WO2018128828A1

WO2018128828A1 - Novel hepcidin mimetics and uses thereof

Info

Publication number: WO2018128828A1
Application number: PCT/US2017/067764
Authority: WO
Inventors: Donald Bierer; Elisabeth Pook; Jan Stampfuss; Richard I. Feldman; Carsten ALT
Original assignee: Bayer Healthcare Llc; Bayer Pharma AG; Bayer Ag
Priority date: 2016-12-23
Filing date: 2017-12-21
Publication date: 2018-07-12

Abstract

The present invention relates to novel peptides acting as hepcidin mimetics, as well as analogues and derivatives thereof. The invention further relates to compositions comprising the peptides of the present invention, and to the use of the peptides in the prophylaxis and treatment of hepcidin-associated disorders, including prophylaxis and treatment of iron overload diseases such as hemochromatosis, iron-loading anemias such as thalassemia, and diseases being associated with ineffective or augmented erythropoiesis, as well as further related conditions and disorders described herein.

Description

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NOVEL HEPCIDIN MIMETICS AND USES THEREOF

DESCRIPTION FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION AND PRIOR ART

Hepcidin Antimicrobial Peptide (HAMP; also known as LEAP-1; further referred to as Hepcidin) is a 25 amino acid peptide (Krause et al., 2000). Hepcidin is produced in the liver and functions as the master iron regulatory hormone controlling intestinal iron uptake, and also regulates iron storage in other organs (Ganz, 2006; Hunter et al, 2002; Park et al., 2001). Hepcidin limits iron-uptake by binding to the iron transport molecule ferroportin and causing its degradation (Sebastiani et al., 2016).

Hepcidin has a hairpin structure with 8 cysteines that form 4 disulfide bridges (Jordan et al., 2009). The N-terminus is critical for the iron-regulatory function since deletion of the first 5 amino acids resulted in complete loss of bioactivity (Nemeth et al, 2006).

Iron overload has been associated with a variety of diseases (Blanchette et al, 2016). Hereditary hemochromatosis is the most common inherited disease in Europe and is caused by lack of, or insensitivity to, hepcidin (Powell et al., 2016). The clinical manifestation of hemochromatosis are hepatic cirrhosis, diabetes, and skin pigmentation (Powell et al., 2016). While this disease can be managed by phlebotomy, this approach may be cumbersome and does not treat the cause of the disease.

Iron-loading anemias such as β-thalassemia are also associated with reduced hepcidin levels (Origa et al., 2007). Treatment of this disease with hepcidin mimetics may not only address the iron overload, but has also been shown to improve the ineffective erythropoiesis that occurs in this disease (Casu et al., 2016). This may be of major benefit for thalassemia patients who may be less dependent on blood transfusions - which majorly contribute to the iron overload in these patients. Myelofibrosis, myelodysplastic syndrome, and sickle cell disease are diseases that are also characterized by ineffective erythropoiesis and that may require frequent blood transfusions (Carreau et al., 2016; Temraz et al., 2014; Walter et al., 2009). Reduced hepcidin levels have been described in some of these patients (Cui et al., 2014; Santini et al, 2011). Hepcidin mimetics may also be beneficial in these patients.

Polycythemia vera is a disease characterized by increased erythropoiesis. It has been shown in animal models that high doses of hepcidin mimetics can ameliorate this disease by diminishing erythropoiesis (Casu et al., 2016). Reduction of iron uptake and thereby serum iron levels may even be beneficial in diseases where iron load is normal, such as kidney diseases (Walker and Agarwal, 2016), infections with iron-dependent bacteria (Arezes et al., 2015), and polymicrobial sepsis (Zeng et al., 2015).

Hepcidin itself is limited in its use as a drug because of its complex structure which requires a complicated manufacturing, and also its limited in vivo duration of action. Continuous efforts have been made to search for hepcidin mimetics and chemical compounds that could be used to increase hepcidin levels - as novel compounds are needed which have hepcidin-like activity and also possess additional beneficial physical properties such as improved solubility, stability, and/or potency.

A common approach relates to small hepcidin-derived or hepcidin-like peptides, which can be produced affordably, and can be used to treat hepcidin-related diseases and disorders such as, e.g., those described herein. Such so-called mini-hepcidins are rationally designed small peptides that mimic hepcidin activity and may be useful for the treatment of iron overload, and also iron overload related disease symptoms.

Such mini-hepcidin peptides are described for example in WO 2010/065815 A2 and WO 2013/086143 Al. WO 2015/157283 Al and the corresponding US 9,315,545 B2 describe hepcidin mimetic peptides and the use thereof in hepcidin-related disorders, such as iron overload, β-thalassemia, hemochromatosis etc. and cover a development compound MO 12 of the company Merganser Biotech, having been under evaluation in a Phase 1 clinical program as a potentially transformative therapy for a number of hematological diseases including β-thalassemia, low risk myelodysplasia and polycythemia vera.

WO 2014/145561 A2 and WO 2015/200916 A2 describe further small hepcidin peptide analogues and the use thereof in the treatment or prevention of a variety of hepcidin-related diseases, including iron overload diseases and iron-loading anemias, and further related disorders.

Further, WO 2015/042515 Al relates to hepcidin and its peptide fragments, which are particularly intended for treating renal ischemia reperfusion injury or acute kidney injury.

Further, mini-hepcidin analogs are described for example by Preza et al., 2011 or in CN 104 011 066 and in WO 2016/109363 Al.

OBJECT TO BE SOLVED

It was the object of the present invention to provide novel peptide-based hepcidin mimetics, having hepcidin activity and other beneficial properties making them suitable as efficient and safe alternatives to hepcidin. In particular the novel hepcidin mimetics should be suitable for the prophylaxis and treatment of the hepcidin-related diseases as described herein. DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to peptides acting as hepcidin mimetics and methods of making and using the same.

In particular, the invention provides peptides, which may be isolated and/or purified, comprising, essentially consisting of, or consisting of, the following structural formula (I):

X0 - X12 - X13 - XI - Thr - His - X2 - X3 - X4 - XI 1 - X5 - X6 - X7 - [X8(- X9)] - -

(I) or pharmaceutically acceptable salts or solvates thereof,

wherein

X0 is Gly, 2,2-dimethylglycine (Aib), sarcosine (Sar), acetyl, C₃-Ci₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), pyroglutamate, Ci-C₆ alkyl, Ci-C₆ dialkyl or X0 is absent;

XI is Ala, Asn, Asp, Gin, Glu, Gly, 2,2-dimethylglycine (Aib), iminodiacetic acid (Ida), orotic acid (OA), dihydroorotic acid (Hoo), sarcosine (Sar), or a group [6-aminohexanoic acid- Asp] ([Ahx-D]), or XI is absent;

X2 is Phe, substituted Phe, diphenylalanine (Dip), Trp, 1-napthylalanine (1-Nal), or 2- napthylalanine (2-Nal);

X3 is Pro, β-proline, a proline mimetic, a proline spacer, Gly-Gly, sarcosine (Sar), or 2,2- dimethylglycine (Aib);

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X6 is Phe, substituted Phe, diphenylalanine (Dip), Trp, 1-napthylalanine (1-Nal), 2- napthylalanine (2-Nal), or Tyr;

X7 is a PEG spacer, having the formula (la)

(la)

- - or having the formula (lb)

(lb)

with n = 1 to 6,

or a group

X7-X7' wherein X7 is a PEG spacer according to formula (la) or (lb) with n = 1 to 6 and

XT Arg (R);

X8 is Lys, or ornithine (Om);

X9 is a C₈ to C₂o fatty acid;

X10 is from 0 up to 15 additional amino acids, with an amide or carboxylic acid C-terminus; XI 1 is Cys, Met, Ser, Cys-Me, N-Me-Cys or Penicillamine (Pen);

X12 is 1, 2 or 3 additional amino acids and/or spacer, wherein the spacer may be selected from 6-aminohexanoic acid (Ahx), 10-aminodecanoic acid, 11-undecanoic acid, 12- aminododecanoic acid, PEG1, β-alanine, and 4-aminobutyric acid, or X12 is absent

X13 is Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the Cys residues, N-Me-Cys or Penicillamine (Pen), or X13 is absent;

or a homodimer thereof, comprising two peptide chains according to formula (I), which are bound to each other via a disulfide bond formed between the Cys residues in the two peptide chains.

In a further aspect, the invention provides peptides, which may be isolated and/or purified, comprising, essentially consisting of, or consisting of, the following structural formula (I):

X0-X12-X13-Xl-Thr-His-X2-X3-X4-Xll-X5-X6-X7-[X8(-X9)]-

X10

(I) or pharmaceutically acceptable salts or solvates thereof, - - wherein

X0 is Gly, 2,2-dimethylglycine (Aib), sarcosine (Sar), acetyl, C₃-C₁ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), pyroglutamate, C[-C₆ alkyl, C[-C₆ dialkyl or X0 is absent;

XI is Ala, Asn, Asp, Gin, Glu, Gly, 2,2-dimethylglycine (Aib), iminodiacetic acid (Ida), orotic acid (OA), dihydroorotic acid (Hoo), sarcosine (Sar), or XI is absent;

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X7 is a PEG spacer, having the formula (la)

(la)

or having the formula (lb)

(lb)

with n = 1 to 6;

X8 is Lys, or ornithine (Orn);

X9 is a C₈ to C₂o fatty acid;

X10 is from 0 up to 15 additional amino acids, with an amide or carboxylic acid C-terminus; XI 1 is Cys, Met, Ser, N-Me-Cys or Penicillamine (Pen);

X12 is 1, 2 or 3 additional amino acids and/or spacer, wherein the spacer may be selected from 6-aminohexanoic acid (Ahx), 10-aminodecanoic acid, 11-undecanoic acid, 12- aminododecanoic acid, PEG1, β-alanine, and 4-aminobutyric acid, or X12 is absent X13 is Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the Cys residues, N-Me-Cys or Penicillamine (Pen), or X13 is absent.

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art. Throughout this specification, the word "comprise" or variations thereof such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components). The singular forms "a," "an," and "the" include the plurals unless the context clearly dictates otherwise. The term "including" is used to mean "including but not limited to", which expressions can be used interchangeably.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. "Essentially consisting of is understood as a peptide being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the peptide it is compared to.

The term "peptide" as used herein, refers broadly to a sequence of two or more amino acids joined together by peptide bonds. It should be understood that this term does not indicate a specific length of a polymer of amino acids, nor is it intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The term peptide may comprise also dimers, such as in particular homodimers, comprising the peptide chains as described herein.

The term "amino acid" or "any amino acid" as used herein refers to any and all amino acids, including naturally occurring amino acids (e.g., a-L-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building- blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The 20 proteinogenic, natural amino acids in the standard genetic code are listed in the below tables. The "non-standard," natural . . amino acids are pyrrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N- formylmethionine (encoded by the start codon AUG in bacteria, mitochondria and chloroplasts). "Unnatural" or "non-natural" amino acids are non-proteinogenic amino acids (i.e., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 natural amino acids are known and thousands of more combinations are possible. Examples of "unnatural" amino acids include β-amino acids (β and β²), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present in the amino acid. In accordance with the understanding of a person skilled in the art, the peptide sequences disclosed herein are shown proceeding 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. Among sequences disclosed herein are sequences incorporating either an "-OH" moiety or an "- NH₂" moiety at the carboxy terminus (C-terminus) of the sequence. An "-OH" or an "-NH₂" moiety at the C-terminus of the sequence indicates a hydroxy group or an amino group, corresponding to the presence of a carboxy group or an amido (-(C=0)-NH₂) group at the C- terminus, respectively. In each sequence of the invention, a C-terminal "-OH" moiety may be substituted for a C-terminal "-NH₂" moiety, and vice-versa. However, among said alternatives a C-terminal "-NH₂" moiety is preferred.

