CN117813313A

CN117813313A - Binding hepcidin mimetics

Info

Publication number: CN117813313A
Application number: CN202280037123.5A
Authority: CN
Inventors: G·T·伯恩; A·班达里
Original assignee: Protagonist Therapeutics Inc
Current assignee: Protagonist Therapeutics Inc
Priority date: 2021-04-01
Filing date: 2022-03-31
Publication date: 2024-04-02

Abstract

The present invention relates, inter alia, to certain hepcidin peptide analogs, including both peptide monomers and peptide dimers, and conjugates and derivatives thereof, as well as compositions comprising the peptide analogs, and to the use of peptide analogs in the treatment and/or prevention of a variety of diseases, conditions or disorders, including in the treatment and/or prevention of erythrocyte-excess (erythrocyte) diseases such as polycythemia vera, iron overload diseases such as hereditary hemochromatosis (hereditary hemochromatosis), iron-excess anemia (iron-loading anemia), and other conditions and disorders described herein.

Description

Binding hepcidin mimetics

Cross reference to related applications

The application claims the following benefits and preferences: U.S. provisional application No. 63/169,545 filed on 1, 4, 2021 and U.S. provisional application No. 63/325,328 filed on 30, 3, 2022, each of which are incorporated herein by reference in their entirety.

Technical Field

Background

Hepcidin (also known as LEAP-1), a peptide hormone produced by the liver, is a regulator of iron balance in humans and other mammals. Hepcidin acts by binding to its receptor (iron export channel membrane iron transporter), leading to its internalization and degradation. Human hepcidin is a 25 amino acid peptide (Hep 25). See Krause et al (2000) European society of Biochemical society of Association flash (FEBS Lett) 480:147-150 and Park et al (2001) journal of biochemistry (J Biol Chem) 276:7806-7810. The biologically active 25 amino acid form of hepcidin structure is a simple hairpin structure with 8 cysteines forming 4 disulfide bonds as described by Jordan et al J.Biochem.284:24155-67. The N-terminal region is essential for iron regulatory function, and deletion of 5N-terminal amino acid residues results in loss of iron regulatory function. See Nemeth et al (2006) Blood (Blood) 107:328-33.

Abnormal hepcidin activity is associated with iron overload diseases, including Hereditary Hemochromatosis (HH) and iron-excess type anemia. Hereditary hemochromatosis is a hereditary iron overload disease, mainly caused by hepcidin deficiency or in some cases by resistance to iron regulation. This can lead to excessive absorption of iron from the diet and cause iron overload. Clinical manifestations of HH may include liver diseases (e.g., cirrhosis NASH and hepatocellular carcinoma), diabetes, and heart failure. Currently, the only treatment for HH is periodic exsanguination, which can place a significant burden on the patient. Iron-excess anemia is a genetic anemia of ineffective erythropoiesis, such as beta thalassemia, accompanied by severe iron overload. Complications of iron overload are a major cause of morbidity and mortality in these patients. Hepcidin deficiency is a major cause of iron overload in non-transfusional patients and causes iron overload in transfusional patients. Current treatment for iron overload in these patients is iron rejection therapy (iron therapy), which is very burdensome, sometimes ineffective, and often accompanied by side effects.

Hepcidin has several limitations that limit its use as a drug, including difficult synthetic processes, in part because proteins aggregate and precipitate during folding, which in turn can lead to low bioavailability, injection site reactions, immunogenicity, and high commodity costs. There is a need in the art for compounds that have hepcidin activity and also have other advantageous physical properties (such as improved solubility, stability and/or potency) such that hepcidin-based compounds can be economically produced and used to treat hepcidin-related diseases and conditions such as those described herein.

The present invention addresses such a need by providing novel peptide analogs, including both peptide monomer analogs and peptide dimer analogs, which possess hepcidin activity and which also possess other beneficial properties, making the peptides of the invention suitable alternatives to hepcidin.

Disclosure of Invention

The present invention relates generally to peptide analogs (including both monomers and dimers) that exhibit hepcidin activity and methods of use thereof.

In one aspect, the invention comprises 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 (I)

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

R ¹ is hydrogen, C ₁ -C ₆ Alkyl, C ₆ -C ₁₂ Aryl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ Alkyl, C ₁ -C ₂₀ Alkanoyl, or C ₁ -C ₂₀ A cycloalkanoyl group;

R ² is NH ₂ Substituted amino, OH, or substituted hydroxy;

x1 is absent, or Asp, isoAsp, asp (OMe), glu, bhGlu, bGlu, gly, N substituted Gly, gla, glp, ala, arg, dab, leu, lys, dap, orn, (D) Asp, (D) Arg, tet1 or Tet2, lys, substituted Lys, (D) Lys, or substituted (D) Lys;

x2 is Ala, t-BuAla, thr, substituted Thr, gly, N substituted Gly, or Ser;

x3 is Ala, t-BuAla, gly, N substituted Gly, his, or substituted His;

x4 is Ala, t-BuAla, phe, dpa, gly, N substituted Gly, bhPhe, a-MePhe, NMe-Phe, D-Phe, or 2Pal;

x5 is Ala, t-BuAla, pro, D-Pro, bhPro, D-bhPro, NPC, D-NPC, gaba, 2-pyrrolidinopropionic acid (Ppa), 2-pyrrolidinobutyric acid (Pba), glu, lys, substituted Lys, (D) Lys, or substituted (D) Lys;

x6 is absent or any amino acid other than Cys, (D) Cys, aMeCys, hCys or Pen;

x7 is absent, or is Ala, t-BuAla, gly, N substituted Gly, ile, val, leu, NLeu, lys, substituted Lys, (D) Lys, or substituted (D) Lys;

X8 is absent, or is Ala, t-BuAla, (D) Ala, ile, gly, N substituted Gly, glu, val, leu, NLeu, phe, bhPhe, lys, substituted Lys, (D) Lys, substituted (D) Lys, aMeLys, or 123 triazole;

x9 is absent, or is Ala, ile, gly, N substituted Gly, val, leu, NLeu, phe, bhPhe, lys, substituted Lys, (D) Lys, or substituted (D) Lys;

x10 is absent, or is Ala, gly, N substituted Gly, ile, phe, bhPhe, lys, substituted Lys, (D) Lys, or substituted (D) Lys;

x11 is absent, or Ala, pro, bhPhe, lys, substituted Lys, or (D) Lys;

and is also provided with

Each of X12 to X14 is absent or independently any amino acid;

the precondition is that:

i) The peptide may be further bound at any amino acid;

ii) any amino acid of the peptide may be the corresponding (D) -amino acid of the amino acid or may be N-substituted; and

iii) At least two of X1 to X14 are independently Ala or aMeAla, and the side chain methyl C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Cyclizing the alkenyl linker to form a macrocycle;

and is also provided with

Wherein alkanyl is an alkyl chain; alkenyl is an alkyl chain with at least one double bond embedded;

Dapa is diaminopropionic acid; dpa or DIP is 3, 3-diphenylalanine or b, b-diphenylalanine; bhpe is b-homophenylalanine; bip is biphenylalanine; bhPro is b-homoproline; tic is L-1,2,3,4, -tetrahydro-isoquinoline-3-carboxylic acid; NPC is L-hexahydronicotinic acid; bhTrp is b-homotryptophan; 1-Nal is 1-naphthylalanine; 2-Nal is 2-naphthylalanine; orn is ornithine; nleu is norleucine; 2Pal is 2-pyridylalanine; ppa is 2- (R) -pyrrolidinopropionic acid; pba is 2- (R) -pyrrolidinebutyric acid; the substituted Phe is phenylalanine, wherein the phenyl group is substituted with: F. cl, br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2, 6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine;

the substituted bhpe is b-homophenylalanine, wherein the phenyl group is substituted as follows: F. cl, br, I, OH, methoxy, dimethoxy, dichloro, dimethyl, difluoro, pentafluoro, allyloxy, azido, nitro, 4-carbamoyl-2, 6-dimethyl, trifluoromethoxy, trifluoromethyl, phenoxy, benzyloxy, carbamoyl, t-Bu, carboxyl, CN or guanidine;

The substituted Trp is N-methyl-L-tryptophan, a-methyl tryptophan or tryptophan substituted by F, cl, OH or t-Bu;

the substituted bhTrp is N-methyl-L-b-homotryptophan, a-methyl-b-homotryptophan or b-homotryptophan substituted with F, cl, OH or t-Bu;

tet1 is (S) - (2-amino) -3- (2H-tetrazol-5-yl) propionic acid; and Tet2 is (S) - (2-amino) -4- (1H-tetrazol-5-yl) butanoic acid;

123 triazole isAnd is also provided with

Dab is

In one embodiment, each of X1 and X6, X1 and X7, or X1 and X8 is Ala, and the pendant methyl group C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

In one embodiment, each of X4 and X6, or X4 and X8 is Ala, and the pendant methyl group C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

In one embodiment, each of X5 and X6 is Ala, and the pendant methyl group C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

In one embodiment, each of X6 and X7, or X6 and X8Each is Ala and the side chain methyl C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

In one embodiment, C ₂ -C ₁₂ The alkanyl radical being-CH ₂ -(CH ₂ ) _q -CH ₂ -; wherein q is 2 to 10.

In one embodiment, C ₂ -C ₁₂ Alkenyl is- (CH) ₂ ) _t1 -(CH＝CH)-(CH ₂ ) _t2 -; wherein each t1 and t2 is independently 0 to 9.

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

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

R ¹ -Ala'-Thr-His-[Dpa]-Pro-X6-X7-Ala'-X9-X10-X11-X12-X13-X14-R ² (II)

wherein R is ¹ 、R ² X6 to X7, and X9 to X14 are as described for formula (I); and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

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

R ¹ -Glu-Thr-His-Ala'-Pro-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R ² (III)

wherein R is ¹ 、R ² And X7 to X14 are as described for formula (I); and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

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

R ¹ -Glu-Thr-His-[Dpa]-Ala'-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R ² (IV)

wherein R is ¹ 、R ² And X7 to X14 are as described for formula (I); and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ AlkanesRadicals or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

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

R ¹ -Glu-Thr-His-[Dpa]-Pro-Ala'-Ala'-X8-X9-X10-X11-X12-X13-X14-R ² (V)

wherein R is ¹ 、R ² And X8 to X14 are as described for formula (I); and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

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

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

R ¹ -Glu-Thr-His-[Dpa]-Pro-Ala'-X7-Ala'-X9-X10-X11-X12-X13-X14-R ² (VI)

In one embodiment, R ¹ Is IVA or isovaleric acid.

In one embodiment, R ² Is NH ₂ . In one embodiment, R ² Is OH.

In a particular embodiment of any of the hepcidin analogs of the invention, the substituted Lys or substituted (D) Lys is Lys or (D) Lys substituted directly or via a linker with an acid selected from the group consisting of: c12 (lauric acid), C14 (myristic acid), C16 (palmitic acid), C18 (stearic acid), C20, C12 diacid, C14 diacid, C16 diacid, C18 diacid, C20 diacid, biotin, isovaleric acid, or residues thereof. In one embodiment, the linker is Ahx, PEG or PEG-Ahx.

In a particular embodiment of any hepcidin analog of the invention, X8 or X10 is Lys substituted with L1Z or (D) Lys; wherein L1 is absent and is Dapa, D-Dapa or isoGlu, PEG, ahx, isoGlu-PEG, PEG-isoGlu, PEG-Ahx, isoGlu-Ahx or isoGlu-PEG-Ahx; ahx is an aminocaproic acid moiety; PEG is- [ C (O) -CH ₂ -(Peg) _n -N(H)] _m -or- [ C (O) -CH ₂ -CH ₂ -(Peg) _n -N(H)] _m -; and Peg is-OCH ₂ CH ₂ -, m is 1, 2 or 3; and n is an integer between 1 and 100K; and Z is a half-life extending moiety. In one embodiment, the half-life extending moiety is C ₁₀ -C ₂₁ Alkanoyl.

In certain embodiments, the peptide analogs or dimers of the invention comprise an isovaleric acid moiety bound to the N-terminal X1 residue. In certain embodiments, the peptide analogs or dimers of the invention comprise an isovaleric acid moiety bound to the N-terminal Asp residue. In certain embodiments, the peptide analogs or dimers of the invention include an isovaleric acid moiety that binds to an N-terminal Glu residue.

In certain embodiments, the peptide analogs of the invention include amidated C-terminal residues.

In a related embodiment, the invention comprises a polynucleotide encoding a peptide (or a monomeric subunit of a dimer) of a hepcidin analog or dimer of the invention.

In another related embodiment, the invention comprises a vector comprising a polynucleotide of the invention. In particular embodiments, the vector is an expression vector comprising, for example, a promoter operably linked to the polynucleotide in a manner that facilitates expression of the polynucleotide.

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

In another embodiment, the invention provides a method of binding to a membrane iron transporter or inducing internalization and degradation of a membrane iron transporter, comprising contacting a membrane iron transporter with at least one hepcidin analog, dimer, or composition of the invention.

In another embodiment, the invention comprises a method for treating an iron metabolic disease in a subject in need thereof comprising providing to the subject an effective amount of a pharmaceutical composition of the invention. In certain embodiments, the pharmaceutical composition is provided to the subject by oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal or topical administration route. In certain embodiments, the pharmaceutical composition is provided to the subject by an oral or subcutaneous route of administration. In certain embodiments, the iron metabolic disease is an iron overload disease. In certain embodiments, the pharmaceutical composition is provided to the subject at up to or about twice daily, up to or about once every two days, up to or about once a week, or up to or about once a month.

In certain embodiments, the hepcidin analog is provided to the subject at a dose of about 1mg to about 100mg or about 1mg to about 5 mg.

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

In yet another embodiment, the invention comprises a kit comprising a pharmaceutical composition of the invention, packaged with an agent, device, or instructional material, or a combination thereof.

Detailed Description

The present invention relates generally to hepcidin analog peptides and methods of making and using the same. In certain embodiments, the hepcidin analogs exhibit one or more hepcidin activities. In certain embodiments, the invention relates to hepcidin peptide analogs comprising one or more peptide subunits that form a cyclized structure via an intramolecular bond (e.g., an intramolecular disulfide bond). In certain embodiments, the potency and selectivity of the cyclized structure is increased as compared to the non-cyclized hepcidin peptide and analogs thereof. In certain embodiments, the hepcidin analog peptides of the invention exhibit increased half-lives, e.g., upon oral delivery, as compared to hepcidin or a previous hepcidin analog.

Definition and naming

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. In general, the nomenclature used in connection with the chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein and the techniques of such disciplines are well known and commonly employed in the art.

As used herein, the following terms have the meanings given, unless otherwise specified.

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

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

The term "including" is used to mean "including but not limited to (including but not limited to)". "including" and "including but not limited to" are used interchangeably.

The terms "patient", "subject" and "individual" are used interchangeably and refer to a human or non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovine, porcine), companion animals (e.g., canine, feline), 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, livestock, and the like.

As used herein, the term "peptide" generally refers to a sequence of two or more amino acids joined together by peptide bonds. It should be understood that this term does not imply a particular length of the amino acid polymer, nor is it intended to suggest or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring.

As used herein, the term "peptide analog" or "hepcidin analog" generally refers to peptide monomers and peptide dimers that include one or more structural features and/or functional activities identical to hepcidin or a functional region thereof. In certain embodiments, peptide analogs comprise peptides that share substantial amino acid sequence identity with hepcidin, e.g., peptides comprising one or more amino acid insertions, deletions, or substitutions as compared to the wild-type hepcidin (e.g., human hepcidin) amino acid sequence. In certain embodiments, the peptide analog includes one or more additional modifications, such as binding to another compound. The term "peptide analog" encompasses any peptide monomer or peptide dimer of the present invention. In certain instances, a "peptide analog" may also or alternatively be referred to herein as a "hepcidin analog", "hepcidin peptide analog" or "hepcidin analog peptide".

As used herein, recitation of "sequence identity", "percent homology" or, for example, including "sequences that are … …% identical" refers to sequences that are identical on a nucleotide-by-nucleotide and amino acid-by-amino acid basis over a comparison window. Thus, the "percent sequence identity" may be calculated by: comparing the two optimally aligned sequences within a comparison window, determining the number of positions at which identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) occur in the two sequences to yield a number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity.

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

The percentage of identity between two sequences is related to the number of identical positions that are common to the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

A mathematical algorithm may be used to achieve a sequence comparison and a percent identity determination between two sequences. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970, journal of molecular biology (j. Mol. Biol.) 48:444-453) algorithm, using the Blossum 62 matrix or PAM250 matrix, and GAP weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, which have been incorporated into the GAP program in the GCG software package. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using the nws gapdna.cmp matrix and GAP weights 40, 50, 60, 70, or 80 and length weights 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap expansion penalty of 4, and a frame shift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E.Meyers and W.Miller (1989, cabios 4:11-17), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4.

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

As used herein, the term "conservative substitution" refers to the replacement of one or more amino acids with another biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. See, for example, the following table. In some embodiments of the invention, one or more Met residues are substituted with norleucine (Nle), which is a bioisostere of Met, but which is less susceptible to oxidation than Met. In some embodiments, one or more Trp residues are substituted with Phe, or one or more Phe residues are substituted with Trp, and 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 conservative substitutions of residues not normally present in endogenous mammalian peptides and proteins is the conservative substitution of Arg or Lys with, for example, ornithine, canavanine, aminoethylcysteine or another basic amino acid. In some embodiments, another conservative substitution is substitution of one or more Pro residues with bhPro or Leu or D-Npc (isonicotinic acid). For more information on the substitution of phenotype silence in peptides and proteins, see, e.g., bowie et al Science 247,1306-1310,1990. In the following schemes, conservative substitutions of amino acids are grouped by physicochemical properties. I: neutral, hydrophilic, II: acid and amide, III: alkaline, IV: hydrophobicity, V: aromatic, large amino acids.

I	II	III	IV	V
					A	N	H	M	F
S	D	R	L	Y
					T	E	K	I	W
P	Q		V
					G			C

In the following schemes, conservative substitutions of amino acids are grouped by physicochemical properties. VI: neutral or hydrophobic, VII: acidity, VIII: alkaline, IX: polarity, X: aromatic compounds.

VI	VII	VIII	IX	X
					A	E	H	M	F
L	D	R	S	Y
					I		K	T	W
P			C
					G			N
V			Q

As used herein, the term "amino acid" or "any amino acid" refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and unnatural amino acids. Which comprises D-amino acids and L-amino acids. Natural amino acids comprise amino acids found in nature, such as 23 amino acids combined into peptide chains to form building blocks of a large number of proteinsAnd (3) a base acid. These are mainly the L stereoisomers, although some D-amino acids are present in bacterial envelopes and in some antibiotics. The table above lists 20 "standard" natural amino acids. "nonstandard" natural amino acids are pyrrolysine (present in methanogens and other eukaryotes), selenocysteine (present in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). An "unnatural" or "(non-natural) amino acid is a naturally occurring or chemically synthesized non-protein amino acid (i.e., an amino acid that is not naturally encoded or found in the genetic code). More than 140 natural amino acids are known, and there are thousands of possible combinations. Examples of "unnatural" amino acids include beta-amino acids (beta ³ Beta and beta ² ) High amino acids, proline and pyruvic acid derivatives, 3 substituted alanine derivatives, glycine derivatives, ring substituted phenylalanine and tyrosine derivatives, linear nuclear amino acids, diamino acids, D-amino acids and N-methyl amino acids. Unnatural or unnatural amino acids also include modified amino acids. "modified" amino acids include amino acids that are chemically modified to include one or more groups or chemical moieties that do not naturally occur on the amino acid (e.g., natural amino acids).

As will be clear to those of skill in the art, the peptide sequences disclosed herein are shown from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C-terminus of the peptide. The sequences disclosed herein incorporate, inter alia, a "Hy-" moiety at the amino-terminus (N-terminus) of the sequence and an "-OH" moiety or an "-NH" moiety at the carboxy-terminus (C-terminus) of the sequence ₂ "sequence of parts". In this case, and unless otherwise indicated, the "Hy-" portion at the N-terminus of the sequence in question indicates a hydrogen atom corresponding to the presence of a free primary or secondary amino group at the N-terminus, while the "-OH" or "-NH" at the C-terminus of the sequence ₂ "moieties respectively indicate that the amino group (CONH) is present at the C-terminus ₂ ) Hydroxyl or amino groups of (a). In each of the sequences of the invention, the C-terminal "-OH" moiety may replace the C-terminal "-NH moiety ₂ "part of, and vice versa. It will be further appreciated that the amino terminusOr the moiety at the carboxy-terminus may be a bond, such as a covalent bond, particularly where the amino-terminus or the carboxy-terminus is bound to a linker or another chemical moiety (e.g., a PEG moiety).

As used herein, the term "NH ₂ "refers to a free amino group present at the amino terminus of a polypeptide. As used herein, the term "OH" refers to the free carboxyl group present at the carboxyl terminus of a peptide. Furthermore, as used herein, the term "Ac" refers to acetyl protection via acylation of the C-or N-terminus of a polypeptide.

The term "carboxy", as used herein, refers to-CO ₂ H。

In most cases, the names of naturally occurring and non-naturally occurring aminoacyl residues as used herein follow the naming convention proposed by the IUPAC organic chemistry nomenclature committee (IUPAC Commission on theNomenclature of Organic Chemistry) and the IUPAC-IUB biochemical nomenclature committee (IUPAC-IUB Commission on Biochemical Nomenclature), as set forth in "alpha-amino acid nomenclature (1974)" Biochemistry (Biochemistry), 14 (2), (1975). If the names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims are different from those suggested, it will be clear to the reader. Some abbreviations that are helpful in describing the present invention are defined in table 1 below.

