herein.

[0378] In addition to the methods described in the examples herein, the hepcidin analogs of the present invention may be prepared using methods known in the art, including chemical synthesis, biosynthesis, or in vitro synthesis using recombinant DNA methods, and solid-phase synthesis. See, for example, Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol. 12, JK Setlow ed., Plenum Press, NY, pp. 1-19; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:5131-5135; and Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce, Rockford, IL. These documents are incorporated herein by reference in their entirety. The hepcidin analogs of the present invention may be purified using, for example, protein purification techniques known in the art, such as reverse-phase high-performance liquid chromatography (HPLC), ion exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem. 12(16):3121-3126; and Scopes (1982) Protein Purification, Springer-Verlag, NY. Those documents are incorporated herein by reference in their entirety. 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 anticipated herein. In certain preferred embodiments, the polynucleotides are isolated. As used herein, “isolated polynucleotide” means a polynucleotide in an environment different from the environment in which the polynucleotide naturally occurs. [Examples]

[0379] The following examples illustrate specific embodiments of the present invention. Unless otherwise described in detail, the following examples are known to those skilled in the art and were carried out using routine, standard techniques. It should be understood that these examples are for illustrative purposes only and are not intended to be entirely definitive regarding the conditions or scope of the present invention. Therefore, they should never be construed as limiting the scope of the present invention. Abbreviation DCM: Dichloromethane DMF: N,N-dimethylformamide NMP: N-methylpyrrolidone HBTU:O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU:2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate DCC: Dicyclohexylcarbodiimide NHS: N-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)

[0380] K( ): In 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 shall be understood to be a side chain conjugated to the lysine residue. Thus, for example, but not limited to, K-[(PEG8)]- indicates that the PEG8 portion is conjugated to the side chain of the lysine.

[0381] Palm: indicates a conjugation of palmitic acid (palmitoyl).

[0382] Synthesis Protocol-1 Synthesis of peptide monomers The peptide monomers of the present invention were synthesized using the Merrifield solid-phase synthesis method in Protein Technology's Symphony multi-channel synthesizer. The peptides were assembled using HBTU (O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate) and diisopropylethylamine (DIEA) coupling conditions. For some amino acid couplings, PyAOP (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) and DIEA conditions were used. Rink Amide MBHA resin (100-200 mesh, 0.57 mmol / g) was used for peptides with a C-terminal amide, and Wang resin pre-loaded with N-α-Fmoc protected amino acids was used for peptides with a C-terminal acid. Coupling reagents (HBTU and DIEA pre-mixed) were prepared at a concentration of 100 mmol. Similarly, amino acid solutions were prepared at a concentration of 100 mmol. The peptide inhibitors of the present invention were identified and screened based on biochemical optimization and / or phage display, and those exhibiting excellent binding and / or inhibitory properties were identified.

[0383] assembly The peptides were assembled using the standard Symphony protocol. The peptide sequences were assembled as follows: In each reaction vial, the resin (250 mg, 0.14 mmol) was washed twice with 4 ml of DMF, followed by treatment with 2.5 ml of 20% 4-methylpiperidine (Fmoc deprotection) for 10 minutes. The resin was then filtered, washed twice with 4 ml of DMF, and retreated with piperidine for 30 minutes. The resin was washed three more times with 4 ml of DMF, followed by the addition of 2.5 ml of amino acids and 2.5 ml of HBTU-DIEA mixture. After 45 minutes of vigorous stirring, the resin was filtered and washed three times with 4 ml of DMF each. For typical peptides of the present invention, double coupling was performed. After the coupling reaction was complete, the resin was washed three times with 4 ml of DMF each, and then the next amino acid coupling was performed.

[0384] Cutting After the peptide assembly was complete, the peptide was cleaved from the resin by treatment with a cleavage reagent such as Reagent K (82.5% trifluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, 2.5% 1,2-ethanedithiol). The cleavage reagent was able to cleave the peptide from the resin, as well as all remaining side-chain protecting groups.

[0385] The cleaved peptides were precipitated in cold diethyl ether and subsequently washed twice with ethyl ether. The filtrate was poured in, a second aliquot of cold ether was added, and this procedure was repeated. The crude peptides were dissolved in a solution of acetonitrile:water (7:3, containing 1% TFA) and filtered. The quality of the linear peptides was then verified using electrospray ionization mass spectrometry (ESI-MS) (Micromass / Waters ZQ) and subsequently purified.

[0386] purification Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed using a Gemini C18 column (4.6 mm × 250 mm) (Phenomenex). Semiparative reversed-phase HPLC was performed using a Gemini 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of buffer A in B (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at flow rates of 1 mL / min (analytical) and 20 mL / min (preparative). Separation was performed using a linear gradient of buffer A in B (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at flow rates of 1 mL / min (analytical) and 15 mL / min (preparative).