It is further understood that the moiety at the amino terminus or carboxy terminus may be a bond, e.g., a covalent bond, particularly in situations where the amino terminus or carboxy terminus is bound to a linker or to another chemical moiety.

The term "NH2," as used herein, refers to the free amino group present at the amino terminus of a polypeptide. The term "OH," as used herein, refers to the free carboxy group present at the carboxy terminus of a peptide. . .

In the present invention the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in "Nomenclature of a- Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975), unless explicitly stated otherwise.

In the present specification naturally occurring proteinogenic amino acids are usually designated by their conventional three-letter abbreviations. Alternatively, they can also be referred to by their single-letter abbreviations (e.g. in particular in the sequence listings) or by their full name as shown in Table 1 below:

Table 1:

In the case of non-proteinogenic or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four-character codes are employed for residues thereof, including those abbreviations as indicated in the abbreviation list below. In the sequence listing ornithine can also be indicated by "O" or "o". The term "L- amino acid," as used herein, refers to the "L" isomeric form of an amino acid, and conversely the term "D-amino acid" refers to the "D" isomeric form of an amino acid. It is further a conventional manner to indicate the L-amino acid with capital letters such as Ala / A, Arg / R, etc. and the D-amino acid with small letters such as ala / a, arg / r, etc.. - -

The three-letter code in the form as indicated in the table above, i.e. Ala, Arg, Asn etc. and as generally used in the present specification, shall generally comprise the D- and L- form as well as homo- and nor-forms, unless explicitly indicated otherwise. The prefix "nor" refers to a structural analog that can be derived from a parent compound by the removal of one carbon atom along with the accompanying hydrogen atoms. The prefix "homo" indicates the next higher member in a homologous series. A reference to a specific isomeric form will be indicated by the capital prefix L- or D- as described above (e.g. D-Arg, L-Arg etc.). A specific reference to homo- or nor-forms will accordingly be explicitly indicated by a respective prefix (e.g. homo-Arg, h-Arg, or hArg, nor- Arg, homo-Cys, h-Cys, or hCys etc.).

The term "cyclized," as used herein, refers to a reaction in which one part of a polypeptide molecule (e.g. Cys) becomes linked to another part of the polypeptide molecule (e.g. another Cys) to form a closed ring, such as by forming a disulfide bridge or other similar bond. C+ in a sequence of a peptide according to the invention refers to a cyclic peptide, wherein two Cys are linked to one another forming a closed ring via a disulfide bridge.

The term "spacer" is used in conventional manner and generally indicates a chemical group or compound, which is introduced into the peptide chain of the present invention providing a connection between two other moieties (e.g. amino acids) of the peptide chain. Examples of spacers as used in the present invention include 5-aminovaleric acid, 6-aminohexanoic acid (Ahx), 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 11-undecanoic acid, 12-aminododecanoic acid, 2,3-diaminopropanoic acid (Dap), 2,4- diaminobutyric acid (Dab), PEG1 to PEG6 (as defined below for the substituent X7), β-alanine, and 4-aminobutyric acid.

In particular the term "proline spacer" as used in context with the definition of X3 indicates a spacer group replacing Pro, such as any group as defined in US 9,315,545 for proline, which is herewith incorporated by reference. Particular examples of a proline spacer according to the present invention comprise: 2,2-dimethylglycine (Aib), cyclopropylglycine (Cppg), cyclobutylglycine (Cbg), cyclopentylglycine (Cpg), aminocyclopropanecarboxylic acid (ACPPC), aminocyclobutanecarboxylic acid (ACBC), aminocyclopentanecarboxylic acid (ACPC), aminocyclohexanecarboxylic acid (ACHC), substituted prolines, such as 4- hydroxyproline (Hyp) and 4-fluoroproline, N-Me-Ala, tranexamic acid (Tran), piperidine-2- carboxylic acid (Pip), piperidine-3-carboxylic acid (Nipecotic acid, Nip), octahydroindole - -

(Oic), piperidine-4-carboxylic acid (Inp or Isn), etc.. Preferred proline spacers are selected from the group consisting of piperidine-2-carboxylic acid (Pip), Piperidine-3-carboxylic acid (Nipecotic acid, Nip), 2,2-dimethylglycine (Aib), 4-hydroxyproline (Hyp), and 4-fluoi proline. Further preferred proline spacers are selected from the group consisting of γ-aminobutyric acid, morpholine-3-carboxylic acid, nipecotic acid (Nip), octahydroindole (Oic), and pipecolic acid (Pip).

The peptide of the present invention is characterized by having a PEG spacer within the peptide chain. As used in context with the definition of X7 the particular term "PEG spacer" represents a PEG group being introduced into the peptide chain, providing a connection between two other moieties (i.e. between the moieties X6 and X8) of the peptide chain according to formula (I), (II) and (III) of the present invention, and having the formula (la)

(la) or having the formula (lb)

(lb)

in each case with n = 1 to 6.

Examples of PEG spacer according to the present invention comprise PEG1 having 1 ethylene glycol unit (n=l; 10 atoms), PEG2 having 2 ethylene glycol units (n=2; 13 atoms), PEG3 having 3 ethylene glycol units (n=3; 16 atoms), PEG4 having 4 ethylene glycol units (n=4; 19 atoms), PEG5 having 5 ethylene glycol units (n=5; 22 atoms), PEG6 having 6 ethylene glycol units (n=6 ; 25 atoms) etc.. In particular for the moiety X7 PEG spacer as defined in formula (la) with n = 2 to 5 are preferred, more preferably with n = 3 to 5, i.e. with n = 3, 4 or 5; such as in particular PEG3, PEG4 and PEG5. - -

The PEG spacer according to formula (lb) are indicated herein as PEGl-acetic, PEG2-acetic, PEG3-acetic, PEG4-acetic, PEG5-acetic, PEG5-acetic, etc. comprising

PEGl-acetic having 1 ethylene glycol unit (n=l; 9 atoms), PEG2-acetic having 2 ethylene glycol units (n=2; 12 atoms), PEG3-acetic having 3 ethylene glycol units (n=3; 15 atoms), PEG4-acetic having 4 ethylene glycol units (n=4; 18 atoms), PEG5-acetic having 5 ethylene glycol units (n=5; 21 atoms), PEG6-acetic having 6 ethylene glycol units (n=6 ; 24 atoms) etc..

In particular for the moiety X7 PEG spacer as defined in formula (la) and (lb) with n = 2 to 5 are preferred, more preferably with n = 3 to 5, i.e. with n = 3, 4 or 5; such as in particular PEG3, PEG4 and PEG5 or PEG3-acetic, PEG4-acetic and PEG5-acetic. In another aspect of the invention for the moiety X7 PEG spacer according to formula (la) and (lb) with n = 2 or 3, such as in particular PEG2, PEG3, PEG2-acetic, and PEG3-acetic are preferred.

The peptide of the present invention is further characterized by comprising at least one C₈ to C₂o fatty acid as a side chain (X9) conjugated to the amino acid moiety X8. Generally, such fatty acid may be branched or cyclic. Preferably the fatty acid side chain conjugated to the amino acid moiety X8 is a fatty acid > C₈, more preferably a fatty acid > C]₂, more preferably a fatty acid > C₁₄. It is further preferred that the fatty acid side chain conjugated to the amino acid moiety X8 is a C₁₂ to Ci₈ fatty acid, preferably a C₁₂ to C₁₆ fatty acid, or a Q₄ to C₁₈ fatty acid, or a Cj₄ to Ci₆ fatty acid. Most preferred is a Ci₆ fatty acid such as palmitic acid (palmitoyl, Palm).

It is noted that the PEG spacer reagents as defined herein may be indicated with different names by commercial suppliers, which shall not exclude such identical compounds with different names from the present invention.

Generally, in the context of the present invention, a compound or chemical group presented in parentheses ( ) directly after an amino acid residue, such as in particular as indicated for the group X8(-X9) or -[X8(-X9)]-, indicates that the compound or chemical group in the parentheses (e.g. (-X9)) represents a group conjugated to the side chain of the amino acid put in front of the parentheses (e.g. X8).

In the peptide according to the present invention an N-terminal amino acid moiety can optionally be capped with one or more groups, which may be the same or different and which may be selected from Gly, 2,2-dimethylglycine (Aib), sarcosine (Sar), acetyl, C₃-C₁₆ fatty acid . . which can be branched or cyclic, such as e.g. isovaleric acid (which may be indicated in the sequences according to the present invention by the term "isovaleric"), orotic acid (OA), dihydroorotic acid (Hoo), pyroglutamate, or Ci-C₆ alkyl, to provide a modified amine terminus of the peptide of the present invention. Preferably a modified amine terminus is obtained by capping with one or more moieties selected from acetyl, C₃-C₁₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), pyroglutamate or Ci_₆ alkyl. Capping with two identical or different Ci_₆ alkyl groups may also be indicated as Ci_₆ dialkyl, including for example dimethyl, diethyl, dipropyl, etc. as well as mixed dialkyl-groups such as for example a methyl-ethyl-, methyl-propyl, ethyl-propyl, etc.

Therein, the term "Ci.₆ alkyl" includes a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 6 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, and the like, while saturated branched alkyls include, without limitation, isopropyl, sec- butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, without limitation, cyclopentenyl, cyclohexenyl, and the like. Preferred is saturated straight chain alkyl. Particularly preferred is methyl, ethyl, n-propyl. Most preferred is methyl.

The term "acetyl" (also abbreviated "Ac") refers to an acetyl protection of the N-terminal moiety through acylation of the N-terminus of a peptide.