TABLE 1 abbreviations for unnatural amino acids and chemical moieties

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Throughout this specification, unless a naturally occurring amino acid is referred to by its full name (e.g., alanine, arginine, etc.), it is denoted by its conventional three-letter or one-letter abbreviation (e.g., ala or a means alanine, arg or R means arginine, etc.). In the case of less common or non-naturally occurring amino acids, unless the amino acid is referred to in its full name (e.g., sarcosine, ornithine, etc.), its residue takes the commonly used three-or four-character code, including Sar or sarcosine (i.e., N-methylglycine), aib (α -aminoisobutyric acid), daba (2, 4-diaminobutyric acid), dapa (2, 3-diaminopropionic acid), γ -Glu (γ -glutamic acid), pGlu (pyroglutamic acid), gaba (γ -aminobutyric acid), β -Pro (pyrrolidine-3-carboxylic acid), 8Ado (8-amino-3, 6-dioxaoctanoic acid), abu (4-aminobutyric acid), bhPro (β -homoproline), bhPhe (β -homol-phenylalanine), bhascp (β -homoaspartic acid), dpa (β, β -diphenylalanine), ida (iminodiacetic acid), ys (homocysteine), bhpa (β -homohcp, β -diphenylalanine).

In addition, R ¹ May be substituted in all sequences with isovaleric acid or equivalent. In some embodiments where the peptides of the invention are combined with acidic compounds (such as isovaleric acid, isobutyric acid, valeric acid, and the like), the presence of such combination is mentioned in the acid form. Thus, for example, but not limiting in any way, in some embodiments, the application may mention such binding as isovaleric acid, rather than indicating binding of isovaleric acid to peptides by reference to isovaleryl.

It is understood that for each of the formulae of the hepcidin analogs provided herein, a bond may be indicated by "-" or implied based on formulae and ingredients. For example, "B7 (L1Z)" should be understood to include a bond between B7 and L1 if L1 is present, or a bond between B7 and Z if L1 is not present. Similarly, "B5 (L1Z)" should be understood to include a bond between B5 and L1 if L1 is present, or a bond between B5 and Z if L1 is not present. Furthermore, it should be understood that when both L1 and Z are present, there is a bond between them. Thus, the definition of certain substituents (such as B7, L1 and J) may include "-" before and/or after the defined substituents, but in each case it is understood that the substituents are bonded to the other substituents via single bonds. For example, where "J" is defined as Lys, D-Lys, arg, pro, -Pro-Arg-, etc., it is understood that J is bound to Xaa2 and Y1 via single bonds. Thus, the definition of a substituent may or may not include "-", but is still understood to be bonded to an adjacent substituent.

As used herein, the term "L-amino acid" refers to the "L" isomeric form of the peptide, while the opposite term "D-amino acid" refers to the "D" isomeric form of the peptide. In certain embodiments, the amino acid residues described herein are in the "L" isomeric form, however, any L-amino acid residue may be substituted with a residue in the "D" isomeric form, so long as the desired function is retained by the peptide.

Unless otherwise indicated, reference is made to the L-isomeric forms of the natural and unnatural amino acids in question, which have chiral centers. Where appropriate, the D-isomer form of an amino acid is represented in the conventional manner by the prefix "D" preceding the conventional three-letter code (e.g.Dasp, (D) Asp or D-Asp; dphe, (D) Phe or D-Phe).

As used herein, "lower Lys homolog" refers to an amino acid having a lysine structure but one or more fewer carbons in its side chain as compared to lysine.

As used herein, "higher Lys homolog" refers to an amino acid having a lysine structure but one or more additional carbon atoms in its side chain as compared to lysine.

As used herein, the term "DRP" refers to disulfide rich peptides.

As used herein, the term "dimer" generally refers to a peptide comprising two or more monomeric subunits. Some dimers include two DRPs. The dimers of the invention comprise homodimers and heterodimers. The monomeric subunits of the dimer may be linked at their C or N termini, or may be linked via internal amino acid residues. The individual monomer subunits of the dimer may be linked via the same site, or the individual monomer subunits may be linked via different sites (e.g., C-terminal, N-terminal, or internal sites).

The terms "isostere substitution" or "isostere substitution" are used interchangeably herein to refer to any amino acid or other analog moiety having similar chemical and/or structural properties to a given amino acid. In certain embodiments, an isostere substitution is a conservative substitution with a natural or unnatural amino acid.

As used herein, the term "cyclization" refers to a reaction in which one portion of a polypeptide molecule becomes linked to another portion of the polypeptide molecule, such as by forming disulfide bridges or other similar bonds, to form a closed loop.

As used herein, the term "subunit" refers to one of a pair of polypeptide monomers that are joined to form a dimeric peptide composition.

As used herein, the term "linking moiety" generally refers to a chemical structure capable of linking or joining two peptide monomer subunits together to form a dimer.

In the context of the present invention, the term "solvate" refers to a complex of defined stoichiometry formed between a solute (e.g. a hepcidin analogue according to the invention or a pharmaceutically acceptable salt thereof) and a solvent. In this regard, the solvent may be, for example, water, ethanol, or another pharmaceutically acceptable, typically small molecule, organic substance such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such solvates are often referred to as hydrates.

As used herein, "iron metabolism disorder" includes the following: wherein abnormal iron metabolism directly causes a disease, or wherein an imbalance in iron blood levels causes a disease, or wherein an imbalance in iron is caused by another disease, or wherein a disease can be treated by modulating iron content and the like. More specifically, iron metabolism disorders according to the present disclosure include iron overload disorders, iron deficiency disorders, iron biodistribution disorders, other iron metabolism disorders, other disorders that may be related to iron metabolism, and the like. Iron metabolic diseases include hemochromatosis, HFE mutant hemochromatosis, membrane iron transporter mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojugglin (hemojuvelin) mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin mutant hemochromatosis, b-cell overload (transfusional iron overload), thalassemia (tharosemia), intermediate thalassemia (thalassemia intermedia), alpha-thalassemia, iron particle young cell anemia (sideroblastic anemia), porphyria (porphyria), bradycardia skin lesion porphyria (porphyria cutanea tarda), african iron overload (African iron overload), hyperferritinmia (hyperferrimia), ceruloplasmin deficiency (ceruloplasmin deficiency), transferrin deficiency (atransferrimia), congenital erythropoiesis abnormal anemia (congenital dyserythropoietic anemia), glomerulonephropathy (hypochromic microcytic anemia), polycythemia (2), polycythemia (sickle cell disease 4), secondary to polycythemia (24) such as polycythemia vera (24), secondary to polycythemia (24) and secondary to polycythemia (24) such as polycythemia; COPD), post-kidney transplant mutations, chuvash mutations, HIF and PHD mutations), as well as idiopathic myelodysplasia (idiopathic myelodysplasia), pyruvate kinase deficiency, iron-deficiency obesity, other anemias, benign or malignant tumors that overproduce or induce overproduction of hepcidin, hepcidin excess conditions, friedreich ataxia (Friedreich ataxia), gracile syndrome, hallervorden-Spatz disease, wilson's disease, pulmonary hemosiderosis (pulmonary hemosiderosis), hepatocellular carcinoma, cancer, hepatitis, cirrhosis, pica (pica), chronic renal failure, insulin resistance (insulin resistance), diabetes, atherosclerosis, neurodegenerative disorders (neurodegenerative disorder), multiple sclerosis (multiple sclerosis), parkinson's disease, huntington's disease, and Alzheimer's disease.

In some embodiments, the diseases and conditions are associated with iron overload diseases such as iron hemochromatosis, HFE mutant hemochromatosis, membrane iron transporter mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojul mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha-thalassemia, sickle cell disease, myelodysplasia, iron granulocyte infection, diabetic retinopathy (diabetic retinopathy), and pyruvate kinase deficiency.

In some embodiments, the hepcidin analogs of the invention are useful in treating diseases and conditions not normally identified as iron-related. For example, hepcidin is highly expressed in murine pancreas, indicating that diabetes (type I or type II), insulin resistance, glucose intolerance, and other conditions can be ameliorated by treatment of underlying iron metabolic disorders. See Ilyin, g. Et al (2003) european society for biochemistry, meeting bulletin 54222-26, which is incorporated herein by reference. Thus, the peptides of the invention are useful in the treatment of these diseases and conditions. One of ordinary skill in the art is readily able to determine whether a given disease can be treated with a peptide according to the present invention using methods known in the art, including the assays of WO 2004092405, incorporated herein by reference, and assays that monitor hepcidin, hemojuvelin, or iron content and expression, such as those described in U.S. patent No. 7,534,764, incorporated herein by reference.

In certain embodiments of the invention, the iron metabolic disease is an iron overload disease, including hereditary hemochromatosis, iron-excess type anemia, alcoholic liver disease, and chronic hepatitis C.

As used herein, the term "pharmaceutically acceptable salt" means a salt or zwitterionic form of a peptide or compound of the invention, which is water-soluble or oil-soluble or dispersible, suitable for use in treating a disease without undue toxicity, irritation, and allergic response commensurate with a reasonable benefit/risk ratio, and effective for its intended use. Salts may be prepared during final isolation and purification of the compounds or by reacting the amino group with a suitable acid, respectively. Representative acid addition salts include: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumaric acid, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, mesitylene sulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Furthermore, the amino groups in the compounds of the invention may be quaternized by: chlorides, bromides and iodides of methyl, ethyl, propyl and butyl groups; dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; chlorides, bromides and iodides of decyl, lauryl, myristyl and stearyl groups; benzyl bromide and phenethyl bromide. Examples of acids that can be used to form the therapeutically acceptable addition salts include: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. The pharmaceutically acceptable salt may suitably be a salt selected from, for example, acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts. Examples of basic salts include salts in which the cation is selected from alkali metal cations (such as sodium or potassium ions), alkaline earth metal cations (such as calcium or magnesium ions), and substituted ammonium ions (such as N (R1) (R2) (R3) (R4) +type ions), wherein R1, R2, R3 and R4 will typically independently represent hydrogen, optionally substituted C1-6 alkyl or optionally substituted C2-6 alkenyl. Examples of related C1-6 alkyl groups include methyl, ethyl, 1-propyl, and 2-propyl. Examples of C2-6 alkenyl groups that may be relevant include vinyl, 1-propenyl, and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in "encyclopedia of formulation technology (Encyclopaedia of Pharmaceutical Technology)", 3 rd edition, james Swarbrick (ed.), informa Healthcare USA (inc.), NY, USA,2007, "Remington's Pharmaceutical Sciences)", 17 th edition, alfonso r.gennaro (ed.), mark Publishing Company, easton, PA, USA,1985 (and newer versions thereof), journal of pharmaceutical science (j.pharm.sci.) 66:2 (1977). Furthermore, for comments on suitable salts, see the manual for pharmaceutically acceptable salts of Stahl and wermth: properties, selection, and Use (Handbook ofPharmaceutical Salts: properties, selection, and Use) (Wiley-VCH, 2002). Other suitable base salts are formed from bases that form non-toxic salts. Representative examples include aluminum, arginine, benzathine (bezathine), calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Semi-salts of acids and bases, such as hemisulfate and hemicalcium salts, may also be formed.

As used herein, the term "N (α) methylation" describes methylation of an α amine of an amino acid, also commonly referred to as N-methylation.

As used herein, the term "symmetrical methylation" or "Arg-Me-sym" describes symmetrical methylation of two nitrogens of an arginine guanidino group. Furthermore, the term "asymmetric methylation" or "Arg-Me-asym" describes the methylation of a single nitrogen of an arginine guanidino group.

As used herein, the term "acylated organic compounds" refers to various compounds having carboxylic acid functionality for acylating the N-terminus of an amino acid subunit prior to formation of a C-terminal dimer. Non-limiting examples of acylated organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorobenzeneacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, 3-trifluoropropionic acid, 3-fluoromethylbutyric acid, tetrahydro-2H-pyran-4-carboxylic acid.

The term "alkyl" comprises straight or branched, acyclic or cyclic, saturated aliphatic hydrocarbons containing from 1 to 24 carbon atoms. The term "C _n-m "indicates a range including endpoints, where n and m are integers and indicates the number of carbon atoms. Examples include C _1-4 、C _1-6 、C _1-8 、C _1-20 And the like. The term "C _n-m Alkyl "refers to an alkyl group having n to m carbon atoms. For example, "C _1-6 Alkyl "refers to a straight or branched hydrocarbon group containing 1 to 6 carbon atoms that is derived by removing one hydrogen atom from a single carbon atom of a parent alkane. Representative saturated straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched chain alkyl groups include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyl groups include, but are not limited toBut are not limited to cyclopentenyl, cyclohexenyl, and the like.

The term "alkenyl" refers to a straight or branched monovalent hydrocarbon group having the number of carbon atoms shown in the prefix and containing at least one double bond. For example, (C) ₂ -C ₆ ) Alkenyl is intended to include ethenyl, propenyl, and the like. In some embodiments, alkenyl groups have 2 to 24 carbon atoms.

The term "alkylene" refers to divalent alkyl groups, especially divalent alkyl groups having 1 to 24 carbon atoms. The term is defined by, for example, methylene (-CH) ₂ (-), ethylene (-CH) ₂ CH ₂ -)、-(CH ₂ ) ₄ -、-(CH ₂ ) ₆ -, propylene isomers (e.g., -CH ₂ CH ₂ CH ₂ -and-CH (CH) ₃ )CH ₂ The (-) and the like are exemplified.

The term "alkenylene" refers to divalent alkenyl groups containing at least one carbon-carbon double bond, especially having 2 to 24 carbon atoms. Such terms as-ch=ch-, -CH ₂ CH＝CH-、-C(CH ₃ )＝CH-、-CH ₂ C(＝CH ₂ )C(＝CH ₂ )CH ₂ -、-CH ₂ CH＝CHCH ₂ -、-(CH ₂ )C(＝CH ₂ )C(＝CH ₂ )(CH ₂ ) ₂ -the groups are exemplified.

The term "administering" or "administering" refers to orally administering, topically contacting, intravenously, intraperitoneally, intramuscularly, intralesionally, intranasally, or subcutaneously to a subject, or implanting a slow release device, such as a micro-osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other modes of delivery include, but are not limited to, use of liposome formulations, intravenous infusion, transdermal patches, and the like.

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

Peptide analogues of hepcidin

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

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

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

In some embodiments, the hepcidin analogs of the invention exhibit a membrane iron transporter binding activity that is at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more than 99% of the binding activity exhibited by a hepcidin reference compound. In some embodiments, the hepcidin analogs of the invention have a lower EC50 or IC than the hepcidin reference compound ₅₀ (i.e., higher binding affinity) to bind to hepcidin (e.g., human membrane iron transporter). In some embodiments, the hepcidin analogs of the invention have an EC50 or IC in membrane iron transporter competitive binding assays ₅₀ At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than the hepcidin reference compound.

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

In some embodiments, the peptide analogs of the invention exhibit an in vitro activity that induces degradation of human membrane iron transporter of at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% of that of a hepcidin reference compound, wherein the activity is measured according to the methods described herein.

In some embodiments, the peptide or peptide dimer of the invention exhibits an in vivo activity that induces a decrease in free plasma iron in an individual of at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% of a hepcidin reference compound, wherein said activity is measured according to a method described herein.

In some embodiments, the activity is an in vitro or in vivo activity, e.g., an in vivo or in vitro activity as described herein. In certain embodiments, the hepcidin analogs of the invention exhibit an 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-fold or at least about 10%, 20%, 30, 40, 50, 60%, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000% greater than the hepcidin reference compound, wherein the activity is an in vitro activity for degrading a membrane iron transporter, e.g., as measured according to the examples herein; or wherein the activity is for reducing in vivo activity of free plasma iron, e.g., as measured according to the examples herein.

In some embodiments, the hepcidin analogs of the invention mimic Hep25 (biologically active human 25-amino acid form) hepcidin activity, 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 is capable of reducing the plasma iron concentration of a subject (e.g., a mouse or human) when administered (e.g., parenterally or orally) to the subject in a dose-dependent and time-dependent manner. See, for example, river et al (2005) blood 106:2196-9. In some embodiments, the peptides of the invention reduce plasma iron concentration in a subject by at least about 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold, or by at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

In some embodiments, the hepcidin analogs of the invention have in vitro activity as determined by the ability to cause internalization and degradation of iron transporters in membrane iron transporter expressing cell lines as taught in Nemeth et al (2006) blood 107:328-33. In some embodiments, in vitro activity is measured by a dose-dependent loss of fluorescence of cells engineered to display membrane iron transporters fused to green fluorescent protein, as described in Nemeth et al (2006) blood 107:328-33. Aliquots of cells were incubated with Hep25 reference formulation or micro hepcidin at fractional concentrations for 24 hours. As provided herein, EC ₅₀ The values are provided as concentrations of the given compound (e.g., the hepcidin analog peptide or peptide dimer of the invention) that elicit 50% of the maximum fluorescence loss generated by the reference compound. In this assay, EC of Hep25 formulation ₅₀ In the range of 5 to 15nM, and in certain embodiments, preferred hepcidin analogs of the invention have an EC of about 1,000nM or less in an in vitro activity assay ₅₀ Values. In certain embodiments, the hepcidin analogs of the invention have less than about any of 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 500nM in an in vitro activity assay (e.g., as described in nemet al (2006) blood 107:328-33 or examples herein)EC of one ₅₀ . In some embodiments, the hepcidin analogs or EC of a biotherapeutic composition (e.g., any of the pharmaceutical compositions described herein) ₅₀ Or IC (integrated circuit) ₅₀ The value is about 1nM or less.

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

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

In certain embodiments, the hepcidin analogs of the invention exhibit a longer half-life than a hepcidin reference compound. In certain embodiments, the hepcidin analogs of the invention have a half-life of at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 4 days, at least about 7 days, at least about 10 days, at least about two weeks, at least about three weeks, at least about 1 month, at least about 2 months, at least about 3 months or more or any intermediate half-life or range therebetween, 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 week, about 2 days, about 2 weeks, about 3 weeks, or more, about 2 months, about 1 month, or more. In some embodiments, the half-life of a hepcidin analog of the invention is extended by its binding to one or more lipophilic substituents or half-life extending moieties (e.g., any of the lipophilic substituents or half-life extending moieties disclosed herein). In some embodiments, the half-life of a hepcidin analog of the invention is extended by its binding to one or more polymeric moieties (e.g., any of the polymeric moieties disclosed herein). In certain embodiments, the hepcidin analogs of the invention have a half-life as described above under a given set of conditions, wherein the temperature is about 25 ℃, about 4 ℃ or about 37 ℃, and the pH is physiological pH or pH is about 7.4.

In certain embodiments, the serum half-life of the hepcidin analogs of the invention comprising a binding half-life extending moiety is extended after oral, intravenous, or subcutaneous administration as compared to the same analog without the binding half-life extending moiety. In particular embodiments, the serum half-life of a hepcidin analog of the invention 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 after any of oral, intravenous, or subcutaneous administration. In particular embodiments, it is between 12 and 168 hours, between 24 and 168 hours, between 36 and 168 hours, or between 48 and 168 hours.

In certain embodiments, the hepcidin analogs of the invention, e.g., comprising a hepcidin analog that binds to a half-life extending moiety, result in a decrease in serum iron concentration upon oral, intravenous, or subcutaneous administration to a subject. In particular embodiments, the serum iron concentration of the subject is reduced to a serum iron concentration of 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% without administration of the hepcidin analog to the subject. In particular embodiments, the reduced serum iron concentration is maintained for at least 1 hour, at least 4 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours after administration to the subject. In particular embodiments, between 12 and 168 hours, between 24 and 168 hours, between 36 and 168 hours, or between 48 and 168 hours. In one embodiment, the serum iron concentration of the subject is reduced to less than 20% about 4 hours or about 10 hours after, for example, intravenous, oral, or subcutaneous administration to the subject. In one embodiment, the serum iron concentration of the subject decreases to less than 50% or less than 60% within about 24 to about 30 hours after, for example, intravenous, oral, or subcutaneous administration.

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 a hepcidin analog of the invention is determined by incubating the hepcidin analog with pre-warmed human serum (Sigma) at 37 ℃. Samples were taken at different time points, typically up to 24 hours, and the stability of the samples was analyzed by separating the hepcidin analog from serum proteins and then analyzing for the presence of the hepcidin analog of interest using LC-MS.

In some embodiments, the stability of the hepcidin analog is measured in vivo using any suitable method known in the art, e.g., 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., mouse), and then samples are taken from the subject by drawing blood at different time points, typically up to 24 hours. The samples were then analyzed as described above with respect to half-life measured in vitro. In some embodiments, the in vivo stability of the hepcidin analogs of the invention is determined by the methods disclosed in the examples herein.

In some embodiments, the present invention provides a hepcidin analog as described herein, wherein the hepcidin analog exhibits improved solubility or improved aggregation characteristics as compared to a hepcidin reference compound. Solubility can be determined by any suitable method known in the art. In some embodiments, methods known in the art to be suitable for determining solubility include incubating a peptide (e.g., a hepcidin analog of the invention) in various buffers (acetate pH4.0, acetate pH5.0, phos/citrate pH5.0, phos citrate pH6.0, phos pH 7.0, phos pH7.5, strong PBS pH7.5, tris pH 8.0, glycine pH 9.0, water, acetic acid (pH 5.0, and others known in the art) and testing for aggregation or solubility using standard techniques, for example, such methods include, but are not limited to, visual precipitation, dynamic light diffusion, circular dichroism, and fluorochromes for measuring surface hydrophobicity and detecting aggregation or fibrillation.

In certain embodiments, the invention provides a hepcidin analog as described herein, wherein the hepcidin analog exhibits an increase in solubility in a particular solution or buffer (e.g., in water or a buffer known in the art or disclosed herein) of 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-fold or an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% over a hepcidin reference compound.