[0387] Synthesis Protocol-1 Synthesis of peptide monomers The peptide monomers of the present invention were synthesized using standard Fmoc solid-phase synthesis techniques in a CEM Liberty Blue® microwave peptide synthesizer. The peptides were assembled using Oxyma / DIC (cyanohydroxyiminoethyl acetate / diisopropylcarbodiimide) under microwave heating. Rink Amide-MBHA resin (100-200 mesh, 0.66 mmol / g) was used for peptides with a C-terminal amide, and Wang resin pre-loaded with N-α-Fmoc protected amino acids was used for peptides with a C-terminal acid. Oxyma was prepared as a 1 M DMF solution containing 0.1 M DIEA. DIC was prepared as a 0.5 M solution in DMF. Amino acids were prepared at 200 mM. The peptide inhibitors of the present invention were identified and screened based on medicinal chemical optimization and / or phage display to identify those with excellent binding and / or inhibitory properties.

[0388] assembly The peptide was prepared using the standard CEM Liberty Blue® protocol. The peptide sequence was assembled as follows: The resin (400 mg, 0.25 mmol) was suspended in 10 ml of 50 / 50 DMF / DCM. The resin was then transferred to a reaction vessel in a microwave cavity. The peptide was assembled by repeating Fmoc deprotection and Oxyma / DIC coupling cycles. For deprotection, a 20% 4-methylpiperidine DMF solution was added to the reaction vessel and heated to 90°C for 65 seconds. The deprotection solution was drained and the resin was washed three times with DMF. For most of the amino acids in the 5 equivalents, Oxyma and DIC were then added to the reaction vessel, and the mixed reaction was rapidly heated at 90°C for 4 minutes by microwave irradiation. For the arginine and histidine residues, racemization was prevented using moderate conditions with temperatures of 75°C and 50°C for 10 minutes. Rare and expensive amino acids were often manually coupled overnight at room temperature using only 1.5–2 equivalents of reagent. Difficult couplings were frequently performed using double coupling at 90°C for 2 × 4 minutes. After coupling, the resin was washed with DMF, and the entire cycle was repeated until the desired peptide assembly was complete.

[0389] Cutting After peptide assembly was complete, the peptide was cleaved from the resin by treating it for 2 hours with a standard 91:5:2:2 TFA / H2O / TIPS / DODT cleavage cocktail. If multiple Arg(Pbf) residues were present, cleavage was performed for an additional hour.

[0390] The cleaved peptides were precipitated in cold diethyl ether. The filtrate was decanted, a second aliquot of cold ether was added, and this procedure was repeated. The quality of the linear peptides was then verified using electrospray ionization mass spectrometry (ESI-MS) (Waters® Micromass® ZQ®), followed by purification.

[0391] purification Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed using a Gemini® C18 column (4.6 mm × 250 mm) (Phenomenex). Half-fill reversed-phase HPLC was performed using a Gemini 10 μm C18 column (22 mm × 250 mm) (Phenomenex), or a Jupiter® 10 μm, 300A. O The analysis was performed using a C18 column (21.2 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of buffer A in buffer B (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at flow rates of 1 mL / min (analytical) and 20 mL / min (preparative).

[0392] Example 1A Synthesis of peptide analogs Unless otherwise specified, the reagents and solvents used below are commercially available in standard laboratory or analytical grade and were used without further purification.

[0393] Procedure for solid-phase synthesis of peptides Method A The peptide analogs of the present invention were chemically synthesized using an optimized 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol. For the C-terminal amide, rink-amide resin was used, but wang resin and trityl resin 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 the formation of selective disulfide crosslinks, Acm (acetamidomethyl) was also used as a Cys protecting group. For coupling, a 4-10-fold excess of a (1:1:1.1) solution containing Fmoc amino acids, HBTU, and DIEA was added to the swelling resin [HBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA: diisopropylethylamine; DMF: dimethylformamide]. Instead of HBTU, HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate) was used to improve coupling efficiency in difficult areas. Removal of the Fmoc protecting group was performed by treatment with a DMF, piperidine (2:1) solution.

[0394] Method B : Alternatively, peptides were synthesized using a CEM Liberty Blue Microwave Auxiliary Peptide Synthesizer. FMOC deprotection was performed using Liberty Blue by adding a 20% 4-methylpiperzine DMF solution containing 0.1 M Oxyma in DMF solution, followed by heating to 90°C for 4 minutes using microwave irradiation. After DMF washing, the FMOC-amino acids were coupled by adding 0.2 M amino acids (4-6 equivalents), 0.5 M DIC (4-6 equivalents), and 1 M Oxyma (containing 0.1 M DIEA) (4-6 equivalents, all in DMF solution). The coupling solution was heated to 90°C for 4 minutes using microwave radiation. A second coupling was performed when coupling with Arg or other sterically hindered amino acids. For coupling with histidine, the reaction product was heated to 50°C for 10 minutes. The cycle was repeated until a full-length peptide was obtained.