Chemical groups or moieties used as spacer, conjugated side-chain, for capping a terminal end, or for other suitable modifications of the peptides are abbreviated herein as follows:

ACBC = aminocyclobutanecarboxylic acid

ACHC = aminocyclohexanecarboxylic acid

ACPC = aminocyclopentanecarboxylic acid

ACPPC = aminocyclopropanecarboxylic acid

Ac = acetyl

Ahx = 6-aminohexanoic acid

Aib = 2,2-dimethylglycine

Cbg = cyclobutylglycine

Cha = cyclohexylalanine

Cpg = cyclopentylglycine Cppg = cyclopropylglycine

C(Me) = Cys(Me) or Cys-Me or Cys-methyl or S-methyl-cysteine

Dap = 2,3-diaminopropanoic acid

Dab = 2,4-diaminobutyric acid

Dip = 3,3-diphenylalanine (may sometimes also be abbreviated as "Dpa")

Hoo = dihydroorotic acid

Hyp = 4-hydroxyproline

Ida = iminodiacetic acid

Inp or Isn = piperidine-4-carboxylic acid

1-Nal = 1-napthylalanine

2-Nal = 2-Napthylalanine

Nip = Nipecotic acid = Piperidine-3-carboxylic acid

Nle = Norleucine

N-Me-Cys = N-methyl-Cys

Nva = Norvaline

OA = orotic acid

Orn = ornithine (also indicated by "O" in the sequence listing)

Palm = palmitoyl

PEG = polyethylene glycol with

PEG1 = 1 ethylene glycol unit (n=l), 10 atoms

PEG2 = 2 ethylene glycol units (n=2), 13 atoms

PEG3 = 3 ethylene glycol units (n=3), 16 atoms

PEG4 = 4 ethylene glycol units (n=4), 19 atoms

PEG5 = 5 ethylene glycol units (n=5), 22 atoms

PEG6 = 6 ethylene glycol units (n=6), 25 atoms

PEG 1 -acetic = PEG 1 -acetic acid = 1 ethylene glycol unit (n=l), 9 atoms

PEG2-acetic = PEG2-acetic acid = 2 ethylene glycol units (n=2), 12 atoms

PEG3-acetic = PEG3-acetic acid = 3 ethylene glycol units (n=3), 15 atoms

PEG4-acetic = PEG4-acetic acid = 4 ethylene glycol units (n=4), 18 atoms

PEG5-acetic = PEG5-acetic acid = 5 ethylene glycol units (n=5), 21 atoms

PEG6-acetic = PEG6-acetic acid = 6 ethylene glycol units (n=6), 24 atoms

Pen = Penicillamine

Pip = piperidine-2-carboxylic acid

Sar = sarcosine

Tran = tranexamic acid

The term "mimetic", used in context with some amino acids in the definition of several moieties of the peptide according to formula (I), (II) or (III) of the present invention, represents a respective amino acid mimetic, such as e.g. an arginine mimetic, an isoleucine mimetic or a - - proline mimetic. Generally, a "protein mimetic" indicates a molecule such as a peptide, a modified peptide or any other molecule that biologically mimics the action or activity of some other protein. In context with the use of the term "mimetic" in connection with a certain amino acid said term "mimetic" analogously indicates any other amino acid, amino acid analogue, amino acid derivative, amino acid conjugate or the like, which biologically mimics the action or activity of the respective amino acid.

Proline mimetics according to the present invention comprise in particular 2,2-dimethylglycine (Aib), 4-hydroxyproline (Hyp), 4-fluoroproline, piperidine-2-carboxylic acid (Pip), piperidine- 3-carboxylic acid (Nipecotic acid, Nip), Octahydroindole-2-carboxylic acid (Oic), cyclopropylglycine (Cppg), aminocyclopropanecarboxylic acid (ACPPC), cyclobutylglycine (Cbg), aminocyclobutanecarboxylic acid (ACBC), aminocyclopentanecarboxylic acid (ACPC), cyclopentyl glycine (Cpg), aminocyclohexanecarboxylic acid (ACHC), N-Me-Ala, and substituted prolines.

Arginine mimetics according to the present invention comprise in particular norarginine, homoarginine, 3-cyanophenylalanine, 4-cyanophenylalanine, 3-amidinophenylglycine, 4- amidinophenylglycine, or a guanidine containing amino acid, such as Gly-4-piperidine[N- amidino] and Gly-3-piperidine[N-amidino] as shown below:

Isoleucine mimetics according to the present invention comprise in particular leucine, allo- isoleucine, cyclopropylalanine, cyclobutylalanine, cyclopentylalanine, cyclohexylalanine (Cha), norleucine (Nle), cyclopropylglycine (Cppg), cyclobutylglycine (Cbg), cyclopentylglycine (Cpg), valine, norvaline (Nva), and phenylalanine.

The invention further comprises analogues and derivatives of the described peptides. The term "analogue" or "derivative" of a peptide or an amino acid sequence according to the present invention comprises in particular any amino acid sequence having a sequence identity of at least 80% or at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably of at least 99% identity to said sequence, and same or comparable properties or activity. Sequence identity can be determined by common techniques, such as visual comparison or by means of any computer tool generally used in the field. Examples comprise BLAST programs used with default parameters.

An analogue or derivative of a peptide or an amino acid sequence of the invention may result from changes derived from mutation or variation in the sequences of peptides of the invention, including the deletion or insertion of one or more amino acids or the substitution of one or more amino acids, or even to alternative splicing. Several of these modifications may be combined. Preferably, an analogue of an amino acid sequence of the invention comprises conservative substitutions relative to the sequence of amino acids.

The term "conservative substitution" as used herein denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. See, for example, the scheme in Table 2 below, wherein conservative substitutions of amino acids are grouped by physicochemical properties. I: neutral, hydrophilic; II: acids and amides; III: basic; IV: hydrophobic; V: aromatic, bulky amino acids, VI: neutral or hydrophobic; VII: acidic; VIII: polar. Table 2: Amino Acids grouped according to their physicochemical properties

A peptide analogue or derivative may also comprise one or more additional modifications such as, e.g., conjugation to another compound to form an amino acid conjugate.

Such a modification may, alternatively or additionally, result from conjugation to the side- chains of one or more amino acid residues in a peptide of the present invention for example a chemical group as defined above in context with X0 or a polymeric moiety. Such modification may, for example, increase solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability of the peptide and are also known to reduce clearance (e.g. renal clearance) of therapeutic proteins and peptides. Suitable modifications are well known to a skilled person and comprise in particular, without being limited thereto, PEGylation of one or more side chains of the peptide of the present invention. Therein, "PEGylation" represents the act of coupling (e.g., covalently) a Polyethylene glycol (PEG) structure to the peptide of the invention. The skilled person knows well possible PEGs for coupling to the amino acid side chains of small peptides for forming a respective conjugate, e.g. from WO 2015/200916 Al, which are herein incorporated by reference. For clarifying reasons it is mentioned that such optional PEGylation of one or more amino acid side chains of the peptide can occur additionally to the incorporation of the PEG spacer X7, which is a mandatory feature of the peptide of the present invention, and which - in contrast to the side chain PEGylation - is incorporated within the peptide chain as explained above. The invention further comprises the pharmaceutically acceptable salts of the peptides as defined herein. Therein, pharmaceutically acceptable salts represent salts or zwitterionic forms of the peptides or compounds of the present invention which are water or oil-soluble or dispersible, which are suitable for treatment of diseases without undue toxicity, irritation, and allergic response; which are commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting an amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethansulfonate (isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Preferred acid addition salts include trifluoroacetate, formate, hydrochloride, and acetate.

Also, amino groups in the compounds of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. A pharmaceutically acceptable salt may suitably be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts.

Examples of basic salts include salts where the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R')(R²)(R³)(R⁴)+, where R¹,

2 3 4

R , R and R independently from each other will typically designate hydrogen, optionally substituted d-₆-alkyl or optionally substituted C₂_6-alkenyl. Examples of relevant Ci.₆-alkyl groups include methyl, ethyl, 1 -propyl and 2-propyl groups. Examples of C₂-₆-alkenyl groups of possible relevance include ethenyl, 1-propenyl and 2-propenyl. Therein, salts where the cation is selected among sodium, potassium and calcium are preferred.

Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). Also, for a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley- VCH, 2002). Other suitable base salts are formed from bases which form non-toxic salts. Representative examples include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases may also be formed, e.g., hemisulphate and hemicalcium salts.

The invention further comprises solvates of the peptides as defined herein. Therein the term "solvate" refers to a complex of defined stoichiometry formed between a solute (e.g., a peptide according to the invention or pharmaceutically acceptable salt thereof) and a solvent. The solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate.

The invention further comprises dimers comprising two of the peptides as defined herein, wherein the two peptide chains forming the dimer may be the same or different, each having the formula (I), (II) or (III) as defined herein. Preferred are homodimers, comprising two identical peptide chains according to formula (I), (II) or (III). The dimers are preferably formed by a disulfide bond between Cys residues in the two peptide chains. Such dimers of the peptides according to the present invention may be suitable pro-drugs in the medical field according to the present invention. The present invention particularly comprises the following embodiments, wherein the substituents or moieties of the peptide according to formula (I), or of formula (II) or (III) as defined below, may independently have the meanings as described below.

One embodiment of the present invention is the peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X7 is a PEG spacer as defined in formula (la) and (lb) with n = 2 to 5. Preferred is a PEG spacer of formula (la) and (lb) for X7 with n = 3 to 5, which includes a PEG spacer with n = 3, 4 or 5, such as PEG3, PEG4 or PEG5, or as PEG3-acetic, PEG4-acetic or PEG5-acetic, each as defined above. More preferably X7 is a PEG spacer as defined in formula (la) and (lb) with n = 2 or 3, such as PEG2, PEG 3, PEG2- acetic or PEG3-acetic. - -

One further embodiment of the present invention is the peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X9 is a fatty acid > C₈, more preferably a fatty acid > C₁₂, more preferably a fatty acid > Ci₄. It is also preferred that the fatty acid side chain conjugated to the amino acid moiety X8 is a Q₂ to Ci₈ fatty acid, preferably a Cj₂ to Cj₆ fatty acid, or a C₁₄ to Q₈ fatty acid, or a Q₄ to Ci₆ fatty acid. It is most preferred, that X9 is a Ci₆ fatty acid such as palmitic acid (palmitoyl, Palm).

One further embodiment of the present invention is the peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X7 is a PEG spacer as defined in formula (la) and (lb), with n = 3, 4 or 5, such as PEG3, PEG4 or PEG5, or as PEG3- acetic, PEG4-acetic or PEG5-acetic, more preferably with n = 2 or 3, such as PEG2, PEG 3, PEG2-acetic or PEG3-acetic.and wherein X9 is a fatty acid > C₈, more preferably a fatty acid > C₁₂, more preferably a fatty acid > C(₄. It is also preferred that the fatty acid side chain conjugated to the amino acid moiety X8 is a Q₂ to Ci₈ fatty acid, preferably a Q₂ to Q₆ fatty acid, or a C₁₄ to C₁₈ fatty acid, or a C₁₄ to C₁₆ fatty acid. It is most preferred, that X9 is a Cj₆ fatty acid such as palmitic acid (palmitoyl, Palm).

In a particular embodiment of the present invention, in the peptide according to formula (I) or (II), or a pharmaceutically acceptable salt or solvate thereof, both X4 and X5 are Arg and are independently selected from L-Arg, D-Arg, homo-arginine, nor-arginine, and an arginine mimetic.

Therein, it is preferred that X4 and X5 are both L-Arg, or X4 and X5 are both D-Arg, or one of X4 and X5 is L-Arg and the other is D-Arg.

Therein, it is even more preferred that X4 and X5 are both L-Arg, or that X4 and X5 are both D-Arg. In a further particular embodiment of the present invention, in the peptide according to formula (I) or (II), or a pharmaceutically acceptable salt or solvate thereof, both X4 and X5 are He and are independently selected from L-Ue, D-Ile, homo-isoleucine, nor-isoleucine, an isoleucine mimetic. Therein, it is preferred that X4 and X5 are both L-Ile, or X4 and X5 are both D-Ile, or X4 and X5 is L-Ile and the other is D-Ile.

Therein, it is even more preferred that X4 and X5 are both L-Ile, or that X4 and X5 are both D- He.

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X8 is Lys, including D- and L-Lys as well as homo- and nor-Lysine.

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X8 is ornithine (Orn).

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein Xl l is Cys, such as preferably L- Cys, D-Cys or homo-Cys.