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

In some embodiments, the invention provides a hepcidin analog as described herein, wherein the hepcidin analog exhibits less degradation (i.e., greater degradation stability) than a hepcidin reference compound, e.g., more or less than about 10%, more or less than about 20%, more or less than about 30%, more or less than about 40%, or more or less than about 50% reduction in degradation. In some embodiments, the degradation stability is determined by any suitable method known in the art. In some embodiments, methods known in the art to be suitable for determining degradation stability include those described in Hawe et al, journal of pharmaceutical science, volume 101, phase 3, 2012, pages 895 to 913, which are incorporated herein in their entirety. In some embodiments, such methods are used to select for effective sequences with an extended shelf life.

In some embodiments, the hepcidin analogs of the invention are synthetically produced. In other embodiments, the hepcidin analogs of the invention are recombinantly produced.

The various hepcidin analog monomers and dimeric peptides of the invention may be constructed solely from natural amino acids. Alternatively, these hepcidin analogs may comprise unnatural or unnatural amino acids, including but not limited to modified amino acids. In certain embodiments, the modified amino acid comprises a natural amino acid that has been chemically modified to comprise one or more groups or chemical moieties that are not naturally present on the amino acid. The hepcidin analogs of the invention may additionally comprise a D-amino acid. Furthermore, the hepcidin analog peptide monomers and dimers of the invention may comprise amino acid analogs. In certain embodiments, the peptide analogs of the invention include any of the peptide analogs described herein, wherein one or more of the natural amino acid residues of the peptide analog is substituted with an unnatural or unnatural amino acid or a D-amino acid.

In certain embodiments, the hepcidin analogs of the invention comprise one or more modified or unnatural amino acids. For example, in certain embodiments, the hepcidin analogs comprise one or more of the following: daba, dapa, pen, sar, cit, pba, cav, HLeu, 2-Nal, 1-Nal, d-2-Nal, bip, phe (4-OMe), tyr (4-OMe), betahTrp, betahpe, phe (4-CF) ₃ ) 2-2-indene, 1-1-indene, cyclobutyl, beta hPhe, hLeu, gla, phe (4-NH) ₂ ) hPhe, 1-Nal, nle, 3-3-diPhe, cyclobutyl-Ala, cha, bip, beta-Glu, phe (4-Guan), homoamino acids, D-amino acids and various N-methylated amino acids. It will be appreciated by those skilled in the art that other modified or unnatural amino acids can be made, and that various other substitutions can be made to natural amino acids with modified or unnatural amino acids to achieve similar desired results, and that such substitutions are within the teachings and spirit of the invention.

The invention includes any of the hepcidin analogs described herein, e.g., in free form or salt form.

The compounds described herein include isotopically-labeled compounds, which are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as respectively ² H、 ³ H、 ¹ 3C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ O、 ¹⁷ O、 ³⁵ S、 ¹⁸ F、 ³⁶ Cl. Certain isotopically-labeled compounds described herein, for example, are incorporated such as ³ H is H ¹⁴ Compounds of the radioisotope of C are suitable for use in drug and/or substrate tissue distribution assays. In addition, the use of a metal such as deuterium (i.e ² H) Certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, may be obtained by isotopic substitution of (c). In certain embodiments, the compound is isotopically substituted with deuterium. In a more particular embodiment, a majority of the labile hydrogen is replaced with deuterium.

The hepcidin analogs of the invention comprise any of the peptide monomers or dimers described herein linked to a linker moiety (comprising any of the specific linker moieties described herein).

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

In certain embodiments, the monomeric subunits of the peptide analogs of the invention or the dimeric peptide analogs of the invention comprise or consist of: 7 to 35 amino acid residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues, 10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25 amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues, and optionally one or more additional non-amino acid moieties such as a binding chemical moiety, e.g., half-life extending moiety, PEG, or linker moiety. In certain embodiments, the monomeric subunits of the hepcidin analogs comprise or consist 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 subunits of the hepcidin analogs of the invention comprise or consist of: 10 to 18 amino acid residues and optionally one or more additional non-amino acid moieties, such as a conjugated chemical moiety, e.g., a PEG or linker moiety. In various embodiments, the monomer subunits comprise or consist of: 7 to 35 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues. In particular embodiments of any of the various formulae described herein, X comprises or consists of: 7 to 35 amino acid residues, 8 to 35 amino acid residues, 9 to 35 amino acid residues, 10 to 35 amino acid residues, 7 to 25 amino acid residues, 8 to 25 amino acid residues, 9 to 25 amino acid residues, 10 to 25 amino acid residues, 7 to 18 amino acid residues, 8 to 18 amino acid residues, 9 to 18 amino acid residues, or 10 to 18 amino acid residues.

In particular embodiments, the hepcidin analogs or dimers of the invention do not comprise any of the compounds described in PCT/US2014/030352 or PCT/US 2015/038370.

Peptide hepcidin analogues

In certain embodiments, the hepcidin analogs of the invention comprise a single peptide subunit optionally conjugated to an acid moiety. In certain embodiments, the acid moiety is bound directly or via a linker.

R ¹ -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R ² (I)

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

R ² is NH ₂ Substituted amino, OH, or substituted hydroxy;

x2 is Ala, t-BuAla, thr, substituted Thr, gly, N substituted Gly, or Ser;

x3 is Ala, t-BuAla, gly, N substituted Gly, his, or substituted His;

x6 is absent or any amino acid other than Cys, (D) Cys, aMeCys, hCys or Pen;

x11 is absent, or Ala, pro, bhPhe, lys, substituted Lys, or (D) Lys;

and is also provided with

Each of X12 to X14 is absent or independently any amino acid;

The precondition is that:

i) The peptide may be further bound at any amino acid;

and is also provided with

123 triazole isAnd is also provided with

Dab is

In one embodiment, each of X6 and X7, or X6 and X8 is Ala, and the pendant methyl group C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

In one embodiment, the linker is- (CH) ₂ ) ₂ -、-(CH ₂ ) ₃ -、-(CH ₂ ) ₄ -or- (CH) ₂ ) ₆ -。

In one embodiment, the linker is- (CH) ₂ ) _t1 -(CH＝CH)-(CH ₂ ) _t2 -, and each t1 and t2 is independently 0, 1, 2 or 3.

In one embodiment, the linker is- (CH) ₂ ) _t1 -(CH＝CH)-(CH ₂ ) _t2 -, and each t1 and t2 is independently 2.

In one embodiment, the linker is- (ch=ch) -or- (CH) ₂ )-(CH＝CH)-(CH ₂ )-。

In one embodiment, the linker is- (CH) ₂ ) ₂ -(CH＝CH)-(CH ₂ ) ₂ -。

In one embodiment of the present invention, in one embodiment,

x1 is Glu, dab, dap, orn, lys, or Tet1;

x2 is Thr;

x3 is His or 1MeHis;

x4 is Dpa;

x5 is Ala or Pro;

x6 is absent, ala, glu, or substituted Lys;

x7 is absent, or Ala, ile, lys, substituted Lys, (D) Lys, or substituted (D) Lys;

x8 is absent, or Ala, ile, glu, asp, 123 triazole, lys, substituted Lys, (D) Lys, substituted (D) Lys, or aMeLys;

X9 is absent, or bhpe;

x10 is absent, or Ala, ile, phe, bhPhe, lys, substituted Lys, (D) Lys, or substituted (D) Lys; and

X11 is absent, or Pro, bhPhe, lys, substituted Lys, or (D) Lys.

In one embodiment, X1 is Ala or Glu.

In one embodiment, X2 is Thr.

In one embodiment, X3 is His.

In one embodiment, X4 is Ala or Dpa.

In one embodiment, X5 is Ala or Pro.

In one embodiment, X6 is Ala or substituted Lys.

In one embodiment, X7 is Ala, ile or substituted Lys.

In one embodiment, X8 is Ala, lys or (D) Lys.

In one embodiment, X9 is absent, or bhF.

In one embodiment, X10 is absent, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

In one embodiment, X11 is absent, arg, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

In one embodiment, each of X12, X13, and X14 is absent.

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

R ¹ -Ala'-Thr-His-[Dpa]-Pro-X6-X7-Ala'-X9-X10-X11-X12-X13-X14-R ² (II)

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

wherein C is Ala side chain carbon and the linker is C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl groups.

In one embodiment, X6 is Ala.

In one embodiment, X6 is Ahx-Palm substituted Lys.

In one embodiment, X6 is absent, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

In one embodiment, X6 is absent.

In one embodiment, X6 is (D) Lys.

In one embodiment, X6 is Lys.

In one embodiment, X6 is Ahx-Palm substituted Lys.

In one embodiment, X6 is Lys (Ahx_palm).

In one embodiment, X6 is a conjugated amino acid.

In one embodiment, X6 is conjugated Lys or (D) Lys.

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

The hepcidin analog of claim 37, wherein L1 is a single bond.

In one embodiment, L1 is iso-Glu.

In one embodiment, L1 is Ahx.

In one embodiment, L1 is iso-Glu-Ahx.

In one embodiment, L1 is PEG.

The hepcidin analog of claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is PEG-Ahx.

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

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

In one embodiment, m is 1.

In one embodiment, m is 2.

In one embodiment, n is 2.

In one embodiment, n is 4.

In one embodiment, n is 8.

In one embodiment, n is 11.

In one embodiment, n is 12.

In one embodiment, n is 20K.

In one embodiment, PEG is 1PEG2; and 1Peg2 is-C (O) -CH2- (Peg) 2-N (H) -.

In one embodiment, PEG is 2PEG2; and 2Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

In one embodiment, PEG is 1PEG2-1PEG2; and each 1Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

In one embodiment, PEG is 1PEG2-1PEG2; and 1Peg2-1Peg2 is- [ (C (O) -CH2- (OCH 2CH 2) 2-NH-C (O) -CH2- (OCH 2CH 2) 2-NH- ] -.

In one embodiment, PEG is 2PEG4; and 2Peg4 is-C (O) -CH2-CH2- (Peg) 4-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 4-NH ] -.

In one embodiment, PEG is 1PEG8; and 1Peg8 is-C (O) -CH2- (Peg) 8-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 8-NH ] -.

In one embodiment, PEG is 2PEG8; and 2Peg8 is-C (O) -CH2- (Peg) 8-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 8-NH ] -.

In one embodiment, PEG is 1PEG11; and 1Peg11 is-C (O) -CH2- (Peg) 11-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 11-NH ] -.

In one embodiment, PEG is 2PEG11; and 2Peg11 is-C (O) -CH2-CH2- (Peg) 11-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 11-NH ] -.

In one embodiment, PEG is 2PEG11' or 2PEG12; and 2Peg11' or 2Peg12 is-C (O) -CH2-CH2- (Peg) 12-N (H) -or- [ C (O) -CH2-CH2- (OCH 2CH 2) 12-NH ] -.

In one embodiment, when PEG is linked to Lys, the-C (O) -of the PEG is linked to Ne of Lys.

In one embodiment, when PEG is linked to isoGlu, the-N (H) -of the PEG is linked to the-C (O) -, of the isoGlu.

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

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

In one embodiment, Z is Palm.

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

R ¹ -Glu-Thr-His-Ala'-Pro-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R ² (III)

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

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

R ¹ -Glu-Thr-His-[Dpa]-Ala'-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R ² (IV)

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

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

R ¹ -Glu-Thr-His-[Dpa]-Pro-Ala'-Ala'-X8-X9-X10-X11-X12-X13-X14-R ² (V)

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

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

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

R ¹ -Glu-Thr-His-[Dpa]-Pro-Ala'-X7-Ala'-X9-X10-X11-X12-X13-X14-R ² (VI)

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

In one embodiment, with respect to formulas (II) to (VI), the linker is-ch=CH-、-CH2-CH＝CH-、-CH＝CH-CH ₂ -、-CH ₂ -CH＝CH-CH ₂ -、-CH ₂ -CH ₂ -CH＝CH-CH ₂ -、-CH ₂ -CH＝CH-CH ₂ -CH ₂ -、-CH ₂ -CH ₂ -CH＝CH-CH ₂ -CH ₂ -、-CH ₂ -CH ₂ -CH ₂ -CH＝CH-CH ₂ -、-CH ₂ -CH ₂ -CH ₂ -CH＝CH-CH ₂ -CH ₂ -、-CH ₂ -CH ₂ -CH ₂ -CH＝CH-CH ₂ -CH ₂ -CH ₂ -、-CH ₂ -CH ₂ -CH＝CH-CH ₂ -CH ₂ -CH ₂ -、-CH ₂ -CH＝CH-CH ₂ -CH ₂ -CH ₂ -or-ch=ch-CH ₂ -CH ₂ -.CH ₂ -。

In one embodiment, X9 is absent.

In one embodiment, X9 is bhF.

In one embodiment, X11 is absent.

In one embodiment, X11 is Arg.

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

In one embodiment, X11 is (D) Lys.

In one embodiment, each of X12, X13 and X14 is independently absent, or any amino acid.

In one embodiment, each of X12, X13, and X14 is absent.

In one embodiment, X10 is absent.

In one embodiment, X10 is (D) Lys.

In one embodiment, X10 is Lys.

In one embodiment, X10 is Ahx-Palm substituted Lys.

In one embodiment, X10 is Lys (Ahx_palm).

In one embodiment, X10 is a conjugated amino acid.

In one embodiment, X10 is conjugated Lys or (D) Lys.

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

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

In one embodiment, L1 is iso-Glu.

In one embodiment, L1 is Ahx.

In one embodiment, L1 is iso-Glu-Ahx.

In one embodiment, L1 is PEG.

The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is PEG-Ahx.

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

In one embodiment, m is 1.

In one embodiment, m is 2.

In one embodiment, n is 2.

In one embodiment, n is 4.

In one embodiment, n is 8.

In one embodiment, n is 11.

In one embodiment, n is 12.

In one embodiment, n is 20K.

In some embodiments of the compounds of formula I, the invention provides peptides of formula (X):

or a pharmaceutically acceptable salt or solvate thereof, wherein the variable R ¹ X2, X3, X4, X5, X6, X7, X9, X10, X11 and R ² As defined herein and in the specification, claims and table 6B for the corresponding amino acid residues. R is R ⁵ Is H or C _1-6 An alkyl group. In one embodiment, R ⁵ H. In another embodiment, R ⁵ Is methyl. L (L) ^x Is a connecting portion. In some embodiments, L ^x Is C _1-8 An alkylene group. In other embodiments, L ^x Is C _2-8 Alkenylene radicals. In one embodiment, R ¹ Is an isovaleric acid residue, i.e., 3-methylbutyryl. In some embodiments, R ² Is phenyl-C _1-6 Alkylene-amino, OH or NH ₂ . In one embodiment, R ² Is OH or NH ₂ . In another embodiment, R ² Is 4-phenylbutylamino. In another embodiment, R ² Is NH ₂ Or 4-phenylbutylamino. In some embodiments, L ^x Is (trans) -CH ₂ CH＝CHCH ₂ -, (cis) -CH ₂ CH＝CHCH ₂ -, (cis) - (CH) ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -, (trans) - (CH) ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -、-(CH ₂ ) ₂ C(＝CH ₂ )C(＝CH ₂ )(CH ₂ ) ₂ -、-(CH ₂ ) ₆ -、-(CH ₂ ) ₄ -or-CH ₂ C(＝CH ₂ )C(＝CH ₂ )CH ₂ -. In some embodiments, X2 is Thr, (NMe) Thr or thr_psi; x3 is His or his_psi; x4 is DIP or dip_psi; x5 is Pro; x6 is Ala, sar, lys (ahx_palm), lys_ahx_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_dap_c18_diacid, lys_1peg2_1peg2_Isoglu_c18_diacid, lys_1peg2_1peg2_Isoglu_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_dap_c18_diacid, -NHCH ₂ CH ₂ N ⁺ (CH ₃ ) ₂ -CH ₂ C (O) -or lys_1peg2_1peg2_ahx_palm. X7 is Arg, tba, tle, ile, ala or Lys (carpine). X9 is Dip, bhF or NMe_Lys_Ahx_palm. X10 is Arg, (D) Arg, lys_ahx_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_isoglu_palm, lys_1peg2_1peg2_isoglu_c18_diacid, lys_1peg2_1peg2_ahx_c18_diacid, dk_betaine, or (D) Lys. X11 is Arg, (D) Lys or Lys_Carnitine. R is R ¹ Is isovaleric acid. R is R ² Is NH ₂ Or n_butyl_phe.

In other embodiments of the peptide compounds of formula I, the invention provides peptides of formula (XI):

Or a pharmaceutically acceptable salt or solvate thereof, wherein the variable R ¹ X2, X3, X4, X5, X6, X8, X9, X10, X11 and R ² As defined herein and in the specification, claims and corresponding amino acid residues in table 6C. L (L) ^x Is C _2-8 Alkenylene radicals. In one embodiment, R ¹ Is an isovaleric acid residue, i.e., 3-methylbutyryl. In some embodiments, R ² Is phenyl-C _1-6 Alkylene-amino, OH or NH ₂ . In certain embodiments, R ² Is 4-phenylbutylamino, OH or NH ₂ . In one embodiment, R ² Is OH or NH ₂ . In another embodiment, R ² Is 4-phenylbutylamino. In one embodiment, R ² Is NH ₂ Or 4-phenylbutylamino. In some embodiments, L ^x Is (trans) -CH ₂ CH＝CHCH ₂ -, (cis) -CH ₂ CH＝CHCH ₂ -, (cis) - (CH) ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -, (trans) - (CH) ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -、-(CH ₂ ) ₂ C(＝CH ₂ )C(＝CH ₂ )(CH ₂ ) ₂ -、-(CH ₂ ) ₆ -、-(CH ₂ ) ₄ -or-CH ₂ C(＝CH ₂ )C(＝CH ₂ )CH ₂ -. In some embodiments, X2 is Thr, (NMe) Thr or thr_psi; x3 is His or his_psi; x4 is DIP or dip_psi; x5 is Pro; x6 is Ala, sar, lys (ahx_palm), lys_ahx_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_dap_c18_diacid, lys_1peg2_1peg2_Isoglu_c18_diacid, lys_1peg2_1peg2_Isoglu_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_dap_c18_diacid, -NHCH ₂ CH ₂ N ⁺ (CH ₃ ) ₂ -CH ₂ C (O) -or lys_1peg2_1peg2_ahx_palm. X8 is Ala, (a-Me) Ala, bhPhe, lys or (D) Lys. X9 is Dip, bhF or NMe_Lys_Ahx_palm. X10 is Arg, (D) Arg, lys_ahx_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_isoglu_palm, lys_1peg2_1peg2_isoglu_c18_diacid, lys_1peg2_1peg2_ahx_c18_diacid, dk_betaine, or (D) Lys. X11 is Arg, (D) Lys or Lys_Carnitine. R is R ¹ Is isovaleric acid. R is R ² Is NH ₂ Or n_butyl_phe.

In one embodiment, PEG is 1PEG2; and 1Peg2 is-C (O) -CH2- (Peg) 2-N (H) -.

In one embodiment, PEG is 2PEG2; and 2Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

In one embodiment, Z is Palm.

In one embodiment, -L1Z is:

PEG11_OMe；

PEG12_c18 acid;

1PEG2_1PEG2_Ahx_Palm；

1PEG2_Ahx_Palm；

Ado_Palm；

Ahx_Palm；

Ahx_PEG20K；

PEG12_Ahx_IsoGlu_Behenic；

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 (b)

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 (b)

-PEG4_PEG4_IsoGlu_C18_Diacid；

Wherein the method comprises the steps of

PEG11_OMe is- [ C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₁₁ -OMe]；

1PEG2 is-C (O) -CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-；

PEG4 is-C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₄ -NH-；

PEG8 is- [ C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₈ -NH-；

1PEG8 is- [ C (O) -CH ₂ -(OCH ₂ CH ₂ ) ₈ -NH-；

PEG12 is- [ C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₁₂ -NH-；

Ado is- [ C (O) - (CH) ₂ ) ₁₁ -NH]-

Cn acid is-C (O) (CH ₂ ) _n-2 -CH ₃ The method comprises the steps of carrying out a first treatment on the surface of the C18 acid is-C (O) - (CH) ₂ ) ₁₆ -Me；

Palm is-C (O) - (CH) ₂ ) ₁₄ -Me；

isoGlu is isoglutamic acid;

isoGlu_palm is

Ahx is- [ C (O) - (CH) ₂ ) ₅ -NH]-；

Cn_diacids are-C (O) - (CH) ₂ ) _n-2 -COOH; where n is 10, 12, 14, 16, 18 or 22.

In one embodiment, X6 or X10 is Lys (1PEG2_1PEG2_IsoGlu_C) _n Diacid); and Lys (1PEG2_1PEG2_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (1PEG2_1PEG2_IsoGlu_C _n Diacid); and (D) Lys (1PEG2_1PEG2_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (1PEG8_IsoGlu_C _n Diacid); and Lys (1PEG8_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (1PEG8_IsoGlu_C) _n Diacid); and (D) Lys (1PEG8_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (1PEG2_1PEG2_Dap_C) _n Diacid); and Lys (1PEG2_1PEG2_Dap_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (IsoGlu_C _n Diacid); and Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (IsoGlu_C _n Diacid); and (D) Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (PEG 12. Mu.IsoGlu. Mu.C) _n Diacid); and Lys (PEG 12_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (PEG 12. Mu.IsoGlu. Mu.C) _n Diacid); and (D) Lys (PEG 12_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (PEG4_IsoGlu_C _n Diacid); and Lys (PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (PEG4_IsoGlu_C) _n Diacid); and (D) Lys (PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (PEG4_PEG4_IsoGlu_C) _n Diacid); and Lys (PEG4_PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (PEG4_PEG4_IsoGlu_C) _n Diacid); and (D) Lys (PEG4_PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 orX10 is Lys (IsoGlu_C) _n Diacid); and Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (PEG 12_Ahx_C _n Diacid); and Lys (PEG 12_Ahx_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (PEG 12_Ahx_C _n Diacid); and (D) Lys (PEG 12_Ahx_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is Lys (PEG 12-C _n _Diacid)The method comprises the steps of carrying out a first treatment on the surface of the And Lys (PEG 12_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X6 or X10 is (D) Lys (PEG 12-C _n Diacid); and (D) Lys (PEG 12_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

In one embodiment, X1 is Ala.