[0395] Procedure for separating peptides from resin Deprotection and cleavage of the side chains of the peptide analogs of the present invention (e.g., compound number 2) were performed by stirring the dry resin for 2-4 hours in a solution containing trifluoroacetic acid, water, ethanedithiol, and tri-isopropylsilane (90:5:2.5:2.5). After TFA removal, the peptides were precipitated using ice-cold diethyl ether. The solution was centrifuged to decant the ether, followed by a second wash with diethyl ether. The peptides were dissolved in a 1:1 solution of acetonitrile and water containing 0.1% TFA (trifluoroacetic acid), and the resulting solution was filtered. The quality of the linear peptides was evaluated using electrospray ionization mass spectrometry (ESI-MS).

[0396] Peptide purification procedure The peptide of the present invention (e.g., compound number 2) was purified using reversed-phase high-performance liquid chromatography (RP-HPLC). Analysis was performed using a C18 column (3 μm, 50 × 2 mm) at a flow rate of 1 mL / min. Linear peptide purification was performed using preparative RP-HPLC with a C18 column (5 μm, 250 × 21.2 mm) at a flow rate of 20 mL / min. Separation was performed using a linear gradient of buffer B in buffer A (buffer A: 0.05% TFA aqueous solution, buffer B: 0.043% TFA, 90% acetonitrile aqueous solution).

[0397] Those skilled in the art will understand that the compounds of the present invention can be produced using standard methods of peptide synthesis.

[0398] Conjugate of the half-life extension portion Peptide conjugation was performed on resin. Lys(ivDde) ​​was used as the major amino acid. After peptide assembly on resin, selective deprotection of the ivDde group was performed for 5 minutes using a 2% hydrazine DMF solution (3 × 5). Linker activation and acylation were performed for 3 hours using 1-2 equivalents of HBTU and DIEA, followed by Fmoc removal and a second acylation with lipid acids to obtain the conjugated peptide. Example 1B Peptide synthesis: Isovaleric acid-Glu-Thr-His-DIP-Pro-Ala-Ile-Lys(Ahx-Palm)-bhF-NH2 (peptide number 9)

[0399] The TFA salt of peptide number 9 was synthesized on a 0.13 mmol scale. Upon completion, 45.31 mg of peptide number 9 with a purity of over 95% was isolated as a white powder. The overall yield was 21.5%.

[0400] Peptide number 9 was synthesized using the Merrifield solid-phase synthesis method on Protein Technology's Symphony multi-channel synthesizer and constructed on Rink Amide MBHA (100-200 mesh, 0.66 mmol / g) resin under standard Fmoc protection synthesis conditions. The constructed peptide was isolated from the resin and protecting groups by cleavage with a strong acid followed by precipitation. The crude precipitate was then purified by RP-HPLC. The final product, peptide number 9, was obtained by lyophilization of the pure fraction.

[0401] Peptide assembly Swelling resin: 200 mg of Rink Amide MBHA solid-phase resin (0.66 mmol / g loading) was transferred to a 25 mL reaction vessel (for Symphony peptide synthesizer). The resin was swelled with 3.75 mL of DMF (3 × 10 mins).

[0402] Step 1: Coupling of FMOC-β-homo-L-Phe-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-β-homo-L-Phe-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0403] Step 2: Coupling of FMOC-L-Lys(IvDde)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Lys(IvDde)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0404] Step 3: Coupling of FMOC-L-Dpa-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Ile-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0405] Step 4: Coupling of FMOC-L-Ala-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Ala-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0406] Step 5: Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-Pro-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0407] Step 6: Coupling of FMOC-L-DIP-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-DIP-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0408] Step 7: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-His(Trt)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0409] Step 8: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Thr(tBu)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0410] Step 9: Coupling of FMOC-L-Glu(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Glu(tBu)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0411] Step 10: Isovaleric Acid Coupling: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of isovaleric acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, a DMF mixed solution of HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0412] Step 11: Removal of IvDde from Fmoc-Ahx-OH and coupling: IvDde was removed from the Lys C-terminus of the resin-bound peptide using a 2-5% hydrazine DMF solution (4 × 30 min), followed by DMF washing. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of amino acid Fmoc-Ahx-OH DMF solution (200 mM) and 2.0 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was initiated.