A further particular embodiment of the present invention relates to a cyclized peptide, wherein in the peptide according to formula (I), as defined above, Xl l is Cys and X13 is Cys, which together form a disulfide bond, thus forming a cyclized peptide of the following formula (II):

X0 - X12 - Cys - XI - Thr - His - X2 - X3 - X4 - Cys - X5 - X6 - X7 - [X8(- X9)] - X10

(Π) or a pharmaceutically acceptable salt or solvate thereof.

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein XI 1 is Met.

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein XI 1 is Ser. The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein XI 1 is Cys-Me.

The invention relates to a peptide according to formula (I) or (II) or a pharmaceutically acceptable salt or solvate thereof, wherein XO is particularly selected from the group consisting of acetyl, a C₃-Ci₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), or XO is absent.

Therein, more particularly, XO is acetyl, isovaleric acid, orotic acid (OA), dihydroorotic acid (Hoo), or XO is absent.

Therein, even more particularly, XO is acetyl, isovaleric acid, dihydroorotic acid (Hoo), or XO is absent. The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein XO has one of the aforesaid meanings and X13 is Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the Cys residues according to formula (II) above. The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein XI is Asp, iminodiacetic acid (Ida), Gly, Ahx-Asp or is absent.

In a further aspect of the invention XI is Asp, iminodiacetic acid (Ida), Ac-Gly, Me-Gly or is absent.

In a further aspect of the invention XI is Asp, Gly, iminodiacetic acid (Ida) or is absent. Therein, XI is preferably Asp or iminodiacetic acid (Ida).

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein XO is absent, and XI is Asp or iminodiacetic acid (Ida). In a further aspect of the invention XO is absent, and XI is Asp, Gly, iminodiacetic acid (Ida) or is absent. The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X2 is Phe, substituted Phe, or diphenylalanine (Dip).

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X3 is Pro, β-proline, a proline mimetic, Gly-Gly, sarcosine (Sar), 2,2-dimethylglycine (Aib), or a proline spacer which is selected from the group consisting of γ-aminobutyric acid, morpholine-3-carboxylic acid, nipecotic acid (Nip), octahydroindole (Oic), or piperidine-2-carboxylic acid (Pip).

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X6 is Phe, substituted Phe, diphenylalanine (Dip), or Tyr.

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein X12 particularly indicates 1, 2 or 3 additional amino acids and/or spacer as defined above, or X12 is absent. Preferably X12 indicates Gly, Ser, Ala, β-alanine, PEG1, or 6-aminohexanoic acid, or X12 is absent.

The invention further relates to a peptide according to formula (I), (II) or (III) or a pharmaceutically acceptable salt or solvate thereof, wherein

X2 is Phe,

X4 is Arg,

X5 is Arg, and

X6 is Phe.

X2 is Phe,

X4 is Arg,

XI 1 is Cys or Met,

X5 is Arg,

X6 is Phe, and

X7 is PEG3. - -

X2 is Phe,

X3 is Pro,

X4 is Arg,

X5 is Arg, and

X6 is Phe.

X2 is Phe,

X3 is Pro,

X4 is Arg,

XI 1 is Cys or Met,

X5 is Arg,

X6 is Phe, and.

X7 is PEG3.

In the aforementioned embodiments the remaining moieties may have the meaning as defined anywhere in the present invention. In particular, the remaining moieties may have the meanings, indicated as particularly preferred.

The invention further relates to a peptide according to formula (I) or (II) or a pharmaceutically acceptable salt or solvate thereof, wherein X10 particularly represents from 0 up to 10, preferably 0 up to 5, more preferably 0 up to 3, i.e. 0, 1, 2 or 3 additional amino acids, preferably 0 or 1 additional amino acids, most preferred is that X10 indicates 0 additional amino acids, resulting in an amide C-terminus (-NH₂), i.e. a peptide according to formula (III):

X0 - X12 - X13 - Xl - Thr - His - X2 - X3 - X4 - Xl l - X5 - X6 - X7 - [X8(- X9)] - NH₂

(III) wherein the remaining moieties X0, XI, X2, X3, X4, X5, X6, X7, X8, X9, XI 1, X12, and X13 may have any meaning as defined herein.

In any of the aforesaid embodiments the remaining substituents or moieties may have any of the meanings as defined above. A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, wherein

X0 is C₃-Ci₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), or X0-Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the Cys residues, or X0 is absent;

XI is Asn, Asp, Glu, Gin, Gly, iminodiacetic acid (Ida), orotic acid (OA), dihydroorotic acid (Hoo), sarcosine (Sar), or a group Ahx-Asp, or X12- XI with X12 having the meaning as defined below, or XI is absent;

X2 is Phe, substituted Phe, diphenyl alanine (Dip), Trp, 1-napthylalanine (1-

Nal), or 2-napthylalanine (2-Nal);

X3 is Pro, β-proline, a proline mimetic, 2,2-dimethylglycine (Aib), or a proline spacer which is selected from the group consisting of γ-aminobutyric acid, morpholine-3-carboxylic acid, nipecotic acid, octahydroindole (Oic), and pipecolic acid;

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X6 is Phe, substituted Phe, diphenylalanine (Dip), Lys, Trp, 1-napthylalanine

(1-Nal), 2-napthylalanine (2-Nal), Tyr;

X7 is a PEG spacer as defined in formula (la) and (lb) of claim 1 , with n = 2 to

5, preferably with n = 3 to 5, more preferably with n = 3, 4 or 5, more preferably with n = 2 or 4, such as PEG2, PEG3, PEG4 or PEG5, or as PEG2-acetic, PEG3-acetic, PEG4-acetic or PEG5-acetic, or a group

XT is Arg (R);

X8 is Lys, or ornithine (Orn);

X9 is a fatty acid > C₈; preferably a fatty acid > C]₂, preferably a fatty acid >

Ci₄, preferably a C₁₂ to Cjg fatty acid, or preferably a Cj₆ fatty acid such as palmitic acid (palmitoyl, Palm);

X10 is 0 up to 15 additional amino acids, with an amide or carboxylic acid C- terminus;

XI I is Cys, Met, or Ser; - -

X12 is 1, 2 or 3 additional amino acids and/or spacer, wherein the spacer may be selected from 6-aminohexanoic acid (Ahx), PEG1, β-alanine, or 4- aminobutyric acid, or XI 2 is absent;

X13 is Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the

Cys residues, or X13 is absent.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, wherein X0 isC₃-Ci₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), or X0-Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the Cys residues, or X0 is absent;

XI is Asn, Asp, Glu, Gin, Gly, iminodiacetic acid (Ida), orotic acid (OA), dihydroorotic acid (Hoo), sarcosine (Sar), or X12-X1 with X12 having the meaning as defined below, or XI is absent;

X2 is Phe, substituted Phe, diphenylalanine (Dip), Trp, 1-napthyl alanine ilNal), or 2-napthylalanine (2-Nal);

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X6 is Phe, substituted Phe, diphenylalanine (Dip), Trp, 1-napthylalanine (1-

Nal), 2-napthylalanine (2-Nal), Tyr;

X7 is a PEG spacer as defined in formula (la) and (lb) of claim 1, with n = 2 to

5, preferably with n = 3 to 5, more preferably with n = 3, 4 or 5, such as

PEG3, PEG4 or PEG5, or as PEG3-acetic, PEG4-acetic or PEG5-acetic;

X8 is Lys, or ornithine (Orn);

X9 is a fatty acid > C₈; preferably a fatty acid > Q₂, preferably a fatty acid >

C_i , preferably a Ci₂ to Ci₈ fatty acid, or preferably a C₎₆ fatty acid such as palmitic acid (palmitoyl, Palm);

XI I is Cys, Met, or Ser; X12 is 1, 2 or 3 additional amino acids and/or spacer, wherein the spacer may be selected from 6-aminohexanoic acid (Ahx), PEG1, β-alanine, or 4- aminobutyric acid, or X12 is absent;

Cys residues, or X13 is absent.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, wherein

X0 is isovaleric acid, or dihydroorotic acid (Hoo), or X0 is absent;

XI is Asp, or iminodiacetic acid (Ida), or XI is absent;

X2 is Phe, substituted Phe, or diphenylalanine (Dip);

X3 is Pro, β-proline, a proline mimetic, 2,2-dimethylglycine (Aib) or a proline spacer which is selected from the group consisting of γ-aminobutyric acid, morpholine-3-carboxylic acid, nipecotic acid, octahydroindole (Oic), and pipecolic acid;

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X6 is Phe, substituted Phe, diphenylalanine (Dip), or Tyr;

X7 is a PEG spacer as defined in formula (la) of claim 1, with n = 2 to 5, preferably with n = 3 to 5, more preferably with n = 3, 4 or 5, more preferably with n = 2 or 3, such as PEG2, PEG3, PEG4 or PEG5;

X8 is Lys, or ornithine (Orn);

Cj₄, preferably a Cj₂ to C₁₈ fatty acid, or preferably a Ci₆ fatty acid such as palmitic acid (palmitoyl, Palm);

X10 is 0 additional amino acids, resulting in an amide C-terminus (-NH₂);

XI 1 is Cys, Met, Cys-Me or Ser;

X12 is absent;

Cys residues, or X13 is absent.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, wherein X0 is isovaleric acid, or dihydroorotic acid (Hoo), or X0 is absent;

XI is Asp, or iminodiacetic acid (Ida), or XI is absent;

X2 is Phe, substituted Phe, or diphenylalanine (Dip);

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X6 is Phe, substituted Phe, diphenylalanine (Dip), or Tyr;

X7 is a PEG spacer as defined in formula (la) of claim 1, with n = 2 to 5, preferably with n = 3 to 5, more preferably with n = 3, 4 or 5, such as PEG3, PEG4 or PEG5;

X8 is Lys, or ornithine (Orn);

X9 is a fatty acid > C₈; preferably a fatty acid > Ci₂, preferably a fatty acid >

C₁₄, preferably a Cj₂ to Cj₈ fatty acid, or preferably a C₁₆ fatty acid such as palmitic acid (palmitoyl, Palm);

X10 is 0 additional amino acids, resulting in an amide C-terminus (-NH₂);

XI I is Cys, Met, or Ser;

X12 is absent;

Cys residues, or X13 is absent.

X0 is absent;

XI is Asp, or iminodiacetic acid (Ida), or XI is absent;

X2 is Phe, substituted Phe, or diphenylalanine (Dip);

X3 is Pro, β-proline, a proline mimetic,, 2,2-dimethylglycine (Aib) or a proline spacer which is selected from the group consisting of γ-aminobutyric acid, morpholine-3-carboxylic acid, nipecotic acid, octahydroindole (Oic), and pipecolic acid;

X4 and X5 are identical and are selected from L-Arg, D-Arg, L-Ile and D-Ile; ₉

X6 is Phe, substituted Phe, diphenylalanine (Dip), or Tyr;

X7 is a PEG spacer as defined in formula (la) of claim 1, with n = 3, 4 or 5, such as PEG3, PEG4 or PEG5, preferably with n = 2 or 3, such as PEG2 or PEG3;

X8 is Lys, or ornithine (Orn);

X9 is a fatty acid > Q₄, preferably a C₁₂ to Ci₈ fatty acid, or preferably a C₁₆ fatty acid such as palmitic acid (palmitoyl, Palm);

X10 is 0 additional amino acids, resulting in an amide C-terminus (-NH₂);

XI 1 is Cys, Met, Cys-Me, or Ser;

XI 2 is absent;

Cys residues, or X13 is absent.