In one embodiment, X2 is Thr, ala, N-MeThr or t-BuAla. In a particular embodiment, X2 is Thr.

In one embodiment, X3 is His, ala, N-MeHis or t-BuAla. In a particular embodiment, X2 is His.

In one embodiment, X4 is Dpa, ala, N-MePhe or t-BuAla. In a particular embodiment, X4 is Dpa.

In one embodiment, X5 is Pro, ala or t-BuAla. In a particular embodiment, X5 is Pro.

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

In one embodiment, X7 is absent, lys, substituted Lys, ala, ile, t-BuAla or N-MeLeu. In a particular embodiment, X7 is Ala.

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

In one embodiment, X9 is absent, ala, bhPhe, phe or substituted Phe. In one embodiment, X9 is Ala. In another embodiment, X9 is Phe. In another embodiment, X9 is N-MePhe. In a particular embodiment, X9 is bhpe.

In one embodiment, X10 is absent, lys, (D) Lys, substituted (D) Lys, or Ala. In another embodiment, X10 is substituted N-MeLys. In a particular embodiment, X10 is substituted Lys. In one embodiment, the substitution on Lys is ahx_palm. In another embodiment, the substitution on Lys is 1peg2_1peg2_dap_c18_diacids.

In one embodiment, X11 is Ala, t-BuAla, lys, (D) Lys or N-Me (D) Lys. In a particular embodiment, X11 is (D) Lys.

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

In certain embodiments, X13 is absent.

In certain embodiments, X14 is absent.

In one embodiment, R ² Is NH ₂ 。

In one embodiment, R ² Is a substituted amino group.

In one embodiment, R ² Is N-alkylamino.

In one embodiment, R ² Is an N-alkylamino group in which the alkyl group is further substituted or unsubstituted.

In one embodiment, R ² Is an N-alkylamino group wherein alkyl is a further substituted aryl or heteroaryl group.

In one embodiment, R ² Is alkylamino wherein alkyl is unsubstituted or aryl substituted; and alkyl is ethyl, propyl, butyl or pentyl.

In one embodiment, R ² Is alkylamino wherein alkyl is unsubstituted or phenyl substituted; and alkyl is ethyl, propyl, butyl or pentyl.

In one embodiment, R ² Is OH.

In one embodiment, R ¹ Is C ₁ -C ₂₀ Alkanoyl.

In one embodiment, R ¹ Is IVA or isovaleric acid.

In one embodiment, the peptide is a linear peptide.

In one embodiment, the peptide is a lactam.

In one embodiment, the peptide is a lactam, any free-NH therein ₂ Is associated with any free-C (O) ₂ H cyclizes.

In one embodiment, the peptide is any one of the peptides listed in table 6A or a variant thereof.

In another embodiment, the invention provides a peptide as shown in table 6B or table 6C, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the peptide is

ID#6

An E or Z isomer; or (b)

ID#16

The E or Z isomer.

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

In one embodiment, R ² Is NH ₂ . In another embodiment, R ² Is a substituted amino group. In another embodiment, R ² Is alkylamino or (substituted alkyl) amino. In another embodiment, R ² Is methylamino, ethylamino, phenylamino, benzylamino, phenyl-C _1-6 Alkylamino, 4-phenylbutylamino or phenethylamino.

In one embodiment, R ² Is OH.

In one embodiment, R ¹ Is C ₁ -C ₂₀ Alkanoyl.

In one embodiment, R ¹ Is IVA or isovaleric acid.

In certain embodiments of any peptide analog having any of the various formulae set forth herein, R ¹ Is selected from: methyl, acetyl, formyl, benzoyl, trifluoroacetyl, isovaleryl, isobutyryl, octyl and laurate, hexadecanoic acid and the combined amides of gamma-Glu-hexadecanoic acid.

In certain embodiments, the substituted Lys is Lys substituted with: 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 Alkanoyl or any of the others described herein. In one embodiment, lys is substituted with Lys N ^ε 。

In certain embodiments, the substituted (D) Lys is a substituted (D) Lys: 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 Alkanoyl or any of the others described herein. In one embodiment, (D) Lys is substituted with N of (D) Lys ^ε 。

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

In a certain embodiment, the invention comprises a polypeptide comprising an amino acid sequence set forth in table 6A, table 6B, or table 6C, or any amino acid sequence having at least 85%, at least 90%, at least 92%, at least 94%, or at least 95% identity to any of these amino acid sequences.

In certain embodiments, the invention comprises hepcidin analogs having the following structure or comprising the amino acid sequence set forth below:

isovalerate-E-T-H- [ Ala (1) ] -P- [ Ala (1) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Ala (2) ] -P- [ Ala (1) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] -P- [ Ala (1) ] -I- [ Ala (2) ] - [ bhPre ] - [ Lys (Ahx_palm) ] [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Ala (1) ] -P- [ Ala (2) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Ala (2) ] -P- [ Ala (2) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] -P- [ Ala (2) ] -I- [ Ala (2) ] -bhpe ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid-E-T-H- [ Ala (1) ] -P- [ Ala (2) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2 (reducing linker)

isovalerate-E-T-H- [ Dpa ] - [ Ala (1) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] - [ Ala (1) ] -Ala (2) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] - [ Ala (2) ] - [ Ala (1) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] - [ Ala (2) ] -I- [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] -P- [ Ala (1) ] - [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] -P- [ Ala (1) ] -Ala (2) ] - [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] -P- [ Ala (2) ] -Ala (1) ] - [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovalerate-E-T-H- [ Dpa ] -P- [ Ala (2) ] - [ (D) Lys ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] -bhpe ] - [ Lys (Ahx_palm) ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] -Lys (Ahx_palm) ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-I- [ Ala (2) ] -bhpe ] - [ Lys (Ahx_palm) ] -NH2;

Isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P- [ Ala (2) ] -bhpe ] - [ Lys (Ahx_palm) ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P- [ Ala (2) ] -Lys (Ahx_palm) ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (1) ] -bhpe ] - [ Lys (Ahx_palm) ] -R-NH2;

isovaleric acid- [ Ala (1) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] -bhpe ] - [ Lys (Ahx_palm) ] -R-NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P- [ Lys (Ahx_palm) ] -I- [ Ala (2) ] -bhpe ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A- [ Lys (Ahx_palm) ] -Ala (2) ] -bhpe ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (1) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (1) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P-A-I- [ Ala (3) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P-A-I- [ Ala (1) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (3) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (1) ] -T-H- [ Dpa ] -P-A-I- [ Ala (3) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P- [ Lys (Ahx_palm) ] -I- [ Ala (3) ] -bhpe ] - [ (D) Lys ] -NH2;

Isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P-A- [ Lys (Ahx_palm) ] - [ Ala (3) ] -bhpe ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P- [ Lys (Ahx_palm) ] - [ Ala (3) ] -bhpe ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P- [ Lys (Ahx_palm) ] -I- [ Ala (2) ] -bhpe ] - [ (D) Lys ] -NH2;

isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P-A- [ Lys (Ahx_palm) ] - [ Ala (2) ] -bhpe ] - [ (D) Lys ] -NH2; or (b)

Isovaleric acid- [ Ala (2) ] -T-H- [ Dpa ] -P- [ Lys (Ahx_palm) ] - [ Ala (2) ] -bhpe ] - [ (D) Lys ] -NH2;

in one embodiment, the invention provides a peptide as set forth in table 7, or a pharmaceutically salt or solvate thereof.

Table 7. Illustrative peptides of the invention.

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In a particular embodiment, the peptide is any peptide wherein FPN activity is <100 nM. In another specific embodiment, the peptide is any peptide wherein FPN activity is <50 nM. In another specific embodiment, the peptide is any peptide wherein FPN activity is <20 nM. In another specific embodiment, the peptide is any peptide wherein FPN activity is <10 nM. In a more specific embodiment, the peptide is any peptide wherein FPN activity is <5 nM.

Peptide analogue conjugates

In certain embodiments, the hepcidin analogs of the invention comprising both monomers and dimers comprise one or more conjugated chemical substituents, such as lipophilic substituents and polymeric moieties, collectively referred to herein as half-life extending moieties. Without wishing to be bound by any particular theory, it is believed that the lipophilic substituent binds to albumin in the blood stream, thereby protecting the hepcidin analog from enzymatic degradation and thus enhancing its half-life. In addition, it is believed that the polymeric moiety may enhance half-life and reduce clearance in the blood stream, and in some cases, may enhance permeability through the epithelium and retention in the underlying basement membrane. In addition, it is speculated that these substituents may in some cases enhance permeability through the epithelium and retention in the underlying basement membrane. The skilled artisan will be well aware of suitable techniques for preparing the compounds used in the context of the present invention. For examples of non-limiting suitable chemistry, see, for example, WO98/08871, WO00/55184, WO00/55119, madsen et al (J.Med. Chem.2007,50,6126-32), and Knudsen et al 2000 (J.pharmaceutical chem. 43, 1664-1669).

In one embodiment, the side chain of one or more amino acid residues (e.g., lys residues) in the hepcidin analogs of the invention are further conjugated (e.g., covalently linked) to a lipophilic substituent or other half-life extending moiety. The lipophilic substituent may be covalently bound to an atom in the amino acid side chain, or alternatively may be bound to the amino acid side chain via one or more spacers or linker moieties. The spacer or linker moiety, if present, may provide a space between the hepcidin analog and the lipophilic substituent.

In certain embodiments, the lipophilic substituent or half-life extending moiety comprises a hydrocarbon chain having 4 to 30C atoms, for example at least 8 or 12C atoms, and preferably 24 or fewer C atoms, or 20 or fewer C 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 moiety (e.g., an acyl group, a sulfonyl group, an N atom, an O atom, or an S atom) that forms part of an amino acid side chain or spacer linkage. In some embodiments, the hydrocarbon chain is acyl substituted, and thus the hydrocarbon chain may form part of an alkanoyl group (e.g., palmitoyl, hexanoyl, lauroyl, myristoyl, or stearoyl).

The lipophilic substituent may be attached to any amino acid side chain in the hepcidin analogs of the invention. In a certain embodiment, the amino acid side chain comprises a carboxyl, hydroxyl, thiol, amido or amino group for forming an ester, sulfonyl ester, thioester, amide or sulfonamide with a spacer or lipophilic substituent. For example, a lipophilic substituent may be bound 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. Amino acids shown as Lys in any of the formulae provided herein may be replaced, for example, with Dbu, dpr or On with the addition of a lipophilic substituent.

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

As used herein, "polyethylene glycol" or "PEG" is of the formula H- (O-CH) ₂ -CH ₂ ) _n -polyether compounds of OH. As used herein, PEG is also referred to as polyethylene oxide (PEO) or Polyoxyethylene (PCE), referring to oligomers or polymers of ethylene oxide, depending on its molecular weight PEO, PEE or POG. Three names are chemically synonymous, but PEG often refers to oligomers and polymers with molecular weights below 20,000g/mol, PEO refers to polymers with molecular weights above 20,000g/mol, and POE refers to polymers of any molecular weight. PEG and PEO are liquids or low melting point solids, depending on their molecular weight. Throughout the present invention, 3 names are used differently. PEG is prepared by polymerization of ethylene oxide and can be in the range of 300g/mol to 10,000,000g/mThe wide range of molecular weights of the ol is commercially available. Although PEG and PEO with different molecular weights can be used in different applications and have different physical properties (e.g., viscosity) due to chain length effects, their chemical properties are nearly identical. The polymeric moiety is preferably water soluble (amphiphilic or hydrophilic), non-toxic and pharmaceutically inert. Suitable polymeric moieties include polyethylene glycol (PEG), homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG) or polyoxyethylene glycerol (POG). See, for example, J.International journal of hematology (int.J.hepatology) 68:1 (1998); bioconjugate chemistry (Bioconjugate chem.) 6:150 (1995); and a review of therapeutic drug Carrier systems (crit.rev.therapeutic. Drug Carrier sys.) 9:249 (1992). PEG prepared for the purpose of extending half-life, e.g., mono-activated, alkoxy-terminated Polyoxyalkylene (POA), such as mono-methoxy-terminated polyethylene glycol (mPEG), are also contemplated; also contemplated are dual activated polyethylene oxides (diols) or other PEG derivatives. Suitable polymers will vary substantially by weight ranging from about 200 to about 40,000, and are generally 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. Different forms of PEG may also be used depending on the initiator used in the polymerization process, for example the common initiator is monofunctional methyl ether PEG or methoxy poly (ethylene glycol), abbreviated mPEG. Other suitable initiators are known in the art and are suitable for use in the present invention.

Low molecular weight PEG may also be provided as a pure oligomer, known as monodisperse, homogeneous or discrete. Which are used in certain embodiments of the present invention.

PEG may also be provided in different geometries: branched PEG has three to ten PEG chains derived from a central core group; star PEG has 10 to 100 PEG chains derived from a central core group; and comb PEG has a plurality of PEG chains typically grafted to the polymer backbone. PEG may also be linear. The numbers often included in PEG names indicate their average molecular weight (e.g., the average molecular weight of PEG with n=9 is about 400 daltons, and labeled PEG 400.

As used herein, "pegylation" is the operation of coupling (e.g., covalently coupling) a PEG structure to a hepcidin analog of the invention, which in certain embodiments is referred to as a "pegylated hepcidin analog". In certain embodiments, the PEG of the pegylated side chain is PEG having a molecular weight of about 200 to about 40,000. In certain embodiments, the PEG moiety of the conjugated half-life extending moiety is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In a particular embodiment, it is PEG11. In certain embodiments, the PEG of the pegylated spacer is PEG3 or PEG8. In some embodiments, the spacer is pegylated. In certain embodiments, the PEG of the pegylated spacer is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In certain embodiments, the PEG of the pegylated spacer is PEG3 or PEG8.

In some embodiments, the invention comprises hepcidin analog peptides (or dimers thereof) conjugated to PEG, covalently linked, e.g., through an amide, thiol, via click chemistry, or via any other suitable means known in the art. In particular embodiments, PEG is a certain PEG derivative used that is linked via an amide bond, and thus will be appropriately functionalized. For example, in certain embodiments, PEG11 is O- (2-aminoethyl) -O' - (2-carboxyethyl) -undecanediol, with amines and carboxylic acids both for attachment to the peptides of the invention. In certain embodiments, PEG25 contains a diacid and 25 diol moieties.

Other suitable polymeric moieties include polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid (see, e.g., gombotz et al, (1995), bioconjugate chemistry (Bioconjugate chem.), volume 6:332-351, hudecz et al (1992), bioconjugate chemistry, volume 3, 49-57, and Tsukada et al (1984), J.S. J.Natl.cancer Inst., volume 73:721-729. Polymeric moieties may be linear or branched, in some embodiments, have a molecular weight of 500 to 40,000Da, e.g., 500 to 10,000Da, 1000 to 5000Da, 10,000 to 20,000Da, or 20,000 to 40,000Da.

In some embodiments, the hepcidin analogs of the invention may comprise two or more such polymeric moieties, in which case the total molecular weight of all such moieties would generally be within the ranges provided above.

In some embodiments, the polymeric moiety may be coupled (via covalent linkage) to an amino, carboxyl, or thiol group of an amino acid side chain. Some examples are thiol groups of Cys residues and epsilon amino groups of Lys residues, and may also involve carboxyl groups of Asp and Glu residues.

The skilled worker will be well aware of suitable techniques that may be used to perform the coupling reaction. For example, a PEG moiety bearing a methoxy group may be coupled to a Cys thiol group via a maleimide linkage using reagents available from NektarTherapeutics AL. See also WO 2008/101017 and the references cited above for details on the appropriate chemistry. Maleimide functionalized PEG may also bind to the side chain sulfhydryl group of Cys residues.

As used herein, disulfide oxidation may occur in a single step or as a two-step process. As used herein, for a single oxidation step, trityl protecting groups are typically employed during assembly to allow deprotection during cleavage, followed by solution oxidation. When a second disulfide bond is desired, either native or selective oxidation may be selected. For selective oxidation requiring orthogonal protecting groups, acm and trityl are used as protecting groups for cysteine. Cleavage results in removal of one cysteine protected pair, allowing oxidation of this pair. Followed by a second oxidative deprotection step of the cysteine-protected Acm group. For natural oxidation, trityl protecting groups are used for all cysteines, taking into account the natural folding of the peptide.

The skilled artisan will be well aware of suitable techniques that may be used to perform the oxidation step.

In particular embodiments, the hepcidin analogs of the invention include half-life extending moieties that may be selected from, but are not limited to, the following: ahx-Palm, PEG2-Palm, PEG11-Palm, isoGlu-Palm, dapa-Palm, isoGlu-lauric acid, isoGlu-myristic acid, and isoGlu-isovaleric acid.

In certain embodiments, the hepcidin analogs include half-life extending moieties having the structures shown below, wherein n=0 to 24 or n=14 to 24:

in certain embodiments, the hepcidin analogs of the invention comprise a binding half-life extending moiety as shown in table 2.

TABLE 2 illustrative half-life extending moieties

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In certain embodiments, the half-life extending moiety binds directly to the hepcidin analog, while in other embodiments, the half-life extending moiety binds to the hepcidin analog peptide via a linker moiety (e.g., any of those depicted in table 3).

TABLE 3 illustrative linker moieties

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* Peg is- (OCH 2CH 2)

Referring to the linker structure shown in table 3, mention of n=1 to 24 or n=1 to 25, etc. (e.g. in L4 or L5) indicates that n may be any integer within the range. Other linker moieties may be used, shown in the "abbreviation" table.

In particular embodiments, the hepcidin analogs of the invention include any of the linker moieties shown in table 3 and any of the half-life extending moieties shown in table 2, including any of the following combinations shown in table 4.

TABLE 4 illustrative combinations of linkers and half-life extending moieties in hepcidin analogs

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In certain embodiments, the hepcidin analogs comprise two or more linkers. In certain embodiments, two or more linkers are in series, i.e., in combination with each other.

In related embodiments, the invention comprises polynucleotides encoding polypeptides having peptide sequences present in any of the hepcidin analogs described herein.

Furthermore, the invention encompasses vectors, e.g. expression vectors, comprising the polynucleotides of the invention.

Therapeutic method

In some embodiments, the invention provides methods for treating a subject suffering from a disease or disorder associated with dysregulation of hepcidin signaling, wherein the methods comprise administering to the subject a hepcidin analog of the invention. In some embodiments, the hepcidin analog administered to the subject is present in a composition (e.g., a pharmaceutical composition). In one embodiment, a method of treating a subject suffering from a disease or disorder characterized by an increase in the activity or expression of a membrane iron transporter is provided, wherein the method comprises administering to the subject an hepcidin analog or composition of the invention in an amount sufficient (partially or fully) to bind and agonize the membrane iron transporter or a mimetic in the subject. In one embodiment, a method of treating a subject suffering from a disease or disorder characterized by deregulated iron metabolism is provided, wherein the method comprises administering to the subject an hepcidin analog or composition of the invention.

In some embodiments, the methods of the invention comprise providing to a subject in need thereof an hepcidin analog or composition of the invention. In particular embodiments, there is a need for a subject to have been diagnosed with or to have been determined to be at risk of developing a disease or disorder characterized by a deregulation of iron content (e.g., an iron metabolism disease or disorder; a disease or disorder associated with iron overload; and a disease or disorder associated with aberrant hepcidin activity or expression). In particular embodiments, the subject is a mammal (e.g., a human).

In certain embodiments, the disease or disorder is an iron metabolism disorder, such as an iron overload disorder, an iron deficiency disorder, an iron biodistribution disorder, another iron metabolic disorder that may be associated with iron metabolism, and others. In particular embodiments, the iron metabolic disorder is hemochromatosis, HFE mutant hemochromatosis, membrane iron transporter mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojugglutinin mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha thalassemia, beta thalassemia, iron particle young cell anemia, porphyria, delayed skin lesion porphyria, african iron overload, hyperferrimia, ceruloplasmin deficiency, transferrin deficiency, congenital erythropoiesis abnormal anemia, glomerulonephrenic anemia, sickle cell disease, polycythemia vera (primary and secondary), secondary erythrocytopenia (such as chronic obstructive pulmonary disease), post renal transplantation mutations, chuva mutations, HIF and PHD mutations, and idiopathic myelodysplasia, pyruvate kinase deficiency, glomerular hypopigmentation anemia, transfusion dependent anemia (hemolytic anemia), iron deficiency obesity, other anemias, benign or malignant tumors that overproduce or induce overproduction of hepcidin, hepcidin excess conditions, friedreich ataxia, gracile syndrome, hallervorden-Spatz disease, wilson's disease, pulmonary ferrioxasis, hepatocellular carcinoma, cancer (e.g., liver cancer), hepatitis, cirrhosis, pica, chronic renal failure, insulin resistance, diabetes, atherosclerosis, neurodegenerative disorders, dementia, multiple sclerosis, parkinson's disease, huntington's disease and Alzheimer's disease.