[0413] Step 12: Palmitic acid coupling: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of isovaleric acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0414] Step 13: TFA cleavage and ether precipitation: 10 ml of the cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT)] was added to the protected resin-bound peptide and shaken for 2 hours. Cold diethyl ether was added to form a white precipitate, which was then centrifuged. The ether was decanted and discarded, and the precipitate was washed with ether two more times. The resulting white precipitate cake was dissolved in acetonitrile / water (7:3), filtered, and then purified.

[0415] Step 14: RP-HPLC purification: Separatory reverse-phase HPLC was performed using a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of buffer A in buffer B (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at a flow rate of 20 mL / min (preparative).

[0416] Step 15: Final Lyophilization and Analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions with a purity exceeding 95% were combined. The combined fraction was lyophilized to obtain peptide number 9 as a white powder with a purity of 97%. Low-resolution LC / MS of the purified peptide number 9 yielded the peptide's 1-charge state, 807.70 M+2 / 2, and 1613.80 molecular ion [M+1]. The experimental mass was consistent with the theoretical mass of 1614.0 Da[M+1]. Example 1C Peptide synthesis: Isovaleric acid-Glu-Thr-His-Dpa-Pro-Ala-Ile-(D)Lys-bhF-Lys(Ahx-Palm)-NH2 (peptide number 4)

[0417] The TFA salt of peptide number 4 was synthesized on a 0.13 mmol scale. Upon completion, 27.74 mg of peptide number 4 with a purity of over 95% was isolated as a white powder. The overall yield was 12.2%.

[0418] Peptide number 4 was synthesized using Merrifield solid-phase synthesis on Protein Technology's Symphony multi-channel synthesizer and constructed on Rink Amide MBHA (100-200 mesh, 0.66 mmol / g) resin under standard Fmoc protection synthesis conditions. The constructed peptide was isolated from the resin and protecting groups by cleavage with a strong acid followed by precipitation. The crude precipitate was then purified by RP-HPLC. The final product, peptide number 4, was obtained by lyophilization of the pure fraction.

[0419] Peptide assembly Swelling resin: 200 mg of Rink Amide MBHA solid-phase resin (0.66 mmol / g loading) was transferred to a 25 mL reaction vessel (for Symphony peptide synthesizer). The resin was swelled with 3.75 mL of DMF (3 × 10 mins).

[0420] Step 1: Coupling of FMOC-L-Lys(IvDde)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Lys(IvDde)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0421] Step 2: Coupling of FMOC-βhomo-L-Phe-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-βhomo-L-Phe-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0422] Step 3: Coupling of FMOC-D-Lys(Boc)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-D-Lys(Boc)-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0423] Step 4: Coupling of FMOC-L-Ile-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Ile-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0424] Step 5: Coupling of FMOC-L-Ala-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Ala-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0425] Step 6: Coupling of FMOC-Pro-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-Pro-OH amino acid DMF solution (200 mM) and 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0426] Step 7: Coupling of FMOC-L-Dpa-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-DIP-OH amino acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0427] Step 8: Coupling of FMOC-L-His(Trt)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-His(Trt)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0428] Step 9: Coupling of FMOC-L-Thr(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Thr(tBu)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0429] Step 10: Coupling of FMOC-L-Glu(tBu)-OH: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of FMOC-L-Glu(tBu)-OH DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0430] Step 11: Isovaleric Acid Coupling: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of isovaleric acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, a DMF mixed solution of HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0431] Step 12: Removal of IvDde from Fmoc-Ahx-OH and coupling: IvDde was removed from the Lys C-terminus of the resin-bound peptide using a 2-5% hydrazine DMF solution (4 × 30 min), followed by DMF washing. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of a 200 mM DMF solution of the amino acid Fmoc-Ahx-OH, and then 2.5 mL of a DMF mixture of the coupling reagent HBTU-DIEA (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was initiated.

[0432] Step 13: Palmitic acid coupling: Deprotection of the Fmoc group was performed by treating the swollen Rink Amide resin twice with 2.5 ml of 20% piperidine DMF solution for 5 minutes and 10 minutes, respectively. After deprotection, the resin was washed with 3.75 mL of DMF (3 × 0.1 min), followed by the addition of 2.5 mL of isovaleric acid DMF solution (200 mM), and then 2.5 mL of the coupling reagent, HBTU-DIEA DMF mixed solution (200 and 220 mM). The coupling reaction was mixed for 1 hour, filtered, and repeated once (double coupling). After the coupling reaction was complete, the resin was washed with 6.25 mL of DMF (3 × 0.1 min), and then the next deprotection / coupling cycle was started.