X0 is absent;

XI is Asp, or iminodiacetic acid (Ida), or XI is absent;

X2 is Phe, substituted Phe, or diphenylalanine (Dip);

X4 and X5 are identical and are selected from L-Arg, D-Arg, L-Ile and D-Ile;

X6 is Phe, substituted Phe, diphenylalanine (Dip), or Tyr;

X7 is a PEG spacer as defined in formula (la) of claim 1, with n = 3, 4 or 5, such as PEG3, PEG4 or PEG5;

X8 is Lys, or ornithine (Orn);

X9 is a fatty acid > Cj₄, preferably a Ci₂ to Ci₈ fatty acid, or preferably a Ci₆ fatty acid such as palmitic acid (palmitoyl, Palm);

X10 is 0 additional amino acids, resulting in an amide C-terminus (-NH₂);

XI I is Cys, Met, or Ser;

X12 is absent; - -

Cys residues, or X13 is absent.

It is further preferred, that in the aforesaid embodiments, X8 is ornithine (Orn). Alternatively, X8 may be Lys.

It is further preferred, that in the aforesaid embodiments XI 1 is Cys.

It is further preferred, that in the aforesaid embodiments XI 1 is Cys and X13 is Cys, which together form a disulfide bond according to formula (II) above.

Alternatively, in the aforesaid embodiments XI 1 is Met. Alternatively, in the aforesaid embodiments XI 1 is Ser.

Alternatively, in the aforesaid embodiments XI 1 is Cys-Me.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 3, SEQ ID 4, SEQ ID 7, SEQ ID 8, SEQ ID 10, SEQ ID 12, SEQ ID 13, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 22, SEQ ID 38 and SEQ ID 45.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 12 and SEQ ID 13.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 3, SEQ ID 4, SEQ ID 7, SEQ ID 8, and SEQ ID 10.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 22, SEQ ID 38 and SEQ ID 45.

A further particular embodiment of the present invention relates to a peptide according to any one of the aforesaid embodiments or a pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ 8, SEQ ID 22, SEQ ID 38 and SEQ ID 45.

A further particular embodiment of the present invention relates to a peptide or a pharmaceutically acceptable salt or solvate thereof, having the sequence ID SEQ ID 22.

A further particular embodiment of the present invention relates to a peptide or a pharmaceutically acceptable salt or solvate thereof, having the sequence ID SEQ ID 45. A further particular embodiment of the present invention relates to a peptide having the structural formula IV)

The peptide according to formula (IV) has the SEQ ID 45.

Preferred examples comprise (using the amino acid 1 -letter code according to Table 1 and the abbreviations above and indicating specific D- and L-isoforms by capital or small letters as explained above in detail) the Example Compounds No. 1 to 40 as shown in Table 3 and 4 in the Examples below. The at least one peptide, or derivative or analogue thereof as defined herein or the pharmaceutically acceptable salt or solvate thereof or the complex or the pharmaceutical composition (as defined below), are hereinafter commonly also referred to as "the/a hepcidin mimetic peptide of the present invention".

In some embodiments, a hepcidin mimetic peptide according to the present invention binds to feiToportin, e.g. human ferroportin. In certain embodiments, the hepcidin mimetic peptide of the present invention specifically binds to human ferroportin. As used herein, "specifically binds" refers to a specific binding agent's preferential interaction with a given ligand over other agents in a sample. For example, a specific binding agent that specifically binds a given ligand binds the given ligand, under suitable conditions, in an amount or a degree that is observable over that of any nonspecific interaction with other components in the sample. Suitable conditions are those that allow interaction between a given specific binding agent and a given ligand. These conditions include pH, temperature, concentration, solvent, time of incubation, and the like, and may differ among given specific binding agent and ligand pairs, but may be readily determined by those skilled in the art. In some embodiments, a hepcidin mimetic peptide of the present invention binds ferroportin with greater specificity than a hepcidin reference compound (e.g. any one of the hepcidin reference compounds provided herein). The invention thus further relates to a complex comprising at least one peptide, derivative or analogue as defined herein bound to ferroportin or to an antibody.

In some embodiments, a hepcidin mimetic peptide of the present invention exhibits specific binding to ferroportin that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, 1000%, or 10,000% higher than a selected hepcidin reference compound. In some embodiments, a hepcidin mimetic peptide of the present invention exhibits specific binding to ferroportin that is at least about 5 fold, or at least about 10, 20, 50, or 100 fold higher than a selected hepcidin reference compound. In some embodiments, a hepcidin mimetic peptide of the present invention exhibits a hepcidin activity. In some embodiments, the activity is an in vitro or an in vivo activity, e.g. an in vitro or in vivo activity described herein. In some embodiments, a hepcidin mimetic peptide of the present invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99% of the activity exhibited by a selected hepcidin reference compound. In some embodiments, a hepcidin mimetic peptide of the present invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99% of the ferroportin binding ability that is exhibited by a selected reference hepcidin. In some embodiments, a hepcidin mimetic peptide of the present invention has a lower IC₅₀ (i.e. higher binding affinity) for binding to ferroportin, (e.g., human ferroportin) compared to a selected reference hepcidin. In some embodiments, a hepcidin mimetic peptide according to the present invention has an IC50 in a ferroportin competitive binding assay which is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 700%, or 1000% lower than that of a reference hepcidin.

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

In some embodiments, a hepcidin mimetic peptide of the present invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200% 300%, 400%, 500%, 700%, or 1000% greater in vitro activity for inducing the degradation of ferroportin protein as that of a selected reference hepcidin.

In some embodiments, a hepcidin mimetic peptide of the present invention exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or greater than 99%, 100%, 200% 300%, 400%, 500%, 700%, or 1000% greater in vivo activity for inducing the reduction of serum iron in an individual as does a reference hepcidin.

In some embodiments, the peptides of the present invention mimic the hepcidin activity of Hep25, the bioactive human 25-amino acid form. As used herein, in certain embodiments, a hepcidin mimetic peptide having a "hepcidin activity" means that the compound has the ability to lower serum iron concentrations in subjects (e.g. mice or humans), when administered thereto (e.g. by the parenteral route, e.g. by injection, or by the pulmonary, nasal, sublingual, lingual, buccal, dermal, transdermal, conjunctival, optic route or as implant or stent orally administered), in a dose-dependent and time-dependent manner. See e.g. as demonstrated in Rivera et al. (2005). In some embodiments, the peptides of the present invention lower the serum 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 at least about 5%, 10%>, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

In particular embodiments, the hepcidin mimetic peptides of the present invention have activity as assayed by the ability to cause the internalization and degradation of ferroportin in a ferroportin-expressing cell line as taught in Nemeth et al. (2006) and as described in detail in the Ferroportin Degradation Assay (FPN Degradation Assay) in the examples of the present invention. Therein, Hepcidin binds, then gets internalized and finally degraded, which is measured by the disappearance of a light signal upon degradation. It is particularly preferred that a hepcidin mimetic peptide of the present invention exhibits a respective activity of < 1,000 nM, preferably < 500 nM, more preferably < 300 nM, more preferably < 250 nM, more preferably < 200 nM, more preferably < 150 nM, more preferably < 100 nM, more preferably < 75 nM, more preferably < 50 nM , more preferably < 45 nM, more preferably < 40nM, more preferably < 35nM, more preferably < 30 nM. In further particular embodiments, the hepcidin activity of the hepcidin mimetic peptides according to the present invention is determined by measurement of their ability to decrease serum iron and their total iron binding capacity, as determined by a Serum Iron and Transferrin Saturation Assay, such as described in detail in the examples of the present invention. It is particularly preferred that a hepcidin mimetic peptide of the present invention lowers serum iron or reduces transferrin saturation at least about 5%, 10%>, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 99%.

In further particular embodiments, the hepcidin activity of the hepcidin mimetic peptides according to the present invention is determined by measurement of their ability to internalize and/or degrade cellular ferroportin, which is determined by immunohistochemistry or by flow cytometry, respectively, using antibodies which recognizes extracellular epitopes of ferroportin, such as described in detail in the Flow Cytometry Test in the examples of the present invention. It is particularly preferred that a hepcidin mimetic peptide of the present invention exhibits a respective activity of < 1,000 nM, preferably < 500 nM, more preferably < 300 nM, more preferably < 250 nM, more preferably < 200 nM, more preferably < 150 nM, more preferably < 100 nM, more preferably < 75 nM, more preferably < 50 nM, more preferably < 45 nM, more preferably < 40nM, more preferably < 35nM, more preferably < 30 nM.

In principle, it is also possible, to measure the in vitro activity of the hepcidin mimetic peptide of the selected reference peptides by their dose-dependent ability to inhibit the efflux of iron from ferroportin-expressing cells that are preloaded with radioisotopes or stable isotopes of iron, as described in Nemeth et al. (2006).

It is furthermore particularly preferred to determine the activity of the peptides of the present invention as a hepcidin mimetic by in vivo studies, such as e.g. in Serum Iron Regulation Studies in mice, such as e.g. in FVB or β-thalassemia mice, such as described in detail in the in vivo Studies in the examples of the present invention.

As provided herein, the IC50 values relate to the average IC₅o derived from the cellular ferroportin degradation assay as described in the examples. It is particularly preferred that a hepcidin mimetic peptide of the present invention exhibits an IC50 value of < 1,000 nM, preferably < 500 nM, more preferably < 300 nM, more preferably < 250 nM, more preferably < 200 nM, more preferably < 150 nM, more preferably < 100 nM, more preferably < 75 nM, more preferably < 50 nM, more preferably < 45 nM, more preferably < 40nM, more preferably < 35nM, more preferably < 30 nM.

It is particularly preferred that a peptide according to the present invention acts as a hepcidin mimetic peptide with its activity being determined in accordance with at least one of the specific assays and/or the in vivo studies according to the examples of the present invention.

In particular, a peptide according to the present invention acts as a hepcidin mimetic peptide when having an average IC₅₀ of not more than 1,000 nM, preferably < 500 nM, more preferably < 300 nM, more preferably < 250 nM, more preferably < 200 nM, more preferably < 150 nM, more preferably < 100 nM, more preferably < 75 nM, more preferably < 50 nM, more preferably < 45 nM, more preferably < 40nM, more preferably < 35nM, more preferably < 30 nM.

The peptides of the present invention (including analogues, derivatives, and pharmaceutically acceptable salts or solvates thereof as well as the above mentioned complex) act as a hepcidin mimetic and are thus particularly suitable for the use in the prophylaxis and treatment of hepcidin-associated disorders.

In particular, the peptides of the present invention (including analogues, derivatives, and pharmaceutically acceptable salts or solvates thereof as well as the above mentioned complex) are able to bind ferroportin or induce ferroportin internalization and/or degradation, thus being particularly suitable for the prophylaxis and treatment of hepcidin-associated disorders.

In particular the dimers of the peptides of the present invention (including analogues, derivatives, and pharmaceutically acceptable salts or solvates thereof as well as the above mentioned complex) may act as pro-drug for a hepcidin mimetic and are thus particularly suitable for the use in the prophylaxis and treatment of hepcidin-associated disorders.