In certain embodiments, the diseases and conditions are associated with iron overload diseases such as iron hemochromatosis, HFE mutant hemochromatosis, membrane iron transporter mutant hemochromatosis, transferrin receptor 2 mutant hemochromatosis, hemojul mutant hemochromatosis, hepcidin mutant hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusion iron overload, thalassemia, intermediate thalassemia, alpha thalassemia, sickle cell disease, myelodysplasia, iron granulomatoid juvenile cell infection, diabetic retinopathy and pyruvate kinase deficiency.

In certain embodiments, the disease or disorder is a disease or disorder that is not normally identified as being associated with iron. For example, hepcidin is highly expressed in murine pancreas, indicating that diabetes (type I or type II), insulin resistance, glucose intolerance, and other conditions can be ameliorated by treatment of underlying iron metabolic disorders. See Ilyin, g. Et al (2003) european society for biochemistry 542-26, which is incorporated herein by reference. Thus, the peptides of the invention are useful in the treatment of these diseases and conditions. One of ordinary skill in the art is readily able to determine whether a given disease can be treated with a peptide according to the present invention using methods known in the art, including the assays of WO 2004092405, incorporated herein by reference, and assays that monitor hepcidin, hemojuvelin, or iron content and expression, such as those described in U.S. patent No. 7,534,764, incorporated herein by reference.

In certain embodiments, the disease or disorder is post-menopausal osteoporosis (postmenopausal osteoporosis).

In certain embodiments of the invention, the iron metabolic disease is an iron overload disease comprising hereditary hemochromatosis, iron-excess type anemia, alcoholic liver disease, heart disease and/or failure, cardiomyopathy, and chronic hepatitis C.

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

In some embodiments, the methods of the invention comprise administering to a subject in need thereof a combination of a hepcidin analog of the invention (i.e., a first therapeutic agent) and a second therapeutic agent. In certain embodiments, the second therapeutic agent is provided to the subject prior to and/or concurrently with and/or after administration of the pharmaceutical composition to the subject. In certain embodiments, the second therapeutic agent is an iron chelator. In certain embodiments, the second therapeutic agent is selected from the following iron chelators: deferoxamine (Deferoxamine) and Deferasirox (exjame) ^TM ). In another embodiment, the method comprises administering a third therapeutic agent to the subject.

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

The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in "Lemmington's pharmaceutical sciences", 17 th edition, alfonso R.Gennaro (ed.), mark Publishing Company, easton, pa., USA, 1985. For example, sterile saline and phosphate buffered saline at slightly acidic or physiological pH may be used. Suitable pH buffers may be, for example, phosphate, citrate, acetate, TRIS (hydroxymethyl) aminomethane (TRIS), N-TRIS (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine or acetate (e.g., in the form of sodium acetate), or mixtures thereof. The term further encompasses any carrier listed in the united states Pharmacopeia (US Pharmacopeia) for use in animals, including humans.

In certain embodiments, the composition comprises two or more hepcidin analogs disclosed herein. In certain embodiments, the combination is one selected from the group consisting of: (i) Any two or more of the hepcidin analog peptide monomers shown therein; (ii) Any two or more of the hepcidin analog peptide dimers disclosed herein; (iii) Any one or more of the hepcidin analog peptide monomers disclosed herein and any one or more of the hepcidin analog peptide dimers disclosed herein.

It will be appreciated that inclusion of the hepcidin analogs of the invention (i.e., one or more hepcidin analog peptide monomers of the invention or one or more hepcidin analog peptide dimers of the invention) in pharmaceutical compositions also encompasses pharmaceutically acceptable salts or solvates comprising the hepcidin analogs of the invention. In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, excipients, or vehicles.

In certain embodiments, the invention provides a pharmaceutical composition comprising a hepcidin analog or a pharmaceutically acceptable salt or solvate thereof for use in treating a variety of conditions, diseases, or disorders as disclosed herein or elsewhere (see, e.g., methods of treatment herein). In certain embodiments, the invention provides a pharmaceutical composition comprising a hepcidin analog peptide monomer or a pharmaceutically acceptable salt or solvate thereof for use in treating a variety of conditions, diseases, or disorders as disclosed elsewhere herein (see, e.g., methods of treatment herein). In a particular embodiment, the invention provides a pharmaceutical composition comprising a hepcidin analog peptide dimer or a pharmaceutically acceptable salt or solvate thereof for use in treating a variety of conditions, diseases, or disorders as disclosed herein.

The hepcidin analogs of the invention may be formulated as pharmaceutical compositions suitable for administration with or without storage and generally include a therapeutically effective amount of at least one hepcidin analog of the invention and a pharmaceutically acceptable carrier, excipient or vehicle.

In some embodiments, the hepcidin analog pharmaceutical compositions of the invention are in unit dosage form. In such forms, the composition is divided into unit doses containing appropriate amounts of one or more active components. The unit dosage form may be in the form of a packaged preparation containing discrete amounts of the preparation, such as packaged tablets, capsules or powders in vials or ampoules. The unit dosage form itself may also be in the form of, for example, a capsule, cachet or tablet, or it may be the appropriate number of any of these packaged forms. The unit dosage form may also be provided in a single dose injectable form, for example in the form of a pen device containing a liquid (usually aqueous) phase composition. The compositions may be formulated for any suitable route and manner of administration, such as any of the routes and manners disclosed herein.

In certain embodiments, the hepcidin analog or pharmaceutical composition comprising the hepcidin analog is suspended in a sustained release matrix. As used herein, a sustained release matrix is a matrix made of a material (typically a polymer) that can be decomposed by enzymatic or acid-base hydrolysis or by dissolution. Once in the body, the matrix is subjected to enzymes and body fluids. The sustained release matrix is desirably selected from biocompatible materials such as liposomes, polylactic acid (polylactic acid), polyglycolide (glycolic acid polymer), polylactic acid-co-glycolide (copolymer 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, amino acids (such as phenylalanine, tyrosine, isoleucine), polynucleotides, polyethylene propylene, polyvinylpyrrolidone, and silicones. One embodiment of the biodegradable matrix is a matrix of one of polylactic acid lactide, polyglycolide or polylactic acid lactide co-glycolide (a copolymer of lactic acid and glycolic acid).

In certain embodiments, the composition is administered parenterally, subcutaneously, or orally. In particular embodiments, the composition is administered orally, intracisternally, intravaginally, intraperitoneally, intrarectally, topically (e.g., by powders, ointments, drops, suppositories, or transdermal patches, including intravitreal, intranasal, and inhaled delivery) or buccally. As used herein, the term "parenteral" refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intra-articular injections and infusions. Thus, in certain embodiments, the compositions are formulated for delivery by any of these routes of administration.

In certain embodiments, the pharmaceutical composition for parenteral injection comprises a pharmaceutically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion or sterile powder for reconstitution into a sterile injectable solution or dispersion immediately prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethyl cellulose and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of delayed absorbers, such as aluminum monostearate and gelatin.

Injectable depot forms include forms made by forming a microencapsulated matrix of an hepcidin analog in one or more biodegradable polymers such as polylactic acid lactide-polyglycolide, poly (orthoesters), poly (anhydrides) and (poly) glycols such as PEG. Depending on the ratio of peptide to polymer and the nature of the particular polymer used, the release rate of the hepcidin analog can be controlled. Depot injectable formulations are also prepared by entrapping hepcidin analogs in liposomes or microemulsions that are compatible with body tissue.

The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium at the point of use.

The hepcidin analogs of the invention may also be administered in the form of liposomes or other lipid-based carriers. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed from a single or multiple layers of hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. In addition to the hepcidin analogs of the invention, the compositions of the invention in liposome form may contain stabilizers, preservatives, excipients, and analogs thereof. In certain embodiments, the lipid comprises a phospholipid comprising phosphatidylcholine (lecithin) and serine, both natural and synthetic. Methods of forming liposomes are known in the art.

Pharmaceutical compositions suitable for parenteral administration in the present invention may comprise sterile aqueous solutions and/or suspensions of peptide inhibitors typically formulated isotonic with the blood of the recipient using sodium chloride, glycerol, glucose, mannitol, sorbitol and the like.

In some aspects, the invention provides a pharmaceutical composition for oral delivery. The compositions and hepcidin analogs of the invention can be prepared for oral administration according to any of the methods, techniques, and/or delivery vehicles described herein. Furthermore, it will be appreciated by those skilled in the art that the hepcidin analogs of the invention may be modified or integrated into systems or delivery vehicles not disclosed herein, but are well known in the art and suitable for oral delivery of peptides.

In certain embodiments, formulations for oral administration may include adjuvants (e.g., resorcinol and/or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyvinyl ether) to artificially increase the permeability of the intestinal wall, and/or enzymatic inhibitors (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) or aprotinin) to inhibit enzymatic degradation.

In particular embodiments, oral dosage forms or unit doses compatible for use with the hepcidin analogs of the invention may comprise a mixture of the hepcidin analog with non-pharmaceutical components or excipients, as well as other non-reusable materials that may be considered ingredients or packaging. The oral composition may comprise at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, an oral dosage form is provided comprising an effective amount of a hepcidin analog, wherein the dosage form comprises at least one of: pills, tablets, capsules, gels, pastes, drinks, syrups, ointments and suppositories. In some cases, an oral dosage form is provided that is designed and configured to achieve delayed release of a hepcidin analog in the small intestine and/or colon of a subject.

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

In one embodiment, the pharmaceutical composition comprising the hepcidin analogs of the invention is provided in the form of an enteric coating designed to protect and release the pharmaceutical composition in a controlled manner within the lower gastrointestinal system of a subject and avoid systemic side effects. In addition to enteric coatings, the hepcidin analogs of the invention can be encapsulated, coated, conjugated or otherwise associated within any compatible oral drug delivery system or component. For example, in some embodiments, the hepcidin analogs of the invention are provided in lipid carrier systems including at least one of polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

To overcome peptide degradation in the small intestine, some embodiments of the invention include a hydrogel polymer carrier system comprising the hepcidin analogs of the invention, wherein the hydrogel polymer protects the hepcidin analogs from proteolysis in the small intestine and/or colon. The hepcidin analogs of the invention may further be formulated for compatible use with a carrier system designed to increase the dissolution kinetics of the peptide and enhance intestinal absorption of the peptide. These methods include the use of liposomes, micelles, and nanoparticles to increase gastrointestinal tract penetration of peptides.

Various bioresponsive systems may also be combined with one or more hepcidin analogs of the invention to provide pharmaceutical agents for oral delivery. In some embodiments, the hepcidin analogs of the invention are conjugated to a bioreaction system (such as hydrogels and mucoadhesive polymers with hydrogen bonding groups (e.g., PEG, poly (meth) acrylic acid [ PMAA)]Cellulose, cellulose,Polyglucamine sugar and alginate) to provide a therapeutic agent for oral administration. Other embodiments include a method for optimizing or extending the drug residence time of a hepcidin analog disclosed herein, wherein the surface of the hepcidin analog surface is modified to include mucoadhesive properties via hydrogen bonding, polymers with attached mucin or/and hydrophobic interactions. According to a desired feature of the invention, these modified peptide molecules may exhibit an increase in drug residence time in a subject. Furthermore, the targeted mucoadhesive system can specifically bind to receptors at the surface of intestinal epithelial cells and M cells, thereby further increasing the uptake of the hepcidin analog-containing particles.

Other embodiments include a method for orally delivering a hepcidin analog of the invention, wherein the hepcidin analog is provided to a subject in combination with a permeation enhancer that facilitates transport of the peptide across the intestinal mucosa by increasing paracellular or intercellular permeation. For example, in one embodiment, a penetration enhancer is combined with the hepcidin analog, wherein the penetration enhancer comprises at least one of a long chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelator. In one embodiment, a permeation enhancer comprising sodium N- [ hydroxybenzoyl) amino ] caprylate is used to form a weaker non-covalent association with the hepcidin analog of the invention, wherein the permeation enhancer facilitates membrane transport and further dissociates upon reaching blood circulation. In another embodiment, the hepcidin analogs of the invention bind to oligoarginines, thereby increasing cellular penetration of peptides into various cell types. Furthermore, in at least one embodiment, a non-covalent bond is located between the peptide inhibitors of the present invention and a permeation enhancer selected from the group consisting of Cyclodextrins (CDs) and dendrimers, wherein the permeation enhancer reduces peptide aggregation and increases the stability and solubility of the hepcidin analog molecules.

Other embodiments of the invention provide a method of treating a subject with an hepcidin analog of the invention having an increased half-life. In one aspect, the invention provides an hepcidin analog having an in vitro or in vivo half-life (e.g., upon administration to a human subject) of at least a few hours to a day sufficient to administer a therapeutically effective amount daily (q.d.) or twice daily (b.i.d.). In another embodiment, the hepcidin analog has a half-life of three days or longer, sufficient to be administered a therapeutically effective amount weekly (q.w.). Furthermore, in another embodiment, the hepcidin analog has a half-life of eight days or longer, sufficient to administer a therapeutically effective amount every two weeks (b.i.w.) or monthly. In another embodiment, the hepcidin analog is derivatized or modified such that it has a longer half-life than the non-derivatized or non-modified hepcidin analog. In another embodiment, the hepcidin analogs contain one or more chemical modifications to increase serum half-life.

The hepcidin analogs of the invention, when used in at least one of the treatment or delivery systems described herein, can be used in pure form or in pharmaceutically acceptable salt form where such forms exist.

Dosage of

The total daily dosage of the hepcidin analogs and compositions of the invention may be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including: a) The condition being treated and the severity of the condition; b) The activity of the particular compound employed; c) The particular composition employed, the age, weight, general health, sex, and diet of the patient; d) The time of administration, route of administration, and rate of excretion of the particular hepcidin analog employed; e) Duration of treatment; f) Drugs used in combination or concurrently with the particular hepcidin analog employed, and like factors well known in the medical arts.

In particular embodiments, the total daily dose of the hepcidin analogs of the invention to be administered in single or divided doses to a human or other mammalian host may be in an amount of, for example, 0.0001 to 300mg per kilogram of body weight per day or 1 to 300mg per kilogram of body weight per day. In certain embodiments, the hepcidin analogs of the invention are administered in a dosage range of about 0.0001 to about 100mg per kilogram of body weight per day, such as about 0.0005 to about 50mg per kilogram of body weight per day, such as about 0.001 to about 10mg per kilogram of body weight per day, for example about 0.01 to about 1mg per kilogram of body weight per day, in one or more doses, such as one to three doses. In particular embodiments, for example, for a human patient, the total dose is about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, or about 10mg, about once or twice a week. In particular embodiments, the total dose is in the range of about 1mg to about 5mg, or about 1mg to about 3mg, or about 2mg to about 3mg, for each human patient, e.g., about once a week.

In various embodiments, the hepcidin analogs of the invention may be administered continuously (e.g., by intravenous administration or another continuous method of drug administration), or may be administered to a subject at intervals, typically at regular intervals, depending on the desired dosage and pharmaceutical composition selected by one of skill in the art for the particular subject. Regular administration intervals include, for example, once daily, twice daily, once every two days, once every three days, once every four days, once every five days, or once every six days, once or twice weekly, once or twice monthly, and the like.

Such regular hepcidin analogue administration regimens of the invention may be advantageously discontinued for a period of time in certain circumstances, such as during prolonged administration, to allow the subject to reduce the level of drug administration or to discontinue administration of the drug, commonly referred to as a drug holiday. Drug holidays are useful, for example, to maintain or restore sensitivity to a drug, especially during long-term sustained treatment, or to reduce adverse side effects of long-term sustained treatment in subjects taking the drug. The timing of drug holidays depends on the timing of the regular dosing regimen and the purpose of the drug holidays (e.g., restoring drug sensitivity and/or reducing non-desirable side effects of continuous long-term administration). In some embodiments, the drug holiday may be a decrease in the drug dose (e.g., less than a therapeutically effective amount for a time interval). In other embodiments, drug administration is stopped for a time interval before administration is initiated again using the same or a different dosing regimen (e.g., at a lower or higher dose and/or frequency of administration). Thus, the drug holidays of the present invention may be selected from a variety of time periods and dosage regimens. Exemplary drug holidays are drug holidays of two or more days, one or more weeks, or one or more months, up to about 24 months. Thus, for example, a conventional daily dosing regimen with a peptide, peptide analog or dimer of the invention may be, for example, interspersed with a one-week or two-week or four-week drug holiday, after which the aforementioned conventional dosing regimen (e.g., daily or weekly dosing regimen) is resumed. A variety of other drug holiday regimens are contemplated for administration of the hepcidin analogs of the invention.

Thus, hepcidin analogs can be delivered via an administration regimen comprising two or more administration phases separated by respective drug holiday phases.

During each administration phase, the hepcidin analog is administered to the recipient subject in a therapeutically effective amount according to a predetermined mode of administration. The mode of administration may include continuing to administer the drug to the recipient subject for the duration of the administration phase. Alternatively, the mode of administration may comprise administering a plurality of doses of the hepcidin analog to the recipient subject, wherein the doses are separated by a dosing interval.

The mode of administration may include at least two administrations per administration phase, at least five administrations per administration phase, at least 10 administrations per administration phase, at least 20 administrations per administration phase, at least 30 administrations per administration phase, or more.

The dosing interval may be a conventional dosing interval as set forth above, including once daily, twice daily, once every two days, once every three days, once every four days, once every five days or once every six days, once or twice weekly, once or twice monthly, or a conventional and even infrequent dosing interval, depending on the particular dosage formulation, bioavailability, and pharmacokinetic profile of the hepcidin analogs of the invention.

The administration phase may have a duration of at least two days, at least one week, at least 2 weeks, at least 4 weeks, at least one month, at least 2 months, at least 3 months, at least 6 months, or more.

When the mode of administration comprises multiple administrations, the duration of the subsequent drug holiday phase is longer than the dosing interval used in the mode of administration. In the case of irregular dosing intervals, the duration of the drug holiday phase may be greater than the average interval between doses over the course of the administration phase. Alternatively, the duration of the drug holiday may be longer than the longest interval between successive administrations during the administration phase.

The duration of the drug holiday phase may be at least twice the relevant dosing interval (or average thereof), at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times the relevant dosing interval or average thereof.

Within these limits, the drug holiday phase may have a duration of at least two days, at least one week, at least 2 weeks, at least 4 weeks, at least one month, at least 2 months, at least 3 months, at least 6 months, or longer, depending on the mode of administration during the aforementioned administration phase.

The administration regimen comprises at least 2 administration phases. The successive administration phases are separated by corresponding drug holiday phases. Thus, an administration regimen may comprise 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 separated by a respective drug holiday phase.

The successive administration phases may utilize the same mode of administration, but may not be ideal or necessary. However, if other drugs or active agents are administered in combination with the hepcidin analogs of the invention, the same combination of drugs or active agents is typically administered in successive administration phases. In certain embodiments, the subject is a human.

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

In some embodiments, the invention provides a method of making a hepcidin analog or a hepcidin analog composition (e.g., a pharmaceutical composition) as disclosed herein.

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

In some embodiments, the invention provides methods of binding to a membrane iron transporter or inducing internalization and degradation of a membrane iron transporter, comprising contacting a membrane iron transporter with at least one hepcidin analog or hepcidin analog composition as disclosed herein.

In some embodiments, the invention provides methods of binding to a ferroportin to block pore and sink functions without causing ferroportin internalization. Such methods comprise contacting a membrane iron transporter with at least one hepcidin analog or hepcidin analog composition as disclosed herein.

In some embodiments, the invention provides kits comprising at least one hepcidin analog or hepcidin analog composition (e.g., a pharmaceutical composition) as disclosed herein, packaged with reagents, devices, instructional materials, or combinations thereof.

In some embodiments, the invention provides a method of administering the hepcidin analogs or hepcidin analog compositions (e.g., pharmaceutical compositions) of the invention to a subject via an implanted or osmotic pump, through a filter cartridge or micropump, or by other means known to the skilled artisan, as is well known in the art.

In some embodiments, the invention provides a complex comprising at least one Hep analog or antibody (such as an antibody that specifically binds to Hep analog, hep25, or a combination thereof as disclosed herein) that binds to a membrane iron transporter (preferably a human membrane iron transporter) as disclosed herein.

In some embodiments, the hepcidin analogs of the invention measure within a FPN internalization assay (e.g., EC ₅₀ ) Less than 500nM. Those skilled in the art will recognize that the function of a hepcidin analog depends on the tertiary structure of the hepcidin analog and the binding surface presented. Thus, it is possible to make minor changes to the sequence encoding the hepcidin analog, which do not affect folding or affect binding surfaces and maintain function. In other embodiments, the invention provides an hepcidin analog having 85% or greater (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) identity or homology to the amino acid sequence of any hepcidin analog described herein that exhibits activity (e.g., hepcidin activity) or alleviates symptoms of a disease or indication in which hepcidin is involved.

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

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

In addition to the methods described in the examples herein, the hepcidin analogs of the invention can be produced using methods known in the art, including chemical synthesis, biosynthesis using recombinant DNA methods, or in vitro synthesis and solid phase synthesis. See, e.g., kelly and Winkler (1990) engineering principles and methods (Genetic Engineering Principles and Methods), vol.12, J.K. Setlow, planum Press, NY, pages 1 to 19; merrifield (1964) American society of chemistry (J Amer chemSoc) 85:2149; houghten (1985) PNAS USA 82:5131-5135; and Stewart and Young (1984) solid phase Synthesis (Solid Phase Peptide Synthesis), 2 nd edition, pierce, rockford, ill, incorporated herein by reference. The hepcidin analogs of the invention can be purified using protein purification techniques known in the art, such as reverse phase High Performance Liquid Chromatography (HPLC), ion exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis. See Olsnes, s. And a.pihl (1973) biochemistry (biochem.) 12 (16): 3121-3126; and scope (1982) protein purification (Protein Purification), springer-Verlag, N.Y., which is incorporated herein by reference. Alternatively, hepcidin analogs of the invention may be made by recombinant DNA techniques known in the art. Thus, polynucleotides encoding the polypeptides of the invention are encompassed herein. In certain preferred embodiments, the polynucleotide is isolated. As used herein, an "isolated polynucleotide" refers to a polynucleotide that is in an environment that is different from the environment in which the polynucleotide naturally occurs.