[0433] Step 14: TFA cleavage and ether precipitation: 10 ml of the cleavage cocktail [TFA cleavage cocktail (90 / 5 / 2.5 / 2.5 TFA / water / Tips / DODT)] was added to the protected resin-bound peptide and shaken for 2 hours. Cold diethyl ether was added to form a white precipitate, which was then centrifuged. The ether was decanted and discarded, and the precipitate was washed with ether two more times. The resulting white precipitate cake was dissolved in acetonitrile / water (7:3), filtered, and then purified.

[0434] Step 15: RP-HPLC purification: Separatory reverse-phase HPLC was performed using a Gemini® 10 μm C18 column (22 mm × 250 mm) (Phenomenex). Separation was performed using a linear gradient of buffer A in buffer B (mobile phase A: water containing 0.15% TFA, mobile phase B: acetonitrile (ACN) containing 0.1% TFA) at a flow rate of 20 mL / min (preparative).

[0435] Step 16: Final Lyophilization and Analysis: The collected fractions were analyzed by analytical RP-HPLC, and all fractions with a purity exceeding 95% were combined. The combined fraction was lyophilized to obtain peptide number 4 as a white powder with a purity of 97%. Low-resolution LC / MS of the purified peptide number 4 yielded the peptide's two charge states: 581.5 M+3 / 3, 871.70 M+2 / 2, and 1741.90[M+1] molecular ions. The experimental mass was consistent with the theoretical mass of 1742.09 Da[M+1]. Example 2A: Activity of peptide analogs

[0436] Peptide analogs were tested in vitro to induce internal translocation of human ferroportin protein. After internal translocation, the ferroportin protein is degraded. The assay used (FPN activity assay) measures the decrease in receptor fluorescence.

[0437] The cDNA encoding human ferroportin (SLC40A1) was cloned from an Origene (NM_014585) cDNA clone. The ferroportin-encoding DNA was amplified by PCR using primers that encoded the terminal restriction site for subcloning but did not contain a stop codon. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker, and the ferroportin reading frame was fused in-frame with the GFP protein. 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 the plasmid was transfected using lipofectamine (manufacturer's protocol, Invitrogen). Cells stably expressing ferroportin-GFP were selected in growth medium using G418 (only cells that incorporated and integrated the cDNA expression plasmid survived), and sorted several times using a Cytomation MoFlo® cell sorter to obtain GFP-positive cells (488nm / 530nm). The cells were grown and frozen in dispensed portions.

[0438] To determine the activity of hepcidin analogs (compounds) against human ferroportin, cells were incubated in 96-well plates in standard medium without phenol red. The compound was added to the desired final concentration for at least 18 hours during incubation. After incubation, residual GFP fluorescence was determined by either whole-cell GFP fluorescence (Envision plate reader, 485 / 535 filter pair) or Beckman Coulter Quanta® flow cytometer (expressed as the geometric mean of fluorescence intensity at 485 nm / 525 nm). The compound was added to the desired final concentration for at least 18 hours, but no more than 24 hours, during incubation.

[0439] 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 retro inverse peptide is a peptide that has the reverse sequence in all D amino acids. For example, Hy-Glu-Thr-His-NH2 becomes Hy-DHis-DThr-DGlu-NH2. The EC of these reference compounds regarding ferroportin internal translocation / degradation... 50 These were determined according to the FPN activity assay described above. These peptides were used as control standards. [Table 11] The EC of various peptide analogs of the present invention has been determined. 50 Potency values ​​(nM) are provided in Tables 6A, 6B, and 6C. These values ​​were determined as described herein. Compound ID numbers are indicated by "Compound ID" and reference compounds by "Reference Compound". The FPN EC determined from these data 50 The values ​​are shown in Tables 6A, 6B, and 6C. T47D(MSA)IC 50 The values ​​are shown in Table 6D. If a value is not shown, the data has not yet been determined. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Table 13] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8] [Table 15-1] [Table 15-2]

[0440] Example 2C: Activity of peptide analogs The efficacy of peptides that induce the internal translocation of ferroportin was evaluated using a T47D cell-based assay. The T47D cell line (HTB 133, ATCC) is an adherent cell line of human breast cancer that endogenously expresses ferroportin. In this internal translocation assay, the efficacy of the test peptide was evaluated in the presence of serum albumin, a major protein component in the blood. T47D cells were maintained in RPMI medium (containing the required amount of fetal bovine serum) and periodically passaged. For assay preparation, cells were seeded in 96-well plates at a density of 80-100k cells per well in 100 μl volume and left to stand overnight. The following day, the test peptides were first prepared in a dilution series (10-point series, starting at a concentration of approximately 5 μM, typically 3-4 times dilution), all containing 0.5% mouse serum albumin (MSA purified from mouse serum; Sigma, A3139). A dilution series of the test peptide was incubated at room temperature for 30 minutes. The culture medium was then aspirated from a 96-cell well plate and the test peptide series was added. After 1 hour of incubation, the culture medium containing the test peptide was aspirated and the AF647-conjugate detection peptide was added at a fixed concentration of 200 nM. The AF647-conjugate detection peptide has already been demonstrated to bind to ferroportin and induce its internal translocation. After 2 hours of incubation, the cells were washed again to prepare for flow cytometry analysis. The median fluorescence intensity (MFI) of the AF647-positive population was measured (after removing dead cells and non-single cells from the analyte). The MFI values ​​were used to construct dose-response curves and obtain the IC50 values ​​of the test peptide. The IC50 values ​​were calculated using a 4-parameter nonlinear fitting function in Graphpad Prism (Table 6D). [Table 16-1] [Table 16-2]