Due to their aforesaid hepcidin activity and activity as a hepcidin mimetic, the peptides of the present invention (including analogues, derivatives, and pharmaceutically acceptable salts or solvates thereof as well as the above mentioned complex) are suitable for the use in in the prophylaxis and treatment of hepcidin-associated disorders.

According to the present invention hepcidin-associated disorders comprise disorders related with reduced hepcidin levels or reduced responsiveness to hepcidin; disorders related with increased serum iron levels, such as in particular iron overload, hemochromatosis (hereditary hemochromatosis); iron- loading anemias such as thalassemia; diseases being associated with ineffective erythropoiesis such as myelofibrosis, myelodysplastic syndrome, and sickle cell disease; diseases with augmented erythropoiesis such as polycythemia vera; reduction of iron levels in patients with chronic kidney diseases; reduction of iron levels in bacterial infections and polymicrobial sepsis.

In particular, the hepcidin-associated disorders include diseases where aberrant iron metabolism directly causes the disease, or where iron blood levels are dysregulated causing disease, or where iron dysregulation is a consequence of another disease, or where diseases can be treated by modulating iron levels, and the like. More specifically, a hepcidin-associated disorder includes: iron overload diseases such as iron hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemojuvelin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, a-thalassemia, β-thalassemia.

The hepcidin mimetics of the invention may further be used to treat diseases and disorders that are not typically identified as being iron-related. For example, hepcidin is highly expressed in the murine pancreas suggesting that diabetes (Type I or Type II), insulin resistance, glucose intolerance and other disorders may be ameliorated by treating underlying iron metabolism disorders. See Ilyin et al. (2003), which is herein incorporated by reference. As such, peptides of the invention may be used to treat these diseases and conditions. Those skilled in the art are 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 2004/092405, which is herein incorporated by reference, and assays which monitor hepcidin, hemojuvelin, or iron levels and expression, which are known in the art such as those described in U.S. Patent No. 7,534,764, which is herein incorporated by reference.

The iron overload diseases also include alcoholic liver diseases and chronic hepatitis C.

It is possible for the hepcidin mimetic peptide of the present invention to have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.

For these administration routes, it is possible for the compounds according to the invention to be administered in suitable administration forms.

For oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally- disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms. Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.

Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents.

The compounds according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia,

• fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel^®), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos^®)),

• ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols),

• bases for suppositories (for example polyethylene glycols, cacao butter, hard fat),

• solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium chain-length triglycerides fatty oils, liquid polyethylene glycols, paraffins),

• surfactants, emulsifiers, dispersants or wetters (for example sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols (such as, for example, Lanette^®), sorbitan fatty acid esters (such as, for example, Span^®), polyoxyethylene sorbitan fatty acid esters (such as, for example, Tween^®), polyoxyethylene fatty acid glycerides (such as, for example, Cremophor^®), polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (such as, for example, Pluronic^®), • buffers, acids and bases (for example phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine),

• isotonicity agents (for example glucose, sodium chloride),

• adsorbents (for example highly-disperse silicas),

• viscosity-increasing agents, gel formers, thickeners and/or binders (for example polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids (such as, for example, Carbopol^®); alginates, gelatine),

• disintegrates (for example modified starch, carboxymethylcellulose-sodium, sodium starch glycolate (such as, for example, Explotab^®), cross- linked polyvinylpyrrolidone, croscarmellose-sodium (such as, for example, AcDiSol^®)),

• flow regulators, lubricants, glidants and mould release agents (for example magnesium stearate, stearic acid, talc, highly-disperse silicas (such as, for example, Aerosil^®)),

• coating materials (for example sugar, shellac) and film formers for films or diffusion membranes which dissolve rapidly or in a modified manner (for example polyvinylpyrrolidones (such as, for example, Kollidon^®), polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates such as, for example, Eudragit^®)),

• capsule materials (for example gelatine, hydroxypropylmethylcellulose),

• synthetic polymers (for example polylactides, polyglycolides, polyacrylates, polymethacrylates (such as, for example, Eudragit^®), polyvinylpyrrolidones (such as, for example, Kollidon^®), polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and blockcopolymers),

• plasticizers (for example polyethylene glycols, propylene glycol, glycerol, triacetine, triacetyl citrate, dibutyl phthalate),

• penetration enhancers,

• stabilisers (for example antioxidants such as, for example, ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate),

• preservatives (for example parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate),

• colourants (for example inorganic pigments such as, for example, iron oxides, titanium dioxide), • flavourings, sweeteners, flavour- and/or odour-masking agents.

The present invention furthermore relates to a pharmaceutical composition comprising at least one peptide, derivative or analogue as defined herein or a pharmaceutically acceptable salt or solvate thereof or a complex as defined above.

In particular, the present invention relates to a pharmaceutical composition comprising at least one peptide, derivative or analogue as defined herein or a pharmaceutically acceptable salt or solvate thereof or a complex as defined above, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention.

A pharmaceutical composition according to the present invention may comprise at least one additional active ingredient, such as preferably an additional active ingredient which is active in the prophylaxis or treatment of the disorders or diseases as defined herein.

Examples of preferred additional active ingredients including (without being limited thereto): active ingredients for treating iron overload conditions, such as for example iron chelating agents, such as e.g. Deferoxamine, Deferoxamine mesylate, Deferasirox (Exjade™, Jadenu™), Deferiprone, and Desferal.

It is also possible to use the peptides of the present invention in a combination therapy for the prophylaxis and treatment of the diseases or disorders described herein. Such combination therapy may comprise providing at least one peptide, derivative or analogue as defined herein or a pharmaceutically acceptable salt or solvate thereof or a complex or a pharmaceutical composition as defined herein as a first therapeutic agent to a subject in need thereof i.e. before and/or simultaneously with and/or after a second therapeutic agent.

The invention further relates to a kit-of-parts combination comprising at least one peptide, derivative or analogue as defined herein or a pharmaceutically acceptable salt or solvate thereof, a complex or a pharmaceutical composition as defined above, and at least one selected from a reagent, medical device, instruction letter or any combination thereof.

The invention further relates to a medical device comprising at least one peptide, derivative or analogue as defined herein or a pharmaceutically acceptable salt or solvate thereof, a complex or a pharmaceutical composition as defined above, for delivery of the peptide, derivative, analogue or complex thereof or of the pharmaceutical composition to a subject.

The pharmaceutical composition, kit-of-parts combination or medical device as defined above is in particular for the use in the prophylaxis or treatment of the disorders or diseases as defined as defined herein.

The invention further relates to a method of treating or ameliorating hepcidin-associated disorders, as defined above, in a subject or patient by administering at least one peptide, derivative or analogue as defined herein or a pharmaceutically acceptable salt or solvate thereof, a complex or a pharmaceutical composition as defined above, to said subject or patient in need thereof.

As used herein, the terms "patient", "subject" or "individual" may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats). The term "mammal" refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like. According to the invention the at least one peptide, derivative or analogue as defined herein or the pharmaceutically acceptable salt or solvate thereof, or the complex as defined above is administered to a patient or subject in a therapeutically effective amount, wherein a "therapeutically effective amount" of the hepcidin mimetic peptide of the invention is meant to describe a sufficient amount of the hepcidin mimetic peptide to treat an hepcidin-related disease or disorder as defined herein. In particular embodiments, the therapeutically effective amount will achieve a desired benefit/risk ratio applicable to any medical treatment.

The at least one peptide, derivative or analogue as defined herein or the pharmaceutically acceptable salt or solvate thereof or the complex or the pharmaceutical compositions as defined above may be administered enterally or parenterally, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal and intraarticular injection and infusion, orally, intravaginally, intraperitoneally, intrarectally, topically or buccally. Suitable formulations for the respective administration routes are well known to a skilled person and include, without being limited thereto: pills, tablets, enteric-coated tablets, film tablets, layer tablets, sustained-release or extended-release formulations for oral administration, plasters, topical extended-release formulations, dragees, pessaries, gels, ointments, syrup, granules, suppositories, emulsions, dispersions, microcapsules, microformulations, nanoformulations, liposomal formulations, capsules, enteric-coated capsules, powders, inhalation powders, microcrystalline formulations, inhalation sprays, powders, drops, nose drops, nasal sprays, aerosols, ampoules, solutions, juices, suspensions, infusion solutions or injection solutions, etc.

The suitable dosage of the hepcidin mimetic peptide of the present invention can be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including: a) the disorder being treated and the severity of the disorder; b) activity of the specific compound employed; c) the specific composition employed, the age, body weight, general health, sex and diet of the patient; d) the time of administration, route of administration, and rate of excretion of the specific hepcidin analogue employed; e) the duration of the treatment; f) drugs used in combination or coincidental with the hepcidin mimetic peptide employed, and like factors well known in the medical arts.

In particular embodiments, the total daily dose of the hepcidin mimetic peptide of the invention to be administered to a subject or patient in single or divided doses may be in amounts, for example, from 0.0001 to 300 mg/kg body weight daily or 1 to 300 mg/kg body weight daily, or from about 0.0001 to about 100 mg/kg body weight per day, such as from about 0.0005 to about 50 mg/kg body weight per day, such as from about 0.001 to about 10 mg/kg body weight per day, e.g. from about 0.01 to about 1 mg/kg body weight per day, administered in one or more doses, such as from one to three doses. Generally, the hepcidin mimetic peptide of the invention may be administered continuously (e.g. by intravenous administration or another continuous drug administration method), or may be administered to a subject at intervals, typically at regular time intervals, depending on the desired dosage and the pharmaceutical composition selected by the skilled practitioner for the particular subject. Regular administration dosing intervals include, e.g., once daily, twice daily, once every two, three, four, five or six days, once or twice weekly, once or twice monthly, and the like.

The invention further comprises the use of the hepcidin mimetic peptide as described herein for the manufacture of a medicament, in particular for the manufacture of a medicament for the prophylaxis or treatment of a disorder or disease as defined herein. The invention further comprises a method of binding ferroportin or inducing ferroportin internalization and/or degradation, comprising contacting the ferroportin with the at least one hepcidin mimetic peptide of the present invention. The invention further comprises a process for manufacturing the peptide, derivative or analogue or the pharmaceutically acceptable salt or solvate thereof or a complex, each as described herein. The process for manufacturing comprises the steps as shown in the examples of the present invention. Generally, The hepcidin mimetic peptide of the present invention may be manufactured synthetically, or semi-recombinantly.

The at least one peptide, derivative or analogue as defined herein or the pharmaceutically acceptable salt or solvate thereof or the complex as defined herein may also be used as a biochemical agent in a biochemical assay, such as e.g. in a diagnostic assay to measure responsiveness to hepcidin or in any biochemical assay being based on hepcidin binding.

The present invention also includes polynucleotides comprising a sequence encoding a hepcidin mimetic peptide according to the present invention, as well as a vector comprising a polynucleotide comprising a sequence encoding a hepcidin mimetic peptide according to the present invention.

The invention is further illustrated by the following examples, which relate to certain specific embodiments of the present invention. The examples were carried out using well known standard techniques within the routine to those of skill in the art, unless indicated otherwise. The following examples are for illustrative purposes only and do not purport to be wholly definitive as to conditions or scope of the invention. As such, they should not be construed in any way as limiting the scope of the present invention. - -

DESCRIPTION OF THE FIGURES

Fig. 1 shows the results of the in vivo evaluation of the serum iron levels in a mouse model, using β-thalassemia mice having been treated subcutaneously with Example Compound 12 (Fig. 1A) and Example Compound 13 (Fig. IB). Serum was collected after 24h and serum iron levels were measured by colorimetric assay.