Examples

The following examples demonstrate certain specific embodiments of the invention. Unless otherwise specifically described, the following examples were carried out using standard techniques well known and commonly used by those skilled in the art. It is to be understood that these examples are for illustrative purposes only and are not intended to fully determine the condition or scope of the present invention. Therefore, it should not be construed as limiting the scope of the invention in any way.

Abbreviations:

DCM: dichloromethane (dichloromethane)

DMF: n, N-dimethylformamide

NMP: n-pyrrolidone

HBTU: o- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

HATU:2- (7-aza-1H-benzotriazol-1-yl) -1, 3-tetramethyluronium hexafluorophosphate

DCC: dicyclohexylcarbodiimide

NHS: n-hydroxysuccinimide

DIPEA: diisopropylethylamine

EtOH: ethanol

Et2O: diethyl ether

Hy: hydrogen gas

TFA: trifluoroacetic acid

TIS: triisopropyl silane

ACN: acetonitrile

HPLC: high performance liquid chromatography

ESI-MS: electron spray ionization mass spectrum

PBS: phosphate buffered saline

Boc: t-Butoxycarbonyl group

Fmoc: fluorenylmethoxycarbonyl radicals

Acm: acetamidomethyl

IVA: isovaleric acid (or isovaleryl)

K (): in the peptide sequences provided herein in which a compound or chemical group is presented in parentheses following a lysine residue, it is understood that the compound or chemical group in parentheses is a side chain that binds to the lysine residue. Thus, for example, but not limited to, in any way, K- [ (PEG 8) ] -indicates that the PEG8 moiety is bound to a side chain of said lysine.

Palm: indicating binding of palmitic acid (palmitoyl).

Synthesis scheme-1

Synthesis of peptide monomers

Peptide monomers of the invention were synthesized on a multichannel synthesizer (SymphonyProtein Technology's Symphony multiple channel synthesizer) of protein technology using Merrifield solid phase synthesis technology. Peptides were assembled using HBTU (O-benzotriazole-N, N' -tetramethyl-uronium-hexafluoro-phosphate), diisopropylethylamine (DIEA) coupling conditions. For some amino acid couplings, pyAOP (7-azabenzotriazol-1-yloxy) tripyrrolidinylphosphonium hexafluorophosphate) and DIEA conditions were used. Rink Amide MBHA resin (100 to 200 mesh, 0.57 mmol/g) was used for peptides with C-terminal Amide and preloaded Wang resin with N- α -Fmoc protected amino acids was used for peptides with C-terminal acid. Coupling reagent (premixed HBTU and DIEA) was prepared at a concentration of 100 mmol. Similarly, 100mmol concentration amino acid solution was prepared. The peptide inhibitors of the invention are identified based on pharmacochemistry optimization and/or phage display and screened to identify peptide inhibitors with excellent binding and/or inhibition properties.

Assembly

Peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: the resin (250 mg,0.14 mmol) in each reaction vial was washed twice with 4ml DMF and then treated with 2.5ml 20% 4-methylpiperidine (removal of Fmoc protection) for 10 minutes. The resin was then filtered and washed twice with DM (4 ml) and treated with N-methylpiperidine for a further 30 minutes. The resin was washed three more times with DMF (4 mL) and then 2.5mL of amino acid and 2.5mL of HBTU-DIEA mixture were added. After frequent stirring for 45 minutes, the resin was filtered and washed three times with DMF (4 ml each). For typical peptides of the invention, double coupling is performed. After the coupling reaction was completed, the resin was washed three times (4 ml each) with DMF and then the next amino acid coupling was continued.

Cleavage of

After peptide assembly, the peptide is 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). Cleavage reagents successfully cleave the peptide from the resin and all remaining side chain protecting groups.

The cleaved peptide was precipitated in low temperature diethyl ether and then washed twice with ethyl ether. The filtrate was filtered off and a second aliquot of the low temperature ether was added and the procedure repeated. The crude peptide was dissolved in acetonitrile: water (7:3 with 1% tfa) and filtered. The mass of the linear peptide was then verified using electrospray ionization mass spectrometry (ESI-MS) (Micromass/Waters ZQ) prior to purification.

Purification

Analytical reverse phase High Performance Liquid Chromatography (HPLC) was performed on a Gemini C18 column (4.6 mm. Times.250 mm) (Phenomnex). Semi-preparative reverse phase HPLC was performed on a Gemini 10. Mu. m C18 column (22 mm. Times.250 mm) (Phenomnex). Separation was achieved using a linear gradient of buffer B in A (mobile phase A: water with 0.15% TFA, mobile phase B: acetonitrile (ACN) with 0.1% TFA) at a flow rate of 1mL/min (analytical) and 20mL/min (preparative). Separation was achieved using a linear gradient of buffer B in A (mobile phase A: water with 0.15% TFA, mobile phase B: acetonitrile (ACN) with 0.1% TFA) at a flow rate of 1mL/min (analytical) and 15mL/min (preparative).

Synthesis scheme-2

Synthesis of peptide monomers

The peptide monomers of the invention are synthesized in CEM Liberty Blue using standard Fmoc solid phase synthesis techniques ^TM And (3) synthesizing on a microwave peptide synthesizer. Peptides were assembled using Oxyma/DIC (cyanoethyl hydroxyiminoacetate/diisopropylcarbodiimide) under microwave heating. Rink Amide MBHA resin (100 to 200 mesh, 0.66 mmol/g) was used for peptides with C-terminal Amide and preloaded Wang resin with N- α -Fmoc protected amino acids was used for peptides with C-terminal acid. Oxyma was prepared as a 1M DMF solution containing 0.1M DIEA. DIC was prepared as a 0.5M DMF solution. 200mM of amino acid was prepared. The peptide inhibitors of the invention are identified based on pharmacochemistry optimization and/or phage display and screened to identify peptide inhibitors with excellent binding and/or inhibition properties.

Assembly

Using standard CEM Liberty Blue ^TM Scheme preparation of peptides. The peptide sequences were assembled as follows: the resin (400 mg,0.25 mmol) was suspended in 10ml 50/50 DMF/DCM. The resin is then transferred to a reaction vessel in a microwave cavity. Using repeated removalFmoc protection and Oxyma/DIC coupling cycles assembled peptides. For deprotection, DMF containing 20% 4-methylpiperidine was added to the reaction vessel and heated to 90 ℃ for 65 seconds. The deprotected solution was drained and the resin was washed three times with DMF. For most of the amino acids, 5 equivalents of amino acids, oxyma and DIC were then added to the reaction vessel and the mixed reactants were rapidly heated to 90 ℃ for 4 minutes by microwave irradiation. For arginine and histidine residues, milder conditions of the respective temperatures 75 ℃ and 50 ℃ were used for 10 minutes to prevent racemization. Usually only 1.5 to 2 equivalents of reagents are used to manually couple rare and expensive amino acids at room temperature overnight. The difficult coupling is usually a double coupling at 90℃for 2X 4 minutes. After coupling, the resin was washed with DMF and the entire cycle was repeated until the desired peptide assembly was complete.

Cleavage of

After completion of peptide assembly, the peptide was then purified by using 91:5:2:2TFA/H ₂ Standard cleavage mixtures of O/TIPS/DODT were treated for 2 hours to cleave from the resin. If more than one Arg (pbf) residue is present, cleavage is allowed to continue for an additional hour.

The cleaved peptide was precipitated in low temperature diethyl ether. The filtrate was decanted and a second aliquot of the low temperature ether was added and the procedure repeated. Followed by electrospray ionization mass spectrometry (ESI-MS) prior to purificationZQ ^TM ) The quality of the linear peptide was verified.

Purification

At the position ofAnalytical reverse phase High Performance Liquid Chromatography (HPLC) was performed on a C18 column (4.6 mm. Times.250 mm) (Phenomnex). At->10 mu m C column (22 mm. Times.250 mm) (Phenomnex) or +.>Semi-preparative reverse phase HPLC was performed on a 10 μm,300A C18 column (21.2 mm. Times.250 mm) (Phenomnex). Separation was achieved using a linear gradient of buffer B in A (mobile phase A: water with 0.15% TFA, mobile phase B: acetonitrile (ACN) with 0.1% TFA) at a flow rate of 1mL/min (analytical) and 20mL/min (preparative).

Example 1A

Synthesis of peptide analogues

Unless otherwise indicated, reagents and solvents employed hereinafter are commercially available as standard laboratory reagents or analytical grades and are used without further purification.

Solid phase peptide synthesis procedure

Method A

The peptide analogues of the invention were chemically synthesized using an optimized 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis scheme. For the C-terminal amide, rink-amide resin was used, but wang and trityl resins were also used to make the C-terminal acid. The side chain protecting groups are as follows: glu, thr and Tyr: O-tButyl; trp and Lys: t-Boc (t-butoxycarbonyl); arg: n-gamma-2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl; his, gln, asn, cys: trityl. For selective disulfide bridge formation, acm (acetamidomethyl) was also used as Cys protecting group. For coupling, a four to ten-fold excess of a solution containing Fmoc amino acid, HBTU and DIEA (1:1:1.1) in DMF was added to the expanded resin [ HBTU: O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate; DIEA: diisopropylethylamine; DMF: dimethylformamide ]. HATU (O- (7-azabenzotriazol-1-yl) -1, 3, -tetramethyluronium hexafluorophosphate) was used instead of HBTU to improve coupling efficiency in difficult areas. Fmoc protecting group removal was achieved by treatment with DMF piperidine (2:1) solution.

Method B

Alternatively, peptides were synthesized using a CEM liberty Blue microwave-assisted peptide synthesizer. FMOC protection was removed using Liberty Blue by adding DMF with 20% 4-methylpiperidine and DMF with 0.1M Oxyma, and then heating to 90 ℃ using microwave irradiation for 4 minutes. After washing with DMF, FMOC-amino acids were coupled by adding 0.2M amino acid (4 to 6 equivalents), 0.5M DIC (4 to 6 equivalents) and 4 to 6 equivalents of 1M Oxyma (containing 0.1M DIEA) (all in DMF). The coupling solution was heated to 90 ℃ using microwave radiation for 4 minutes. When Arg or other sterically hindered amino acid is to be coupled, a second coupling is used. When coupled with histidine, the reaction was heated to 50 ℃ for 10 minutes. The cycle is repeated until full length peptide is obtained.

Procedure for cleavage of peptides from resins

Side chain deprotection and cleavage of peptide analogs (e.g., compound No. 2) of the invention is achieved by stirring the anhydrous resin in a solution containing trifluoroacetic acid, water, ethanedithiol, and triisopropylsilane (90:5:2.5:2.5) for 2 to 4 hours. After TFA removal, the peptide was precipitated using ice-cold diethyl ether. The solution was centrifuged and the ether decanted, followed by a second diethyl ether wash. The peptide was dissolved in acetonitrile in water (1:1) containing 0.1% tfa (trifluoroacetic acid), and the resulting solution was filtered. Linear peptide mass was assessed using electrospray ionization mass spectrometry (ESI-MS).

Peptide purification procedure

Purification of the peptides of the invention (e.g., compound No. 2) is accomplished using reverse phase high performance liquid chromatography (RP-HPLC). Analysis was performed using a C18 column (3 μm, 50X 2 mm) at a flow rate of 1 mL/min. Purification of the linear peptide was achieved with a C18 column (5 μm, 250X 21.2 mm) using preparative RP-HPLC at a flow rate of 20 mL/min. The separation was achieved using a linear gradient of buffer B in A (buffer A:0.05% aqueous TFA; buffer B:0.043% TFA,90% aqueous acetonitrile).

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

Binding of half-life extending moieties

Peptide binding was performed on the resin. Lys (ivDde) was used as a key amino acid. After the peptide was assembled on the resin, the ivDde group protection was selectively removed using DMF containing 3×5min 2% hydrazine for 5 min. The linker was activated and acylated with 1 to 2 equivalents of HBTU, DIEA for 3 hours, and Fmoc was removed, followed by a second acylation with lipid to give the binding peptide.

Example 1B

Synthesis of ID #16 peptide:

isovaleric acid [ Ala (2) ] -T-H- [ Dpa ] -P-A-I- [ Ala (2) ] - [ bhPre ] - [ Lys (Ahx_palm) ] - [ (D) Lys ] -NH2; and side chain C of Ala (2) cyclize via-CH 2-CH=CH-CH 2- (S/S isomer)

TFA salts of the ID #16 peptide were synthesized on Rink amide resin. Upon completion 269mg of peptide ID#16 was isolated as a white powder with a purity of > 93.5%.

The ID #16 peptide was constructed on Rink Amide MBHA (100 to 200 mesh, 0.27 mmol/g) resin using standard Fmoc protected synthesis conditions. The peptide constructed is separated from the resin and protecting group by cleavage with a strong acid followed by precipitation. The crude precipitate was then purified by RP-HPLC. The pure fractions were lyophilized to give the final product id#16 peptide.

Peptide assembly

Expansion resin: 3703mg Rink Amide MBHA solid phase resin (0.27 mmol/g load) was transferred to a 250mL reaction vessel. The resin was swelled with 60mL DMF (2 hours).

Step 1: coupling of FMOC- (D) Lys (Boc) -OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC- (D) Lys (Boc) -OH (400 mM) and 7.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 2: coupling of FMOC-L-Lys (Dde) -OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-L-Lys (Dde) -OH (400 mM) and 7.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 3: coupling of FMOC-. Beta.homo-L-Phe-OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-. Beta.homo-L-Phe-OH (400 mM) and 7.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 4: coupling of (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-hexenoic acid: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF (400 mM) containing the amino acid (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-hexenoic acid and 7.5mL of DMF (400 and 800 mM) containing the coupling reagent HATU-DIEA mixture were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60ml dmf (3 x 0.1 min) and then the next deprotection/coupling cycle was started.

Step 5: coupling of FMOC-Ile-OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-Ile-OH (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 6: coupling of FMOC-Ala-OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL DMF containing the amino acid FMOC-Ala-OH (400 mM) and 7.5mL DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 6': coupling of FMOC-Pro-OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL DMF containing the amino acid FMOC-Pro-OH (400 mM) and 7.5mL DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 7: coupling of FMOC-L-DIP-OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-L-DIP-OH (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 8: coupling of FMOC-L-His (Trt) -OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-L-His (Trt) -OH (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 9: coupling of FMOC-L-Thr (tBu) -OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-L-Thr (tBu) -OH (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 10: coupling of (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-hexenoic acid: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF (400 mM) containing the amino acid (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-hexenoic acid and 7.5mL of DMF (400 and 800 mM) containing the coupling reagent HATU-DIEA mixture were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60ml dmf (3 x 0.1 min) and then the next deprotection/coupling cycle was started.

Step 11: coupling of isovaleric acid: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL of LDMF (5X 0.1 min), followed by the addition of 7.5mL of DMF containing the amino acid isovaleric acid (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM). The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 12: dde removal and Fmoc-Ahx-OH coupling: ivDde was removed from the Lys C-terminus of the resin bound peptide using DMF containing 3% hydrazine (3 x 20 min) followed by DMF washing. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid Fmoc-Ahx-OH (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 13: coupling of palmitic acid: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL of LDMF (5X 0.1 min), then 7.5mL of DMF containing the amino acid palmitic acid (400 mM) and 7.5mL of DMF containing the coupling reagent HATU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 14: closed loop metathesis. The resin was washed with 60ml DCM (3X 1 min) and then treated with 50ml of a solution of 6mM Grubbs' first generation catalyst in DCE (5.01 mg ml-1; 30mol% relative to resin substitution). The solution was reacted twice under microwave conditions at 60 c (2 h) under nitrogen and then discharged. The resin was washed three times with DMF (60 ml each); washed with DCM (60 ml) before drying and cleavage.

Step 15: TFA cleavage and isopropyl ether precipitation: 60ml of cleavage mix [ TFA cleavage mix (90/2.5/2.5/5 TFA/water/Tips/DTT) was added to the protected resin binding peptide and shaken for three hours. Low temperature isopropyl ether was added to form a white precipitate, followed by centrifugation. The isopropyl ether was decanted into waste and the precipitate was again subjected to 2 isopropyl ether washes. The resulting white precipitate cake was dissolved in acetonitrile/water (1:1) and filtered prior to purification.

Step 16: RP-HPLC purification: semi-preparative reverse phase HPLC was performed on an Xtime 10. Mu. m C18 column (50 mm. Times.250 mm) (SHIMADZU LC-8A). Separation was achieved using a linear gradient of buffer B in A (mobile phase A: water with 0.075% TFA, mobile phase B: acetonitrile (ACN)) at a flow rate of 80mL/min (preparative).

Step 17: final lyophilization and analysis: the fractions collected were analyzed by analytical RP-HPLC and all fractions >95% pure were pooled. The combined fractions were lyophilized to give #16 peptide as a white powder with a purity of 93.5%. The purified #16 peptide was subjected to low resolution LC/MS to give 1 charged state of the peptide, m+2/2 was 904.9, and molecular ion [ m+1] was 1808.4. The experimental mass is consistent with the theoretical mass 1808.4Da [ M+1 ].

Example 1C

Synthesis of id#33 peptide:

isovaleric acid- [ Ala (3) ] -T-H- [ Dpa ] -P- [ Lys (Ahx_palm) ] -I- [ Ala (3) ] -bhpe ] -NH2; and side chain C of Ala (3) cyclize via-CH 2-ch=ch-CH 2- (S/S isomer)

TFA salts of the id#33 peptide were synthesized on Rink amide resin. After completion, 17.2mg of ID#33 peptide was isolated as a white powder with a purity of > 87.1%.

The ID #33 peptide was constructed on Rink Amide MBHA (100 to 200 mesh, 0.27 mmol/g) resin using standard Fmoc protected synthesis conditions. The peptide constructed is separated from the resin and protecting group by cleavage with a strong acid followed by precipitation. The crude precipitate was then purified by RP-HPLC. The pure fractions were lyophilized to give the final product ID#33 peptide.

Peptide assembly

Expansion resin: 200mg Rink Amide MBHA solid phase resin (0.27 mmol/g load) was transferred to a 250mL reaction vessel. The resin was swelled with 60mL DMF (2 hours).

Step 1: coupling of FMOC-. Beta.homo-L-Phe-OH: protection against removal of Fmoc groups was achieved by adding 60ml of 20% piperidine in DMF to the swollen Rink Amide resin for 30 min. After deprotection, the resin was washed with 60mL DMF (5X 0.1 min) and then 7.5mL of DMF containing the amino acid FMOC-. Beta.homo-L-Phe-OH (400 mM) and 7.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (400 and 800 mM) were added. The coupling reaction was mixed for 1 hour and filtered. After the coupling reaction was completed, the resin was washed with 60mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 2: coupling of (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -6-heptenoic acid: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL of DMF (3X 0.1 min) and then 2.5mL of DMF (200 mM) containing the amino acid (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-hexenoic acid and 2.5mL of DMF (200 and 220 mM) containing a mixture of coupling reagents HBTU-DIEA were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 3: coupling of FMOC-Ile-OH: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid FMOC-Ile-OH (200 mM) and 2.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 4: coupling of FMOC-L-Lys (IVDde) -OH: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL of DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid FMOC-L-Lys (IVDde) -OH (200 mM) and 2.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 5: coupling of FMOC-Pro-OH: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid FMOC-Pro-OH (200 mM) and 2.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 6: coupling of FMOC-L-DIP-OH: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid FMOC-L-DIP-OH (200 mM) and 2.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 7: coupling of FMOC-L-His (Trt) -OH: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid FMOC-L-His (Trt) -OH (200 mM) and 2.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 8: coupling of FMOC-L-Thr (tBu) -OH: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid FMOC-L-Thr (tBu) -OH (200 mM) and 2.5mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 9: coupling of (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-heptenoic acid: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL of DMF (3X 0.1 min) and then 2.5mL of DMF (200 mM) containing the amino acid (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl ] amino ] -5-hexenoic acid and 2.5mL of DMF (200 and 220 mM) containing a mixture of coupling reagents HBTU-DIEA were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 10: coupling of isovaleric acid: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL DMF containing isovaleric acid (200 mM) and 2.5mL DMF (200 and 220 mM) containing coupling reagent HBTU-DIEA mixture were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 11: ivDde removal and Fmoc-Ahx-OH coupling: ivDde was removed from the Lys C-terminus of the resin binding peptide using DMF containing 2 to 5% hydrazine (4 x 30 min) followed by DMF washing. After deprotection, the resin was washed with 3.75mL of DMF (3X 0.1 min) and then 2.5mL of DMF containing the amino acid Fmoc-Ahx-OH (200 mM) and 2.0mL of DMF containing the coupling reagent HBTU-DIEA mixture (200 and 220 mM) were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 12: coupling of palmitic acid: fmoc group removal protection was achieved by treating twice with 2.5ml of 20% piperidine in DMF to swell the Rink Amide resin for 5 and 10 minutes, respectively. After deprotection, the resin was washed with 3.75mL DMF (3X 0.1 min) and then 2.5mL DMF containing isovaleric acid (200 mM) and 2.5mL DMF (200 and 220 mM) containing coupling reagent HBTU-DIEA mixture were added. The coupling reaction was mixed for 1 hour, filtered and repeated once (double coupling). After the coupling reaction was completed, the resin was washed with 6.25mL DMF (3X 0.1 min) and then the next deprotection/coupling cycle was started.