[0441] Example 2D LAD2 activity of peptide analogs In anaphylactic-like reactions, the main mechanism involves the release of anaphylaxis mediators, such as histamine and β-hexosaminidase, due to direct stimulation of mast cells or basophils. Recent research by McNeil et al. (McNeil BD et al., 2015) has reported that MrgprX2, a specific membrane receptor on human mast cells, induces anaphylactic-like reactions. LAD2 (Laboratory of Allergic Diseases 2) is a human mast cell line derived from human mast cell sarcoma / leukemia (Kirshenbaum et al., 2003), and is frequently used in studies of anaphylactic-like reactions because it shares the same biological characteristics as primary human mast cells, including overexpression of the MrgprX2 receptor and sensitivity to degranulation peptides (Kulka et al., 2008). For example, the release of anaphylaxis mediators such as β-hexosaminidase is evaluated by fluorescence quantification.

[0442] The degranulation ability of hepcidin mimetic compounds was evaluated in LAD2 cells. On the day of the assay, serial dilutions of the compound were added to LAD2 cells seeded at 20,000 cells / well in 96-well plates. After 30 minutes of incubation, the amount of β-hexosaminidase released into the supernatant and in the cell lysates was quantified using the fluorescent substrate 4-methylumbelliferyl-N-acetyl-bD-glucosaminide. Dose-response curves were created by plotting the percentage (y-axis) of β-hexosaminidase release against the concentration (x-axis) of the test peptide. 50 The values ​​and standard errors were calculated using XLfit 5.5.0.5 based on the following formula: 4-parameter sigmoid model: f=(A+((BA) / (1+((C / x)^D)))), where A=Emin, B=Emax, C=EC50, and D=slope. References: McNeil BD et al., Nature, 12, 519 (2015); Kirshenbaum et al. Leukemia Res. 27, 677 (2003); Kulka et al. Immunology 123, 398 (2008).

[0443] Example 3 In vivo validation of peptide analogs The hepcidin analog of the present invention was tested for in vivo activity, and its ability to reduce free Fe2+ in serum was determined.

[0444] Hepcidin analogs or vehicle controls were administered intravenously or subcutaneously to mice (n=3 mice / group) at a concentration of 1000 nmol / kg. Serum samples were collected from the hepcidin analog-treated mouse group at 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 30 hours, 36 hours, and 48 hours after administration. Iron content in plasma / serum was measured using colorimetric analysis with Cobas c 111, according to the instructions of the assay manufacturer (Assay: IRON2: ACN 661).

[0445] In another experiment, various hepcidin analogs or vehicle controls were subcutaneously administered to mice (n=3 mice / group) at a dose of 1000 nmol / kg. Serum samples were collected from the groups of mice that received either the vehicle or the hepcidin analog 30 and 36 hours after administration. Iron content in plasma / serum was measured using colorimetric analysis with Cobas c 111, according to the instructions of the assay manufacturer (Assay: IRON2: ACN 661).

[0446] These studies have shown that the hepcidin analog of the present invention reduces serum iron levels for at least 30 hours, thus demonstrating increased serum stability.

[0447] Example 4 In vitro validation of peptide analogs Based in part on the structure-activity relationship (SAR) determined from the experimental results described herein, various hepcidin-like peptides of the present invention were synthesized using the method described in Example 1, and their in vitro activity was verified as described in Example 2. Reference compounds included natural hepcidin, minihepcidin, R1-minihepcidin, reference compound 1, and reference compound 2. (EC of the peptide) 50 The values ​​are shown in Tables 6A-C of the summary.

[0448] Example 5 plasma stability To complement in vivo results and aid in the design of potent and stable ferroportin agonists, plasma stability experiments were conducted. In vitro stability tests were initially performed on rat and mouse plasma to predict stability in these substances.