Fig. 2 shows the results of the in vivo evaluation of the effects of the treatment of β- thalassemia mice in a mouse model with Example Compound 12, wherein thalassemia mice were treated twice weekly for 6 weeks with Example Compound 12 (3mg/kg, SC).

Complete blood counts performed at the end of the 6 week study showed reduced reticulocyte numbers, increased RBC counts, and hemoglobin, and hematocrit values indicating a more effective erythropoiesis (Fig. 2A).

Average spleen size was significantly reduced in the treated group suggesting a reduction in extramedullary hematopoiesis (Fig. 2B).

Extramedullary hematopoiesis in the spleen, as well as liver glycogen, and liver vacuolation were reduced as observed by histopathology analysis (Fig. 2C)

Spleen iron was increased, liver iron unchanged, and kidney iron reduced (Fig. 2D).

Serum iron was significantly reduced at the 24h after last dose (Fig. 2E).

Weekly blood collection demonstrated significant improvement in the percentage of mature CD44-/CD71-, and a reduction in reactive oxygen species (ROS) in Terl l9+ red blood cells (RBCs) (Fig. 2F). EXAMPLES

Analytical Methods

Method MCW-LTQ-POROSHELL-TFA98-18min

Equipment type MS: ThermoFisherScientific LTQ-Orbitrap-XL; Equipment type HPLC: Agilent 1200SL; Column: Agilent, POROSHELL 120, 3 x 150 mm, SB - C18 2.7 μιη; Eluent A: 1 1 Water + 0.1% Trifluoroacetic acid; Eluent B: 1 1 Acetonitrile + 0.1% Trifluoroacetic acid; Gradient: 0.0 min 2% B→ 1.5 min 2% B→ 15.5 min 95% B → 18.0 min 95% B; Oven: 40°C; Flow rate: 0.75 ml/min; UV-Detection: 210 nm

Method MCW-LTQ-POROSHELL-TFA95-10min

Equipment type: ThermoFisherScientific LTQ-Orbitrap-XL; Equipment type HPLC: Agilent 1200SL; Column: Agilent, POROSHELL 120; 3 x 150 mm, SB - C18 2.7 μπι; Eluent A: 1 1 Water + 0.1% Trifluoroacetic acid; Eluent B: 1 1 Acetonitrile + 0.1% Trifluoroacetic acid; Gradient: 0.0 min 5% B→ 0.3 min 5% B→ 7.0 min 98% B→ 10 min 98% B; Oven: 40°C; Flow rate: 0.75 ml/min; UV-Detection: 210 nm

Example 1 Preparation Example

General Method for Synthetic Peptide Synthesis

Solid Phase Peptide Synthesis (SPPS) according to Method A-1, A-2, B, C or D as described below was carried out either using an automatic peptide synthesizer or performed manually by hand. Peptide synthesis was carried out in scale ranges from 0.1 to 0.25 mmol. When peptide synthesis was carried out by hand, the general procedure according to Method A-2 described below was used. Automatic peptide synthesis was performed on a Symphony X peptide synthesizer (Protein Technologies) by Method A-1 or carried out by Method B (JPT Peptide Technologies GmbH, VolmerstraBe 5, 12489 Berlin) or by Method C (Wuxi AppTec Co, Ltd, Nol Building, 288 FuTe ZhongLu, WaiGaoQiao Free Trade Zone, Shanghai, P.R. China).

Method A-1 - General Method for Automatic Peptide Synthesis

Fmoc-protected amino acids were purchased from Novabiochem, Bachem or Protein Technologies (Fluorenylmethoxycarbonyl = Fmoc) Solid-phase resins were purchased from Novabiochem or from Bachem. The resin loading was 0.4 - 0.74 mmol/g. Peptides were synthesized on Wang or Rink amide resin depending on the desired C-terminus. Cleavage of the fluorenylmethoxycarbonyl (Fmoc) protecting group was achieved using 20% piperidine in dimethylformamide. Each Fmoc cleavage step was carried out twice. Amino acids were coupled using 8 equivalents of the Fmoc-amino acid, with 8 equivalents of DIC (0.5 M in DMF) and 8 equivalents of Oxyma (0.5M in DMF). Amino acid couplings were conducted at room temperature and under a nitrogen atmosphere, when the Symphony X was used. Peptides were completely deprotected using trifluoroacetic acid (TFA)/H20/ triisopropylsilane (Tis) (94:3:3) and TFA/thioanisole (TA)/l,2-ethanedithiol (EDT) (90:7:3) for peptides containing Cys and Met. Oxidized methionine was reduced using 1.6% EDT and 1.2% trimethylsilylbromide in TFA for 2 h at room temperature. Optional Cvclization:

Disulfide bridges were formed by shaking peptides in 0.1 M ammonium bicarbonate buffer (pH

7.83) at a concentration of 0.5 mg/ml overnight. The solution was then lyophilized. Optional Acetylation:

N-terminal acetylation was performed using 10 equivalents acetic anhydride in DMF (2 mL) and 2.5 equivalents DIPEA by shaking the suspension at RT for 1 h on an orbital shaker. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was then repeated again. Peptide cleavage:

A cleavage cocktail containing TFA/EDT/Thioanisol (90.3.7) was prepared. The cleavage cocktail (2 mL) was added to the peptide containing resin and the suspension was shaken on an orbital shaker for 2.5 hours. Cold ether (-20°C) was added to precipitate the peptide. The resulting solution was centrifuged under nitrogen (Sigma 2-16KL), and the resulting solid obtained after decantation was washed with cold ether 3 more times, by centrifugation and decantation. The resulting solid was purified by preparative HPLC.

Preparative HPLC:

An Agilent 1260 Prep reversed-phase HPLC was used for purification. The column is chosen based on the results of a column screen. The peptide is dissolved in 10 - 30% ACN/water (typically the starting point of the gradient). Water and acetonitrile both contain 0.1% TFA. A Waters X-Bridge column (X-Bridge C18 5μηι OBD, 19mm x 250 mm) was often used. Flow rate 20mL/min, 10-30% ACN/water to 85-90% ACN/water was typically used. Fractions were analysed by HPLC (Agilent 1260 Infinity) using using a Chromolith Speedrod column, 5-95% ACN/water gradient over 8 min) and by LCMS using methods described previously. - -

Method A-2 - General Method for Manual Peptide Synthesis

For the preparation of Ac-GTHFPRCRF-PEG3-K(Palmitoyl)-NH₂ representative reagents are used:

0.5M DIC in DMF

0.5M Oxyma in DMF

20% Piperidine in DMF

Acetic anhydride (10 equivalents) in 2mL DMF (0.1 mmol scale) for N-terminal acylation of peptides.

Rink amide resin (Loading = 0.74 mmol) was placed into a syringe and washed with DMF and DCM. 20% Piperidine solution in DMF was added and the suspension was shaken on a shaking apparatus for 2 hours at room temperature to remove the Fmoc group. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc-Lys(Palmitoyl)-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

20% Piperidine solution in DMF was added and the suspension was shaken on a shaking apparatus for 2 hours at room temperature to remove the Fmoc group. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc-NH-PEG3-COOH (PEG with 16 atoms) (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

20% Piperidine solution in DMF was added and the suspension was shaken on a shaking apparatus for 2 hours at room temperature to remove the Fmoc group. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again. . _

Fmoc-Phe-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage;

Fmoc-Arg(Pbf)-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

Fmoc-Cys(Trt)-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

Fmoc-Arg(Pbf)-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again. Fmoc cleavage:

Fmoc-Pro-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

Fmoc-His(Trt)-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

20% Piperidine solution in DMF was added and the suspension was shaken on a shaking apparatus for 2 hours at room temperature to remove the Fmoc group. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again. ^

Fmoc-Thr(tBu)-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

Fmoc-Gly-OH (3 equivalents), 0.5 M DIC solution (3 equivalents), 0.5 M Oxyma solution (3 equivalents) was added and the DMF suspension was shaken on a shaking apparatus for 2 hours at room temperature. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Fmoc cleavage:

Acetylation:

Acetic anhydride (10 equivalents) and diisopropylethyl amine (DIPEA, 2.5 equivalents) were added to the resin and the suspension was shaken for 15 min on a shaking apparatus. The solvent was removed and the resin was washed with DMF (5x) and DCM (5x). The procedure was repeated again.

Cleavage from the resin:

The resin was shaken with a solution of TFA/EDT/Thioanisole (90:3:7, 3 mL). The solution was collected and the peptide was precipitated by the addition of cold ether. The suspension was centrifuged on an orbital centrifuge under nitrogen. The pellet of the crude peptide was collected.

The crude peptide was dissolved in water/ACN (30% ACN) and purified on an Agilent 1260 Prep reversed-phase HPLC using an Waters X-Bridge column (X-Bridge C18 5μπι OBD, 19mm x 250 mm) using a 30% to 85% ACN with a 45 minute run time (water and acetonitrile each contain 0.10% TFA). The highest purity fractions were collected and lyophilized. 1 mg of the final peptide was obtained (97.5% pure, 0.56% yield) as a TFA salt and as a white lyophilized powder. Two other fractions of 96% purity were also obtained.

Method B - Alternative Peptide Synthesis

Resins:

TentaGel HL RAM (Capacity: 0.35 mmol/g): The peptides with C-terminal amide were synthesized using TentaGel HL RAM resin. A pre-loading of this resin did not take place. The coupling of the first amino acid was carried out on synthesis robot.

PHB resin (Capacity: 0.40 mmol/g) was loaded by a standard loading protocol: The unloaded resin was filled in a syringe, 5 equivalents amino acid relative to the resin loading resin were dissolved in dichloromethane (DCM) and added. For 1 g resin approximately 10 mL of the resulting solution were used. Afterwards 5 equivalents diisopropyl carbo->diimide (DIC) and N- methylimidazole (NMI) were added and the mixture was intensively mixed with a mechanical shaker for at least 4 h at ambient temperature. Then the solvent was removed in vacuo. The resin was washed three times using dimethyHformamide (DMF), dichloromethane (DCM) and diethyl ether and was then dried under vacuo to a constant weight. The Fmoc group remains on the loaded resin. The loading of the resin was determined via UV-detection of the Fmoc protecting group. Only when the loading of the resin was in the desired range (0.25 - 0.40 mmol/g) the resin was used for the synthesis.

2-Chlorotritylchloride resin (Capacity: 1.60 mmol/g): For the synthesis of a peptide with a C- terminal cysteine as carboxylic acid we used a pre-loaded cysteine 2 chlorotrityl resin. The following loading procedure was used: The resin was swollen in DCM and then the solvent was drained. Based on the loading capacity of the resin 0.9 equivalents of the first amino acid dissolved in DCM were added (70 mL DCM were used for 10 g of resin) followed by the addition of 3 equivalents NMI (based on resin capacity) and mixing for 3 h. The loading of the resin was determined via UV-detection of the Fmoc protecting group. Only when loading of the resin was in the desired range (0.4 - 0.5 mmol/g) the resin was washed with DCM and the remaining reactive trityl groups were end-capped by using methanol. This was achieved by adding 2 ml of a methanol/DIPEA-mixture (9 : 1) to 1 g of resin and shaking for 30 minutes. Afterwards the resin was washed with DCM before a solution of 25% piperidine in DMF (10 ml/g) was added and the mixture shaken for 20 min. Finally, the solvent was removed and the resin washed three times with DMF, DCM and diethyl ether and dried under vacuo overnight.