Step 13: closed loop metathesis. The resin was washed with 2ml DCM (3X 1 min) and then with 2ml DCE (3X 1 min), then treated with 2ml of a solution of 6mM Grubbs' first generation catalyst in DCE (4.94 mg ml-1; 20mol% relative to resin substitution). The solution was refluxed overnight (12 h) under nitrogen and then vented. The resin was washed three times with DMF (4 ml each); washed with DCM (4 ml) before drying and lysis

Step 14: TFA cleavage and ether precipitation: 10ml of cleavage mix [ TFA cleavage mix (90/5/2.5/2.5 TFA/water/Tips/DODT) was added to the protected resin binding peptide and shaken for two hours. Low temperature diethyl ether was added to form a white precipitate, followed by centrifugation. The diethyl ether was decanted into the waste material and the precipitate was subjected to a further 2 diethyl ether washes. The resulting white precipitate cake was dissolved in acetonitrile/water (7:3) and filtered prior to purification.

Step 15: RP-HPLC purification: at the position ofSemi-preparative reverse phase HPLC was performed on a 10. Mu. m C18 column (22 mm. Times.250 mm) (Phenomnex). Separation was achieved using a linear gradient of buffer B in A (mobile phase A: water with 0.15% TFA, mobile phase B: acetonitrile (ACN) with 0.1% TFA) at a flow rate of 20mL/min (preparative).

Step 16: final lyophilization and analysis: the fractions collected were analyzed by analytical RP-HPLC and all fractions >95% pure were pooled. The combined fractions were lyophilized to give the id#33 peptide as a white powder with a purity of 87.1%. The purified ID #33 was subjected to low resolution LC/MS to give 2 charged states of the peptide, M+2/2 was 883.2, and molecular ion [ M+1] was 1765.4. The experimental mass is consistent with theoretical mass 1765.4Da [ M+1 ].

Example 2

Activity of peptide analogues

Peptide analogs were tested in vitro to induce internalization of human membrane iron transporters. After internalization, the membrane iron transporter is degraded. The assay used (FPN activity assay) measures the decrease in fluorescence of the receptor.

The cDNA encoding the human membrane iron transporter (SLC 40A 1) was cloned from a cDNA clone from origin (NM-014585). DNA encoding the ferroportin was amplified by PCR using primers that also encoded terminal restriction sites for subcloning but no stop codon. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker such that the ferroportin reading frame was fused in-frame with the GFP protein. The fidelity of the DNA encoding the protein was confirmed by DNA sequencing. Transfection of membrane iron transport using HEK293 cellsprotein-GFP receptor expressing plastids. Cells were grown in growth medium according to standard protocols and transfected by plastid using Lipofectamine (Lipofectamine) (manufacturer's protocol, invitrogen). Selection of cells stably expressing Membrane iron Transporter-GFP (where only cells that have taken up and bound to cDNA expressing plastids survive) in growth medium using G418 and in Cytomation MoFlo ^TM The cells were sorted several times on a cell sorter to obtain GFP positive cells (488 nm/530 nm). Cells were propagated in aliquots and frozen.

To determine the activity of hepcidin analogs (compounds) on human membrane iron transporters, cells were incubated in 96-well plates in standard medium without phenol red. Compounds were added to the incubator to reach the desired final concentration for at least 18 hours. After incubation, by whole-cell GFP fluorescence (Envision plate reader, 485/535 filter pair) or by Beckman Coulter Quanta ^TM The residual GFP fluorescence (expressed as the geometric mean of the fluorescence intensities at 485nm/525 nm) was measured by flow cytometry. The compound is added to the incubator to achieve the desired final concentration for at least 18 hours, but not more than 24 hours.

In some experiments, the reference compounds include natural hepcidin, micro-hepcidin, and R1-micro-hepcidin as an analog of micro-hepcidin. "RI" in RI-micro hepcidin refers to the reverse reaction. A retro peptide is a peptide with a retro sequence in all D amino acids. For example, hy-Glu-Thr-His-NH ₂ Will become Hy-DHis-DThr-DGlu-NH ₂ . EC of these reference compounds for membrane iron transporter internalization/degradation was determined according to the FPN activity assay described above ₅₀ . These peptides were used as control standards.

TABLE 5 reference Compounds

Efficacy IC determined for various peptide analogs of the invention ₅₀ Or EC (EC) ₅₀ Values (nM) are provided in tables 6A-6C. These values were determined as described herein. Compound ID is numbered by "Compd ID "and the reference compound is indicated by" ref. FPN EC determined from these data ₅₀ The values are shown in tables 6A to 6C. Where not shown, the data has not been determined.

TABLE 6A illustrative hepcidin analogs

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no-CH is present between the side chain carbon of the @ Ala (1) -alanine and the carbon of the vinyl group ₂ -; ala (2) -alanine has a-CH between the side chain carbon and the vinyl carbon ₂ -; two-CH exist between the side chain carbon of Ala (3) -alanine and the carbon of vinyl ₂ -。

In tables 6B and 6C, IC is represented for FPN and T47D internalization assays ₅₀ The sign of the value has the following meaning: * IC =1 nM ₅₀ ≤10nM；***＝10nM<IC ₅₀ ≤100nM；**＝100nM<IC ₅₀ ≤500nM.；*＝>500NM; where not shown, the data is not yet available.

TABLE 6B illustrative hepcidin analogs represented by formula (X)

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TABLE 6C illustrative hepcidin analogs represented by formula (XI)

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Example 2C

Activity of peptide analogues

The efficacy of the peptides to cause internalization of the membrane iron transporters was evaluated in T47D cell-based assays. The T47D cell line (HTB 133, atcc) is a human breast cancer adhesion cell line endogenously expressing the membrane iron transporter. In this internalization assay, the efficacy of the test peptide is assessed in the presence of serum albumin, which is the major protein component in blood. T47D cells were maintained in RPMI medium (containing the required amount of fetal bovine serum) and periodically subcultured. When ready for analysis, cells were seeded in 96-well plates at a density of 80 to 100k cells per well in a volume of 100 μl and allowed to stand overnight. The next day, the test peptides were first prepared in a dilution series (10-point series, starting at a concentration of about 5. Mu.M, usually 3X 4 dilution steps), all using 0.5% mouse serum albumin (MSA purified from mouse serum; sigma, A3139). The test peptide dilution series were incubated for 30 minutes at room temperature. Media was then aspirated from the 96 well cell plates and test peptide dilution sequences were added. After 1 hour incubation, the medium containing the test peptide was aspirated and AF 647-conjugated test peptide was added at a fixed concentration of 200 nM. The AF 647-bound detection peptide was previously validated to bind to and cause internalization of the membrane iron transporter. Cells were washed again after 2 hours incubation to prepare for flow cytometry analysis. The Median Fluorescence Intensity (MFI) of AF647 positive populations (after removal of dead cells and non-singlets from the assay) was measured. The MFI values were used to generate dose response curves and to obtain IC50 efficacy of the test peptides. IC50 efficacy was calculated using a 4-parameter nonlinear fitting function in GraphpadPrism (table 6A).

Example 2D

LAD2 Activity of peptide analogues

In an allergic-like response, the main mechanism involves direct stimulation of mast cells or basophils, leading to the release of allergic-like mediators (such as histamine and β -hexosidase). Recent studies by McNeil et al (McNeil BD et al 2015) reported that MrgprX2 is a specific membrane receptor on human mast cells, inducing an allergic-like response. LAD2 (allergic disease laboratory 2) human mast cell line derived from human mast cell sarcoma/leukemia (Kirshenbaum et al, 2003) is commonly used to study allergic-like reactions because its biological properties are consistent with those of the original human mast cells, including overexpression of the MrgprX2 receptor and sensitivity to degranulation peptides (Kulka et al, 2008). The release of an allergic-like mediator (such as beta-hexosidase) is assessed quantitatively by fluorescence.

The degranulation potential of hepcidin mimics was assessed in LAD2 cells. On the day of analysis, serial dilutions of the compounds were added to LAD2 cells seeded in 96-well plates at 20000 cells/well. After 30 minutes incubation, the fluorogenic substrate 4-methylcoumarin-N-acetyl-b-D-glucose was used to quantify the amount of β -hexosidase released into the supernatant and cell lysate. Dose response curves were generated by plotting the release% (y-axis) of β -hexosaminidase versus the concentration of the peptide tested (x-axis). EC was calculated using XLfit 5.5.0.5 based on the following equation ₅₀ Value and standard error: 4 parameter Sigmoidal model: f= (a+ ((B-ase:Sub>A)/(1+ ((C/x)/(D)))), where = Emin, B = Emax, C = EC50 and D = slope.

Reference is made to: mcNeil BD et al, nature, 12,519 (2015); kirshenbaum et al Leukemia study (Leukemia Res.) 27,677 (2003); kulka et al Immunology 123,398 (2008).

Example 3

In vivo validation of peptide analogs

The hepcidin analogs of the invention were tested for in vivo activity to determine their ability to reduce free fe2+ in serum.

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

In another experiment, mice (n=3/group) were subcutaneously administered various hepcidin analogs or vehicle controls at 1000 nmol/kg. Serum samples were collected from groups of mice administered vehicle or hepcidin analog 30 hours and 36 hours after administration. The IRON content in the plasma/serum was measured on Cobas c 111 using a colorimetric assay (assay: IRON2: ACN 661) according to the assay manufacturer's instructions.

These studies demonstrate that the hepcidin analogs of the invention reduce serum iron content for at least 30 hours and thus exhibit increased serum stability.

Example 4

In vitro validation of peptide analogs

Based in part on the Structural Activity Relationship (SAR) determined from the results of the experiments described herein, a variety of hepcidin-like peptides of the invention were synthesized using the methods described in example 1 and tested for in vitro activity as described in example 2. The reference compound comprises natural hepcidin, micro hepcidin, R1-micro hepcidin, reference compound 1 and reference compound 2. IC of peptide ₅₀ Or EC (EC) ₅₀ Values are shown in summary tables 6A-6C.

Example 5

Plasma stability

Plasma stability experiments were performed to supplement in vivo results and to help design a potent and stable membrane iron transporter promoter. To predict the stability of rat and mouse plasma, an ex vivo stability study was first performed in these matrices.

Peptides of interest (20. Mu.M) were incubated with pre-warmed plasma (Bioreclamatino IVT) at 37 ℃. Aliquots were removed at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with 4 volumes of organic solvent (acetonitrile/methanol (1:1) and 0.1% formic acid, containing 1 μm internal standard). The quenched samples were stored at 4℃until the end of the experiment and centrifuged at 17,000g for 15 min. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The remaining percentage at each time point was calculated from the peak area ratio (analyte to internal standard) relative to the initial level at time zero. Half-life was calculated by fitting a first order exponential decay equation using Graphpad.

Example 6

Reduction of serum iron in mice

Systemic absorption of hepcidin mimetic compounds designed for oral stability was tested by PO dosing in a wild-type mouse model C57 BL/6. Animals were acclimatized to normal rodent diet 4 to 5 days prior to the start of the study and fasted overnight prior to the start of the study. Each group of 4 animals received vehicle or compound. The compound was formulated in saline at a concentration of 5 mg/mL. Mice received dosing solution via oral gavage at a volume of 200 μl per animal body weight of 20 g. Each group received 1 dose of 50 mg/kg/dose of compound. The panel labeled vehicle received only the formulation. Blood was withdrawn 4 hours after dosing and serum was prepared for PK and PD measurements. The compound concentration was measured by mass spectrometry and the iron concentration in the sample was measured on the Roche cobas c system using a colorimetric method.

Example 7

Reduction of serum iron in mice

In another experiment, a new set of compounds were tested for systemic absorption by PO dosing in a wild type mouse model C57 BL/6. Animals were acclimatized to normal rodent diet 4 to 5 days prior to study initiation. The mice were changed to a low iron diet (containing 2ppm iron) one night prior to the first dose and maintained on this diet for the remainder of the study. Each of 5 animals in each group received vehicle or compound. The compound concentration was 30mg/mL, formulated in 0.7% NaCl+10mM sodium acetate buffer. Food was removed about 2 hours prior to each administration to ensure that no food particles were present in the stomach prior to PO administration. Mice received dosing solution via oral gavage at a volume of 200 μl per animal body weight of 20 g. Each group received 2 doses of 300 mg/kg/dose of compound for several consecutive days. The panel labeled vehicle received only the formulation. Blood was withdrawn 4.5 hours after the last dose and serum was prepared for PD measurement. Serum iron concentrations were measured on the Roche cobas c system using a colorimetric method.

Example 8

Pharmacodynamic effects of representative Compounds on the ability of mice to reduce serum iron

In the second in vivo study, the pharmacodynamic effect of representative compounds was tested with 300 mg/kg/dose of single dose versus 300 mg/kg of 2 dose over two days of QD (once per day). C57BL/6 mice were acclimatized to normal rodent diet 4 to 5 days prior to study initiation. Mice were changed to a low iron diet (containing 2ppm iron) one night prior to the first dose and maintained on this diet for the remainder of the study. Each of 5 animals in each group received vehicle or compound. The compound was formulated at a concentration of 30mg/mL in 0.7% NaCl+10mM sodium acetate buffer. Food was removed about 2 hours prior to each administration to ensure that no food particles were present in the stomach prior to PO administration. Mice received dosing solution via oral gavage at a volume of 200 μl per animal body weight of 20 g.

Example 9

PK/PD effects of representative compounds of the invention orally administered in mice

In another in vivo study against healthy wild-type mouse model C57/BL6, the PK and PD effects of representative compounds were tested over three days for multiple dosing. Mice were maintained on normal rodent diet during acclimation and switched to an iron-deficient diet (with about 2ppm iron) overnight prior to the first administration. Each of the 5 mice in each group was in BID format, receiving a total of 6 doses of vehicle or representative compound of the invention at different dose concentrations over three days. Mice were given representative compounds formulated in 0.7% saline and 10mM sodium acetate via oral gavage. The different groups received vehicle at 150 mg/kg/dose BID, 75 mg/kg/dose BID, 37.5 mg/kg/dose BID, or 18.75 mg/kg/dose BID. Another group received 100 mg/kg/dose BID in addition to total 100 mg/kg/day of compound in Drinking Water (DW), and thus received a total dose of 300 g/kg/day. 3 hours after the last dose, the vehicle group was labeled iron challenge, and all compound-dosed groups received an iron solution via oral gavage at 4mg/kg of iron per animal. Blood was collected 90 minutes after iron challenge to prepare serum for PK and PD measurements. The compound concentration was measured by mass spectrometry and the iron concentration in the sample was measured on the Roche cobas c system using a colorimetric method.

Example 10

Reduction of serum iron in mice

In another injury classification, the pharmacodynamic effects of a group of novel compounds upon oral administration were tested in a wild-type mouse model C57 BL/6. Animals were acclimatized to normal rodent diet 4 to 5 days prior to study initiation. One group of 5 animals designated to receive two doses of representative compound received an iron-deficiency diet (with 2ppm iron) one night prior to the first administration, and all other groups designated to receive a single dose of the different compounds were treated with an iron-deficiency diet two night prior to the administration of the compounds. The concentration of the compound in the dosing solution was 30mg/mL and was formulated in 0.7% NaCl+10mM sodium acetate buffer. Food was removed about 2 hours prior to any administration to ensure that no food particles were present in the stomach prior to PO administration. Mice were dosed via oral gavage with a volume of 200 μl of dosing solution per animal body weight of 20 g. The panel labeled vehicle received only the formulation. Blood was withdrawn 4.5 hours after the last dose and serum was prepared for PD measurement. Serum iron concentrations were measured on the Roche cobas c system using a colorimetric method.

Example 11

Stability of simulated gastric fluid

Blank SGF was prepared by adding 2g sodium chloride, 7mL hydrochloric acid (37%) to the final volume of 1L water and adjusting the pH to 1.2.

By mixing 320mg pepsin%P6887 from porcine gastric mucosa) was dissolved in 100mL of Blank SGF and stirred at room temperature for 30 minutes to prepare SGF. The solution was filtered through a 0.45 μm membrane and aliquoted and stored at-20 ℃.

The test compound of interest (concentration 20. Mu.M) was incubated with pre-warmed SGF at 37 ℃. Aliquots were removed at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with 4 volumes of organic solvent (acetonitrile/methanol (1:1) and 0.1% formic acid, containing 1 μm internal standard). The quenched samples were stored at 4 ℃ until the end of the experiment and centrifuged at 4,000rpm for 10 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The remaining percentage at each time point was calculated from the peak area ratio (analyte to internal standard) relative to the initial level at time zero. Half-life was calculated by fitting a first order exponential decay equation using Graphpad.

Example 12

Stability of simulated intestinal juice

The Blank FaSSIF (pH adjusted to 6.5) was prepared by dissolving 0.348g NaOH, 3.954g monobasic sodium phosphate monohydrate, 6.186g NaCl in a final volume of 1 liter water.

FaSSIF was prepared by dissolving 1.2g porcine pancreatic juice (Chem-supply, PL 378) in 100mL blancfassif and stirring at room temperature for 30 minutes. The solution was filtered through a 0.45 μm membrane and aliquoted and stored at-20 ℃.

The test compound of interest (20. Mu.M) was incubated with prewarmed FaSSIF (1% pancreatic juice in the final incubation mixture) at 37 ℃. Aliquots were removed at various time points up to 24 hours (e.g., 0, 0.25, 1, 3, 6, and 24 hours) and immediately quenched with 4 volumes of organic solvent (acetonitrile/methanol (1:1) and 0.1% formic acid, containing 1 μm internal standard). The quenched samples were stored at 4 ℃ until the end of the experiment and centrifuged at 4,000rpm for 10 minutes. The supernatant was diluted 1:1 with deionized water and analyzed using LC-MS. The remaining percentage at each time point was calculated from the peak area ratio (analyte to internal standard) relative to the initial level at time zero. Half-life was calculated by fitting a first order exponential decay equation using Graphpad.

Example 13

Modification experiments of peptides susceptible to "nonspecific binding

The compound of interest (concentration 20 μm) was mixed with prewarmed FaSSIF (1% pancreatic juice in the final working solution). The solution mixture was aliquoted and incubated at 37 ℃. The number of required aliquots is equal to the number of time points (e.g., 0, 0.25, 1, 3, 6, and 24 hours). At each time point, an aliquot was removed and immediately quenched with 4 volumes of organic solvent (acetonitrile/methanol (1:1) and 0.1% formic acid, containing 1. Mu. MM internal standard). The rest steps are the same as the general experiment.

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 mentioned in this specification and/or listed in the application data sheet are incorporated herein by reference, in their entirety.

At least some of the chemical names or sequences of the compounds of the invention presented and set forth in this application may have been automatically generated using commercially available chemical naming software programs and have not been independently validated. In the event that the chemical name or sequence indicated is different from the depicted structure, the depicted structure is subject to control. In chemical structures where the chiral center is present in the structure but does not exhibit a particular stereochemistry for the chiral center, the structure encompasses both enantiomers associated with the chiral structure. Similarly, for peptides in which E/Z isomers are present but not specifically mentioned, both isomers are explicitly disclosed and contemplated.

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

Claims

1. An hepcidin analog comprising a peptide according to formula I:

R ¹ -X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-R ² (I)

Or a pharmaceutically acceptable salt or solvate thereof,

wherein:

R ² is NH ₂ Substituted amino, OH or substituted hydroxy;

x2 is Ala, t-BuAla, thr, substituted Thr, gly, N substituted Gly, or Ser;

x3 is Ala, t-BuAla, gly, N substituted Gly, his, or substituted His;

x6 is absent or any amino acid other than Cys, (D) Cys, aMeCys, hCys or Pen;

X8 is absent, or is Ala, t-BuAla, (D) Ala, a-MeAla, ile, gly, N substituted Gly, glu, val, leu, NLeu, phe, bhPhe, lys, substituted Lys, (D) Lys, substituted (D) Lys, aMeLys, or 123 triazole;

x11 is absent, or Ala, pro, bhPhe, lys, substituted Lys, or (D) Lys;

and is also provided with

Each of X12 to X14 is absent or independently any amino acid;

the precondition is that:

i) The peptide may be further bound at any amino acid;

iii) X1 to X14At least two are independently Ala or aMeAla, and each Ala has a pendant methyl group C via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Cyclizing the alkenyl linker to form a macrocycle;

and is also provided with

123 triazole isAnd is also provided with

Dab is

2. The hepcidin analog of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein each of X1 and X6, X1 and X7, or X1 and X8 is Ala, and the pendant methyl C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

3. The hepcidin analog of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein each of X4 and X6, or X4 and X8 is Ala, and the pendant methyl C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

4. The hepcidin analog of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein each of X5 and X6 is Ala, and the pendant methyl C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

5. The hepcidin analog of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein each of X6 and X7, or X6 and X8 is Ala, and the pendant methyl C of each Ala is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

6. The hepcidin analog of any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, wherein C ₂ -C ₁₂ The alkanyl radical being-CH ₂ -(CH ₂ ) _q -CH ₂ -; wherein q is 2 to 10.

7. The hepcidin analog of any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, wherein C ₂ -C ₁₂ Alkenyl is- (CH) ₂ ) _t1 -(CH＝CH)-(CH ₂ ) _t2 -; wherein each t1 and t2 is independently 0 to 9.

8. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, wherein the linker is- (CH) ₂ ) ₂ -、-(CH ₂ ) ₃ -、-(CH ₂ ) ₄ -or- (CH) ₂ ) ₆ -。

9. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, wherein the linker is- (CH) ₂ ) _t1 -(CH＝CH)-(CH ₂ ) _t2 -, and each t1 and t2 is independently 0, 1, 2 or 3.

10. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, wherein the linker is- (CH) ₂ ) _t1 -(CH＝CH)-(CH ₂ ) _t2 -, and each t1 and t2 is independently 2.

11. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, wherein the linker is- (ch=ch) -or- (CH) ₂ )-(CH＝CH)-(CH ₂ )。

12. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, wherein the linker is- (CH) ₂ ) ₂ -(CH＝CH)-(CH ₂ ) ₂ -。

13. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 12, wherein

X1 is Glu, dab, dap, orn, lys, or Tet1;

x2 is Thr;

x3 is His or 1MeHis;

x4 is Dpa;

x5 is Ala or Pro;

x6 is absent, ala, glu, or substituted Lys;

x9 is absent, or bhpe;

X11 is absent, or Pro, bhPhe, lys, substituted Lys, or (D) Lys.

14. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-13, wherein X1 is Ala or Glu.

15. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, wherein X2 is Thr.

16. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-15, wherein X3 is His.

17. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-16, wherein X4 is Ala or Dpa.

18. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-17, wherein X5 is Ala or Pro.

19. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 18, wherein X6 is Ala or substituted Lys.

20. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-19, wherein X7 is Ala, ile, or substituted Lys.

21. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 20, wherein X8 is Ala, lys, or (D) Lys.

22. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 21, wherein X9 is absent or bhF.

23. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 22, wherein X10 is absent, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

24. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 23, wherein X11 is absent, arg, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

25. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 24, wherein each of X12, X13 and X14 is absent.

26. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, wherein the peptide is according to formula II:

R ¹ -Ala'-Thr-His-[Dpa]-Pro-X6-X7-Ala'-X9-X10-X11-X12-X13-X14-R ² (II)

Wherein R is ¹ 、R ² X6 to X7, and X9 to X14 are as defined in claim 1; and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

27. The hepcidin analog of claim 26, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is Ala.

28. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is Ahx-Palm substituted Lys.

29. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is absent, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

30. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is absent.

31. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is (D) Lys.

32. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is Lys.

33. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is Ahx-Palm substituted Lys.

34. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is Lys (ahx_palm).

35. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is a conjugated amino acid.

36. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein X6 is conjugated Lys or (D) Lys.

37. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-26, wherein X6 is Lys (L1Z) or (D) Lys (L1Z), wherein L1 is a linker, and wherein Z is a half-life extending moiety.

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

39. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein L1 is iso-Glu.

40. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein L1 is Ahx.

41. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein L1 is iso-Glu-Ahx.

42. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein L1 is PEG.

43. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein L1 is PEG-Ahx.

44. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein L1 is iso-Glu-PEG-Ahx.

45. The hepcidin analog of claim 41, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is- [ C (O) -CH2- (PEG) N-N (H) ] m-, or- [ C (O) -CH2-CH2- (PEG) N-N (H) ] m-; and Peg is-OCH 2CH2-, m is 1, 2 or 3; and n is an integer between 1 and 100, or 10K, 20K or 30K.

46. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein m is 1.

47. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein m is 2.

48. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein n is 2.

49. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein n is 4.

50. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein n is 8.

51. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein n is 11.

52. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein n is 12.

53. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein n is 20K.

54. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 1PEG2; and 1Peg2 is-C (O) -CH2- (Peg) 2-N (H) -.

55. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 2PEG2; and 2Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

56. The hepcidin analog of claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG2-1PEG2; and each 1Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

57. The hepcidin analog of claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG2-1PEG2; and 1Peg2-1Peg2 is- [ (C (O) -CH2- (OCH 2CH 2) 2-NH-C (O) -CH2- (OCH 2CH 2) 2-NH- ] -.

58. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 2PEG4; and 2Peg4 is-C (O) -CH2-CH2- (Peg) 4-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 4-NH ] -.

59. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 1PEG8; and 1Peg8 is-C (O) -CH2- (Peg) 8-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 8-NH ] -.

60. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 2PEG8; and 2Peg8 is-C (O) -CH2- (Peg) 8-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 8-NH ] -.

61. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 1PEG11; and 1Peg11 is-C (O) -CH2- (Peg) 11-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 11-NH ] -.

62. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 2PEG11; and 2Peg11 is-C (O) -CH2-CH2- (Peg) 11-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 11-NH ] -.

63. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein PEG is 2PEG11' or 2PEG12; and 2Peg11' or 2Peg12 is-C (O) -CH2-CH2- (Peg) 12-N (H) -or- [ C (O) -CH2-CH2- (OCH 2CH 2) 12-NH ] -.

64. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein when PEG is linked to Lys, the-C (O) -of PEG is linked to Ne of Lys.

65. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein when PEG is linked to isoGlu, PEG-N (H) -is linked to-C (O) -, of isoGlu.

66. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein when PEG is attached to Ahx, the-N (H) -of PEG is attached to-C (O) -, of Ahx.

67. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein when PEG is attached to Palm, the-N (H) -of PEG is attached to-C (O) -, of Palm.

68. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein Z is Palm.

69. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, wherein the peptide is according to formula III:

R ¹ -Glu-Thr-His-Ala'-Pro-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R ² (III)

Wherein R is ¹ 、R ² And X7 to X14 are as claimed in claim 1; and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

70. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, wherein the peptide is according to formula IV:

R ¹ -Glu-Thr-His-[Dpa]-Ala'-Ala'-X7-X8-X9-X10-X11-X12-X13-X14-R ² (IV)

71. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, wherein the peptide is according to formula V:

R ¹ -Glu-Thr-His-[Dpa]-Pro-Ala'-Ala'-X8-X9-X10-X11-X12-X13-X14-R ² (V)

wherein the method comprises the steps ofR ¹ 、R ² And X8 to X14 are as claimed in claim 1; and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

72. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 69-71, wherein X8 is Lys or (D) Lys.

73. The hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, wherein the peptide is according to formula VI:

R ¹ -Glu-Thr-His-[Dpa]-Pro-Ala'-X7-Ala'-X9-X10-X11-X12-X13-X14-R ² (VI)

Wherein R is ¹ 、R ² And X8 to X14 are as claimed in claim 1; and wherein Ala 'is alanine and the side chain methyl C of each Ala' is via C ₂ -C ₁₂ Chain alkyl or C ₂ -C ₁₂ Alkenyl linkers cyclize to form macrocycles.

74. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-73, wherein X9 is absent.

75. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 73, wherein X9 is bhF.

76. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 75, wherein X11 is absent.

77. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-75, wherein X11 is Arg.

78. A hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 75 wherein X11 is Lys, substituted Lys, (D) Lys, or substituted (D) Lys.

79. A hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 75 wherein X11 is (D) Lys.

80. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 79, wherein each of X12, X13, and X14 is independently absent, or is any amino acid.

81. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 79, wherein each of X12, X13, and X14 is absent.

82. The hepcidin analog of any one of claims 1 to 81, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is absent, lys, substituted Lys, (D) Lys, or substituted (D) Lys.

83. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-81, wherein X10 is absent.

84. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 81, wherein X10 is (D) Lys.

85. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-81, wherein X10 is Lys.

86. The hepcidin analog of any one of claims 1-81, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is Ahx-Palm substituted Lys.

87. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-81, wherein X10 is Lys (ahx_palm).

88. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-81, wherein X10 is a conjugated amino acid.

89. A hepcidin analogue or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 81 wherein X10 is conjugated Lys or (D) Lys.

90. The hepcidin analog of any one of claims 1-81, or a pharmaceutically acceptable salt or solvate thereof, wherein X10 is Lys (L1Z) or (D) Lys (L1Z), wherein L1 is a linker, and wherein Z is a half-life extending moiety.

91. The hepcidin analog of claim 90, wherein L1 is a single bond.

92. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is iso-Glu.

93. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is Ahx.

94. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is iso-Glu-Ahx.

95. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is PEG.

96. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is PEG-Ahx.

97. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is iso-Glu-PEG-Ahx.

98. The hepcidin analog of claim 41, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is- [ C (O) -CH2- (PEG) N-N (H) ] m-, or- [ C (O) -CH2-CH2- (PEG) N-N (H) ] m-; and Peg is-OCH 2CH2-, m is 1, 2 or 3; and n is an integer between 1 and 100, or 10K, 20K or 30K.

99. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein m is 1.

100. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein m is 2.

101. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 2.

102. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 4.

103. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 8.

104. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 11.

105. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 12.

106. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 20K.

107. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG2; and 1Peg2 is-C (O) -CH2- (Peg) 2-N (H) -.

108. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2PEG2; and 2Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

109. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG2-1PEG2; and each 1Peg2 is-C (O) -CH2-CH2- (Peg) 2-N (H) -.

110. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG2-1PEG2; and 1Peg2-1Peg2 is- [ (C (O) -CH2- (OCH 2CH 2) 2-NH-C (O) -CH2- (OCH 2CH 2) 2-NH- ] -.

111. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2PEG4; and 2Peg4 is-C (O) -CH2-CH2- (Peg) 4-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 4-NH ] -.

112. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG8; and 1Peg8 is-C (O) -CH2- (Peg) 8-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 8-NH ] -.

113. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2PEG8; and 2Peg8 is-C (O) -CH2- (Peg) 8-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 8-NH ] -.

114. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 1PEG11; and 1Peg11 is-C (O) -CH2- (Peg) 11-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 11-NH ] -.

115. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2PEG11; and 2Peg11 is-C (O) -CH2-CH2- (Peg) 11-N (H) -or- [ C (O) -CH2- (OCH 2CH 2) 11-NH ] -.

116. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein PEG is 2PEG11' or 2PEG12; and 2Peg11' or 2Peg12 is-C (O) -CH2-CH2- (Peg) 12-N (H) -or- [ C (O) -CH2-CH2- (OCH 2CH 2) 12-NH ] -.

117. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein-C (O) -of PEG is linked to Ne of Lys when PEG is linked to Lys.

118. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of claim 90, wherein when PEG is linked to isoGlu, PEG-N (H) -is linked to-C (O) -, of isoGlu.

119. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of claim 90, wherein when PEG is attached to Ahx, the-N (H) -of PEG is attached to-C (O) -, of Ahx.

120. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein when PEG is attached to Palm, the-N (H) -of PEG is attached to-C (O) -, of Palm.

121. The hepcidin analog of claim 90, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is Palm.

122. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 37 or 90, wherein-L1Z is:

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 (b)

-PEG4_PEG4_IsoGlu_C18_Diacid；

Wherein the method comprises the steps of

PEG11_OMe is- [ C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₁₁ -OMe]；

1PEG2 is-C (O) -CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-；

PEG4 is-C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₄ -NH-；

PEG8 is- [ C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₈ -NH-；

1PEG8 is- [ C (O) -CH ₂ -(OCH ₂ CH ₂ ) ₈ -NH-；

PEG12 is- [ C (O) -CH ₂ -CH ₂ -(OCH ₂ CH ₂ ) ₁₂ -NH-；

Ado is- [ C (O) - (CH) ₂ ) ₁₁ -NH]-

Palm is-C (O) - (CH) ₂ ) ₁₄ -Me；

isoGlu is isoglutamic acid;

isoGlu_palm is

Ahx is- [ C (O) - (CH) ₂ ) ₅ -NH]-；

123. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 37 or 90, wherein X6 or X10 is

Lys(1PEG2_1PEG2_IsoGlu_C _n Diacid); and is also provided with

Lys(1PEG2_1PEG2_IsoGlu_C _n Diac) is

And n is 10, 12, 14, 16 or 18.

124. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 37 or 90, wherein X6 or X10 is

(D)Lys(1PEG2_1PEG2_IsoGlu_C _n Diacid); and is also provided with

(D)Lys(1PEG2_1PEG2_IsoGlu_C _n Diac) is

And n is 10, 12, 14, 16 or 18.

125. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is Lys (1peg8_isoglu_c _n Diacid); and Lys (1PEG8_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

126. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (1peg8_isoglu_c) _n Diacid); and (D) Lys (1PEG8_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

127. The hepcidin analog of claim 37 or 90, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 or X10 is Lys (1peg2_1peg2_dap_c _n Diacid); and Lys (1PEG2_1PEG2_Dap_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

128. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is Lys (isoglu_c _n Diacid); and Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

129. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (isoglu_c) _n Diacid); and (D) Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

130. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is Lys (peg12_isoglu_c _n Diacid); and Lys (PEG 12_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

131. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (PEG 12_isoglu_c) _n Diacid); and (D) Lys (PEG 12_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

132. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is Lys (peg4_isoglu_c) _n Diacid); and Lys (PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

133. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (peg4_isoglu_c) _n Diacid); and (D) Lys (PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

134. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is Lys (peg4_pe4_isoglu_c) _n Diacid); and Lys (PEG4_PEG4_IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

135. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 37 or 90, wherein X6 or X10 is

(D)Lys(PEG4_PEG4_IsoGlu_C _n Diacid); and is also provided with

(D)Lys(PEG4_PEG4_IsoGlu_C _n Diac) is

And n is 10, 12, 14, 16 or 18.

136. The hepcidin analogue or pharmaceutically acceptable thereof according to any one of claims 37 or 90Salts or solvates, wherein X6 or X10 is Lys (IsoGlu_C) _n Diacid); and Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

137. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (isoglu_c) _n Diacid); and (D) Lys (IsoGlu_C) _n Diac) is

And n is 10, 12, 14, 16 or 18

138. The hepcidin analog of any one of claims 37 or 90, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 or X10 is Lys (peg12_ahx_c _n Diacid); and Lys (PEG 12_Ahx_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

139. The hepcidin analog of any one of claims 37 or 90, or a pharmaceutically acceptable salt or solvate thereof, wherein X6 or X10 is Lys (peg12_ahx_c _n Diacid); and Lys (PEG 12_Ahx_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

140. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (peg12_ahx_c _n Diacid); and (D) Lys (PEG 12_Ahx_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

141. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is Lys (peg12_c _n Diacid); and Lys (PEG 12_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

142. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 37 or 90, wherein X6 or X10 is (D) Lys (peg12_c) _n Diacid); and (D) Lys (PEG 12_C) _n Diac) is

And n is 10, 12, 14, 16 or 18.

143. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is NH ₂ 。

144. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is a substituted amino group.

145. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is N-alkylamino.

146. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is an N-alkylamino group in which the alkyl group is further substituted or unsubstituted.

147. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is an N-alkylamino group wherein alkyl is a further substituted aryl or heteroaryl group.

148. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is alkylamino wherein alkyl is unsubstituted or aryl substituted; and alkyl is ethyl, propyl, butyl or pentyl.

149. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is alkylamino wherein alkyl is unsubstituted or phenyl substituted; and alkyl is ethyl, propyl, butyl or pentyl.

150. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-142, wherein R ² Is OH.

151. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-150, wherein R ¹ Is C ₁ -C ₂₀ Alkanoyl.

152. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-150, wherein R ¹ Is IVA or isovaleric acid.

153. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-150, wherein the peptide is a linear peptide.

154. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-150, wherein the peptide is a lactam.

155. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 1-150, wherein the peptide is a lactam, wherein any free-NH ₂ With any free-C (O) ₂ H cyclizes.

156. An hepcidin analogue comprising or consisting of a peptide, or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide is any one of the peptides listed in table 6A.

157. The hepcidin analog of claim 1, or a pharmaceutically acceptable salt or solvate thereof, comprising or consisting of a peptide having formula (X) or (XI):

R ¹ is C ₁ -C ₂₀ An alkanoyl group;

R ² is OH, NH ₂ Or phenyl-C _1-8 An alkylene-amino group;

R ⁵ is H or C _1-6 An alkyl group;

L ^x is-CH ₂ CH＝CHCH ₂ -、-CH ₂ CH＝CHCH ₂ -、-(CH ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -、-(CH ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -、-(CH ₂ ) ₂ C(＝CH ₂ )C(＝CH ₂ )(CH ₂ ) ₂ -、-(CH ₂ ) ₆ -、-(CH ₂ ) ₄ -or-CH ₂ C(＝CH ₂ )C(＝CH ₂ )CH ₂ -；

X2 is Thr, (NMe) Thr or Thr_psi;

x3 is H or his_psi;

x4 is DIP or dip_psi;

x5 is Pro;

x6 is Ala, sar, lys (ahx_palm), lys_ahx_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_dap_c18_diacid, lys_1peg2_1peg2_Isoglu_c18_diacid, lys_1peg2_1peg2_Isoglu_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_dap_c18_diacid, -NHCH ₂ CH ₂ N ⁺ (CH ₃ ) ₂ -CH ₂ C (O) -or lys_1peg2_1peg2_ahx_palm;

x7 is Arg, tba, tle, ile, ala or Lys (carpine);

x8 is Ala, (a-Me) Ala, bhPhe, lys or (D) Lys;

x9 is Dip, bhF or NMe_Lys_Ahx_palm;

x10 is Arg, (D) Arg, lys_ahx_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_isoglu_palm, lys_1peg2_1peg2_isoglu_c18_diacid, lys_1peg2_1peg2_ahx_c18_diacid, dk_betaine, or (D) Lys; and is also provided with

X11 is Arg, (D) Arg or (D) Lys.

158. The hepcidin analog of claim 157, or a pharmaceutically acceptable salt or solvate thereof, wherein R ¹ Is isovaleric acid.

159. The hepcidin analog of claim 157 or 158, or a pharmaceutically acceptable salt or solvate thereof, wherein R ² Is OH, NH ₂ Or 4-phenylbutylamino.

160. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157-159, wherein R ⁵ Is H or methyl.

161. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 157 to 160, wherein X2 is Thr or (NMe) Thr.

162. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157-161, wherein X3 is His, X4 is DIP, and X5 is Pro.

163. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157 to 162, wherein X7 is Arg, tba, tle, ile or Lys (carpine).

164. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157 to 163, wherein X8 is (D) Lys or bhF.

165. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157 to 164, wherein X10 is (D) Arg, lys_ahx_palm, lys_1peg2_1peg2_ahx_c18_diacid, lys_1peg2_dmg_n_2ae_c18_diacid, lys_1peg2_1peg2_isoglu_palm, lys_1peg2_1peg2_isoglu_c18_diacid, lys_1peg2_peg2_ahx_c18_diacid, dk_betaine, or (D) Lys.

166. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157-165, wherein X11 is (D) Arg or (D) Lys.

167. The hepcidin analog or pharmaceutically acceptable salt or solvate thereof of any one of claims 157 to 1667, wherein L ^x Is (trans) -CH ₂ CH＝CHCH ₂ -, (cis) -CH ₂ CH＝CHCH ₂ -, (cis) - (CH) ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -, (trans) - (CH) ₂ ) ₂ CH＝CH(CH ₂ ) ₂ -、-(CH ₂ ) ₂ C(＝CH ₂ )C(＝CH ₂ )(CH ₂ ) ₂ -、-(CH ₂ ) ₆ -、-(CH ₂ ) ₄ -or-CH ₂ C(＝CH ₂ )C(＝CH ₂ )CH ₂ -。

168. The hepcidin analog of claim 1 or 157, or a pharmaceutically acceptable salt or solvate thereof, comprising or consisting of a peptide, wherein the peptide is any one of the peptides listed in table 6B or table 6C.

169. The hepcidin analog of claim 1, or a pharmaceutically acceptable salt or solvate thereof, comprising or consisting of a peptide, wherein the peptide has the formula

ID#6

Or (b)

ID#16

170. The hepcidin analog of claim 1 or 157, or a pharmaceutically acceptable salt or solvate thereof, comprising or consisting of a peptide, wherein the peptide is any one of the peptides listed in table 7.

171. A polynucleotide encoding a peptide present in the hepcidin analog of any one of claims 1 to 170 or a pharmaceutically acceptable salt or solvate thereof.

172. A vector comprising the polynucleotide of claim 171.

173. A pharmaceutical composition comprising the hepcidin analog of any one of claims 1 to 170, or a pharmaceutically acceptable salt or solvate thereof, the polynucleotide of claim 171, or the carrier of claim 172, and a pharmaceutically acceptable carrier, excipient, or vehicle.

174. A method of binding to a ferroportin or inducing internalization and degradation of a ferroportin comprising contacting the ferroportin with at least one hepcidin analog of any one of claims 1-170, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of claim 173.

175. A method of treating a disorder of iron metabolism in a subject in need thereof, comprising providing to the subject an effective amount of a hepcidin analog of any one of claims 1-170 or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of claim 173.

176. A method of treating a disease or disorder associated with dysregulation of hepcidin signaling in a subject in need thereof, comprising providing to the subject an effective amount of an hepcidin analog or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-170, or a pharmaceutical composition according to claim 173.

177. The method of claim 175 or 176, wherein the pharmaceutical composition is provided to the subject by an oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, spray, sublingual, buccal, parenteral, rectal, vaginal, or topical route of administration.

178. The method of claim 177, wherein the pharmaceutical composition is provided to the subject by an oral or subcutaneous route of administration.

179. The method of any one of claims 175-178, wherein the disease or disorder is a disease of iron metabolism.

180. The method of claim 179, wherein the iron metabolic disease is an iron overload disease.

181. The method of any one of claims 175-180, wherein the disease or condition is hemochromatosis, thalassemia, or polycythemia vera.

182. The method of any one of claims 175-181, wherein the hepcidin analog or pharmaceutically acceptable salt or solvate thereof or the pharmaceutical composition is provided to the subject up to twice daily, up to once every two days, up to once a week, or up to once a month.

183. The method of any one of claims 175-182, wherein the hepcidin analog or pharmaceutically acceptable salt or solvate thereof or the pharmaceutical composition is provided to the subject at a dose of about 1mg to about 100 mg.

184. A device comprising the pharmaceutical composition of claim 173 for delivering, optionally orally or subcutaneously, a hepcidin analog or a pharmaceutically acceptable salt or solvate thereof to a subject.

185. A kit comprising the pharmaceutical composition of claim 173, packaged with reagents, devices, or instructional materials, or a combination thereof.