[0449] The target peptide (20 μM) was incubated with plasma (BioreclamationIVT) preheated to 37°C. Dispenses were taken at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, including a 1 μM internal standard). Quenched samples were stored at 4°C until the end of the experiment and centrifuged at 17,000 g for 15 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The residual percentage at each time point was calculated based on the peak area ratio (analyte relative to the internal standard) compared to the initial value at zero. The half-life was calculated by fitting a first-order exponential decay equation using GraphPad.

[0450] Example 6 Decreased serum iron in mice A hepcidin mimetic compound designed for oral stability was tested for systemic absorption via PO administration in a wild-type mouse model C57BL / 6. Animals were acclimatized to a standard rodent diet for 4-5 days prior to the start of the study and fasted overnight. Four animal groups were administered either the vehicle or the compound. The compound was formulated in saline at a concentration of 5 mg / mL. Mice were administered the solution by forced oral administration at a volume of 200 μl per 20 g animal. Each group received one dose of the compound at 50 mg / kg / dose. Groups labeled "vehicle" received only the formulation. Blood was collected 4 hours after administration, and serum was prepared for PK and PD measurements. Compound concentrations were measured by mass spectrometry, and iron concentrations in the samples were measured using colorimetric analysis on a Roche-cobas c system.

[0451] Example 7 Decreased serum iron in mice In another experiment, a new set of compounds was tested for systemic absorption via polyoxygenated solution (PO) administration in the wild-type mouse model C57BL / 6. The animals were acclimatized to a standard rodent diet for 4-5 days prior to the start of the study. The night before the first dose, the mice were switched to a low-iron diet (2 ppm iron), which was maintained for the remainder of the study. Each of the five animal groups received either the vehicle or the compound. The compounds were formulated at a concentration of 30 mg / mL in 0.7% NaCl + 10 mM sodium acetate buffer. Food was collected approximately 2 hours before each dose to ensure there was no food residue in the stomach before PO administration. The solution was administered to the mice by forced oral administration at a volume of 200 μl per 20 g animal. Each group received two doses of the compound at 300 mg / kg / dose, on consecutive days. Groups labeled "vehicle" received only the formulation. Blood was collected 4.5 hours after the last dose, and serum was prepared for PD measurement. Serum iron concentration was measured using colorimetric analysis on a Roche-Cobas c system.

[0452] Example 8 Pharmacodynamic effects of representative compounds on their ability to reduce serum iron in mice. In the second in vivo trial, representative compounds were tested for their pharmacodynamic effects, comparing a single dose of 300 mg / kg with two doses of 300 mg / kg once daily over two days (QD). C57BL / 6 mice were acclimatized to a normal rodent diet for 4-5 days prior to the start of the trial. The night before the first dose, the mice were switched to a low-iron diet (2 ppm iron), which was maintained for the remainder of the trial. Each of the five animal groups received either the vehicle or the compound. The compounds were formulated at a concentration of 30 mg / mL in 0.7% NaCl + 10 mM sodium acetate buffer. Food was collected approximately 2 hours before each dose to ensure that there was no food residue in the stomach before PO administration. The solution was administered to the mice by forced oral administration at a volume of 200 μl per 20 g animal.

[0453] Example 9 PK / PD effect in mice upon oral administration of a representative compound of the present invention. In a separate in vivo study using a healthy wild-type mouse model C57 / BL6, representative compounds were administered multiple times over three days to test their PK and PD effects. During acclimatization, mice were maintained on a normal rodent diet and switched to an iron-deficient diet (containing approximately 2 ppm iron) overnight before the first dose. Each of the five mouse groups received a total of six doses over three days, either the representative compound of the present invention or a vehicle at varying dose intensities. Mice were force-fed the representative compound, formulated in 0.7% saline and 10 mM sodium acetate, orally. Each group received either the vehicle, a BID of 150 mg / kg / dose, a BID of 75 mg / kg / dose, a BID of 37.5 mg / kg / dose, or a BID of 18.75 mg / kg / dose. The additional group received 100 mg / kg / day of BID in addition to a total of 100 mg / kg / day of compounds in drinking water (DW). This resulted in a total dose of 300 g / kg / day. Three hours after the final dose, the vehicle group that received iron and all compound-administered groups were force-administered an iron solution containing 4 mg / kg of iron per animal. Blood was collected 90 minutes after iron administration to prepare serum for PK and PD measurements. Compound concentrations were measured by mass spectrometry, and iron concentrations in the samples were measured using colorimetric analysis on a Roche-Cobas c system.

[0454] Example 10 Decreased serum iron in mice In a separate selection process, a new set of compounds was tested for their pharmacodynamic effects when orally administered to wild-type mouse model C57BL / 6. The animals were acclimatized to a normal rodent diet for 4-5 days prior to the start of the study. Five animal groups, designated to receive two doses of a representative compound, were fed an iron-deficient diet (containing 2-ppm iron) the night before the first dose. All other groups, designated to receive a single dose of a different compound, were treated with an iron-deficient diet for two nights prior to compound administration. The compounds were formulated in 0.7% NaCl + 10mM sodium acetate buffer at a concentration of 30 mg / mL in the administration solution. Food was collected approximately 2 hours before each administration to ensure that there was no food residue in the stomach before PO administration. The solution was administered to mice by forced oral administration at a volume of 200 μl per 20 g animal. Groups labeled "vehicle" received only the formulation. Blood was collected 4.5 hours after the last dose, and serum was prepared for PD measurement. Serum iron concentration was measured using colorimetric analysis on a Roche-Cobas c system.

[0455] Example 11 Stability in simulated gastric juice Blank SGF was prepared by adding 2 g of sodium chloride and 7 mL of hydrochloric acid (37%) to a final volume of 1 L of water and adjusting the pH to 1.2.

[0456] SGF was prepared by dissolving 320 mg of pepsin (Sigma®, P6887, derived from porcine gastric mucosa) in 100 mL of blank SGF and stirring at room temperature for 30 minutes. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at -20°C.

[0457] The experimental compound (at a concentration of 20 μM) was incubated with SGF preheated to 37°C. Dispenses were taken at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, including a 1 μM internal standard). Quenched samples were stored at 4°C until the end of the experiment and centrifuged at 4,000 rpm for 10 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The residual percentage at each time point was calculated based on the peak area ratio (analyte relative to the internal standard) compared to the initial value at zero. The half-life was calculated by fitting a first-order exponential decay equation using GraphPad.

[0458] Example 12 Stability in artificial intestinal fluid A blank FaSSIF was prepared by dissolving 0.348 g of NaOH, 3.954 g of sodium phosphate monobasic monohydrate, and 6.186 g of NaCl in 1 liter of water (adjusting the pH to 6.5).

[0459] FaSSIF was prepared by dissolving 1.2 g of porcine pancreatin (Chem-supply, PL378) in 100 mL of blank FaSSIF and stirring at room temperature for 30 minutes. The solution was filtered through a 0.45 μm membrane, aliquoted, and stored at -20°C.

[0460] The experimental compound (20 μM) was incubated with FaSSIF (1% pancreatin in the final incubation mixture) preheated to 37°C. Dispenses were taken at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with four volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, including a 1 μM internal standard). Quenched samples were stored at 4°C until the end of the experiment and centrifuged at 4,000 rpm for 10 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The residual percentage at each time point was calculated based on the peak area ratio (analyte relative to the internal standard) compared to the initial value at zero. The half-life was calculated by fitting a first-order exponential decay equation using GraphPad.

[0461] Example 13 Modification experiments on peptides that tend to "non-specifically bind". The target compound (at a concentration of 20 μM) was mixed with pre-warmed FaSSIF (1% pancreatin in the final working solution). The solution mixture was dispensed and incubated at 37°C. The number of required dispensings was the same as the number of time points (e.g., 0, 0.25, 1, 3, 6, and 24 hours). One dispensing sample was taken at each time point and immediately quenched with 4 volumes of organic solvent (acetonitrile / methanol (1:1) and 0.1% formic acid, containing a 1 μM internal standard). The remaining steps were the same as in the comprehensive experiment.

[0462] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application data sheets are incorporated herein by reference in their entirety.

[0463] At least some of the chemical names and sequences of the present invention presented and described herein may have been automatically generated using commercially available chemical naming software programs and have not been independently verified. In the event of any discrepancy between a shown chemical name or sequence and an illustrated structure, the illustrated structure shall prevail. In chemical structures where a chiral center is present but no specific stereochemistry is shown for the chiral center, both enantiomers associated with the chiral structure are included in the structure. Similarly, for peptides where E / Z isomers exist but are not specifically mentioned, both isomers shall be specifically disclosed and covered.

[0464] As stated above, specific embodiments of the present invention are described herein for illustrative purposes, but it will be understood that various modifications can be made without departing from the spirit and scope of the invention.

Claims

[Claim 1] A hepcidin analog, or a pharmaceutically acceptable salt or solvate thereof, wherein the hepcidin analog is ID number 321 【Chemistry 24】 ； ID number 319 【Chemistry 25】 ； ID number 322 【Chemistry 26】 ； ID number 318 【Chemistry 27】 ； ID number 320 【Chemistry 28】 ； ID number 56 【Chemistry 29】 ； ID number 286 【Transformation 30】 ； ID number 58 【Chemistry 31】 ； ID number 287 【Chemistry 32】 ； ID number 156 【Transformation 33】 ,or ID number 292 【Transformation 34】 ； A peptide, a hepcidin analog, or a pharmaceutically acceptable salt or solvate thereof.

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