Solid Phase Peptide Synthesis (SPPS):

The peptide syntheses were applied using the conventional Fmoc/tBu Solid Phase Peptide Synthesis (SPPS) and carried out on a fully automated peptide synthesizer (Syro II, MultiSynTech) equipped with two reactor blocks for the synthesis of 48 peptides in parallel. The resins were used according to the C-terminus of the peptide. In case of peptide amides TentaGel HL RAM resins, in case of cysteine and MePhe the preloaded Cl-Trt resin and in case of the other peptide acids preloaded PHB resins were used. All amino acids used for the synthesis were fully protected, i.e. a amino function was protected by fluorenylmethyloxycarbonyl (Fmoc) and the functional groups of the side chains were protected by tert-butylether (Ser, Thr, Tyr), by tert-butylester (Asp, MeAsp, Glu), by trityl (Cys, His, Asn), by butyloxycarbonyl (Boc) (Lys, Trp), by pbf (Arg). All other amino acids or building blocks, namely MeHis, Melle, 9-Anthryl, Dip, Oic, Aib, Hyp, hPro, hCys, Cys(StBu), Pen, hDip, 1-Nal, Bip, 2-Nal, MeThr, Lys(palmitoyl), PEG3, PEGU and Ida were used as Fmoc derivatives without further protection. The synthesis was started with a swelling program. 4 ml of DMF was added to each reactor and the solvent was drained after 10 min. In order to guarantee that every amino on the resin is deprotected, the synthesis of each peptide started with removal of the Fmoc group. The resin was treated with a solution of piperidine DMF (1 : 9, 1.9 mL) for 10 min and then the solution was drained. This procedure was repeated once again and the resin was washed with DMF (5 x 2 mL). Every amino acid coupling step was carried out with a double coupling protocol. 0.3 mL DMF was first delivered in every reaction vessel followed by additiona of 0.675 mL of amino acid (0.5 M), 0.675 mL HATU (0.5 M) and 0.34 mL (2 M) DIPEA. The reaction was allowed to run for 60 min, then the solvent was drained and the resin was washed once with 2.1 ml DMF. For the second coupling the procedure was repeated.

In order to prevent deletion sequences an acetylation step was carried out, before the Fmoc deprotection was executed. Therefore, each reaction vessel was filled with 1.425 mL DMF followed by addition of 0.475 mL of a mixture of acetic acid anhydride/DIPEA/Oxyma dissolved in DMF (7: 1.75:0.2 equivalents related to the loading of the resin). After a reaction time of 5 min the reaction mixture was drained and the resin was washed four times with 2.0 mL DMF.

Fmoc group was removed by adding a solution of piper idine/DMF (1 : 9, 1.9 mL). After 10 min the solution was drained and the resin washed with 4 mL DMF. This procedure was repeated and resin washed with DMF (5 x 2 mL).

The reaction cycles including coupling, washing, deprotection and washing steps were repeated until the desired peptide sequence was completed.

Finally, after the last deprotection step, the resin was washed with DMF (4 x 2 mL) and diethyl ether (4 x 2 mL).

The peptides were cleaved off manually by addition of TFA EDT/H₂0 (94/3/3, 8 mL, 3 h) to the resin. Afterwards the peptide containing cleavage solution was separated and the peptide was precipitated by addition of cold diethyl ether. The precipitated peptides was washed twice with cold diethyl ether, dried overnight and analyzed.

Purification:

The crude peptides were purified via preparative HPLC. Therefore the peptides were dissolved in DMSO or TFA and purified with an appropriate eluent mixture (eluent A: H₂0 with 0.1% TFA; Eluent B: ACN with 0.1% TFA; linear gradient). The selected ratio of A to B depended on the quality and physicochemical properties of the crude peptides. The following columns were used for the purification:

Waters SunFire C18 OBD; 5 μπι; 150 x 50 mm

Agilent PLRP-S 100 A; 8 μπι; 150 x 25 mm

Agilent PLRP-S 100 A; 8 μηι; 300 x 50 mm

All purified peptides were freeze dried.

Method C - Alternative Peptide Synthesis

The synthesis of Example 33 (Ac-C+-DTHFPr-C+-rF-PEG2-K(Palmitoyl)-NH2) is representative: The following reagents were used:

MB HA rink amide resin (1 equiv)

Fmoc_Lys(Dde)-OH (3 equiv)

Fmoc-Phe-OH (3 equiv)

Fmoc-D-Arg(Pbf)-OH (3 equiv)

Fmoc-Cys(Trt)-OH (3 equiv)

Fmoc-D-Arg(Pbf)-OH (3 equiv)

Fmoc-Pro-OH (3 equiv)

Fmoc-Phe-OH (3 equiv)

Fmoc-His(Trt)-OH (3 equiv)

Fmoc-Thr(OtBu)-OH (3 equiv)

Fmoc-Asp(OtBu)-OH (3 equiv)

Fmoc-Cys(Trt)-OH (3 equiv)

Fmoc-PEG2-OH (2.5 equiv)

Palmitic acid (3 equiv)

The peptide was synthesized using standard Fmoc chemistry by manual synthesis. 1. Swell the resin for 30 min in DMF and push out the DMF out of column with nitrogen.

2. The Fmoc group was cleaved from the resin by adding 20% (v/v) piperidine in DMF.

The resin was allowed to react with the 20% piperidine solution for 20 min. 3. After the Fmoc cleavage from the resin is complete, the 20% piperidine solution is pushed out of the column. The resin is washed 3 times with DMF:

4. Preparing (or activating) the amino acid: 3 eq of the amino acid and 2.95 eq of HBTU were weighed and were then dissolved in DMF. 6 eq of DIEA was added to the above solution. The activated solution was then added to the column containing the resin and reacted for about 2 h.

5. The resin was drained and the loaded resin was wasged 3 times with DMF. 6. Steps 2-5 were repeated for each amino acid coupling.

7. The acetyl group protection was conducted by adding the cocktail of 5% Ac₂O/10% NMM 85% DMF. The solution was reacted for about 0.5 h. 8. Boc group protection when necessary was conducted by adding the 3 eq of (Boc)₂0 and 6 eq of DIEA to the resin in DMF. The reaction mixture was reacted for about 0.5 h.

9. The resin was drained and washed with DMF 3 times and with MeOH 3 times. Cleavage and disulfide bond formation:

The resulting residue was treated with cocktail of 90%TFA/5%TIPS/2.5%H2O/2.5%EDT (10 mL) and swelled for about 2 h. The crude peptide was precipitated out by ether. Cleavage:

The resulting residue was treated with cocktail of 90%TFA/5%TIPS/2.5%H₂O/2.5%EDT (10 mL) and swelled for about 2 h. The crude peptide was precipitated out by ether. If no disulfide bond formation was required, the crude peptide was purified by reversed-phase HPLC.

Optional Cyclization (disulfide bond formation):

The crude peptide was dissolved in H₂0/ACN (1:1) to adjust the concentration to 1 mM. Then 1 M NH₄HC0₃ was added to the above solution to adjust the pH to about 8-9. The solution was allowed to react for about 8 h at room temperature. The reaction was monitored by LCMS. After the reaction was completed, the reaction was quenched by acetic acid to adjust the pH to about 6. The reaction mixure was lyophilized and the resulting solid was purified by reversed- phase HPLC. Method D - General Method for the Preparation of Disulfide Dimers (SEP ID 45)

The crude or purified monomelic peptide was dissolved in H₂0/ACN = 1: 1. To the above solution was slowly added 0.5 M I₂/MeOH solution until the solution was turned to yellow. After that, the reaction was monitored by LCMS. After the reaction had gone to completion, the reaction was quenched using 1M Na₂S₂0₃ until the solution turned to colorless. The mixture was lyophilized to dryness and the resulting residue was purified by Prep-HPLC using procedures described above.

With the aforesaid manufacturing methods, the following peptides according to the present invention have been prepared: Table 3: Peptides prepared according to the invention

Example 2 Biological in vitro Testings

Ferroportin internalization assay

Test compounds were dissolved in DMSO. Functional internalization of ferroportin protein was measured using a stably-transfected CHO cell line expressing the human ferroportin tagged to a luciferase reporter. Cells were plated for 24h in the presence of ferric ammonium citrate (FAC). Ferroportin protein expression was induced with doxycycline for 24 h. The next day, cells were incubated with the test compounds for 6 h, and subsequently luciferase activity was measured using the Nano-Glo Luciferase Assay System and Glomax according to manufacturer's instructions (Promega, Madison, WI).

The average IC₅₀ has been determined for several Example Compounds according to the present invention: Table 4: Average IC50 of several Example Compounds according to Ferroportin Internalization Assay

Example SEQ ID Average IC50

Sequence

Compound No No. [nM]

1 1 Ida-THFPrCrF-PEG3-K-NH₂ >2000

2 2 Ida-THFPrCrF-PEG3-K(Ac)-NH₂ >2000

3 3 Ac-GTHFPRCRF-PEG3-K(Palmitoyl)-NH₂ 21

4 4 THFPRCRF-PEG3 -K(Palmitoyl)-NH₂ 32

5 5 DTHFPR-M-RF-PEG2-K(Palmitoyl)-NH₂ 124

6 6 DTHFPR-Nle-RF-PEG2-K(Palmitoyl)-NH₂ >2000

7 7 DTHFPRSRF-PEG2-K(Palmitoyl)-NH₂ 114 (102, 259)

Ac-C+-DTHFPR-C+-RF-PEG2-K(Palmitoyl)-

8 8 15 (25, 191)

NH₂

9 9 Ac-DTHFPRCRF-PEG3-K(Octanoyl)-NH₂ 1,009

10 10 Hoo-DTHFPRCRF-PEG3-K(Palmitoyl)-NH₂ 24

11 11 DTHFPRCRF-PEG3-R-K(Palmitoyl)-NH₂ 36

12 12 Ida-THFPrCiF-PEG3-K(Palmitoyl)-NH₂ 23 (45)

13 13 Ida-THFPRCRF-PEG3-K(Palmitoyl)-NH₂ 26 (50)

14 14 Ida-THFPrCrF-PEG3-k(Palmitoyl)-NH₂ 38 Example SEQ ID Average IC50

Sequence

Compound No No. [nM]

15 15 Ida-THFPRCRF-PEG3-0(Palmitoyl)-NH₂ 28

16 16 Ida-THF-Oic-rCiF-PEG3-K(Palmitoyl)-NH₂ 41

17 17 Ida-THFPr-hC-iF-PEG3-K(Palmitoyl)-NH₂ 42

18 18 Ida-THFPrCiF-PEG3-K(Dodecanoyl)-NH₂ 288

19 19 Ida-THF-Pip-rCrF-PEG3-K(Palmitoyl)-NH₂ 60

Ida-THFPrCiF-PEG3-E(l-Aminohexadecane)-

20 20 50

NH₂

21 21 Ida-TH-Dip-PrCiF-PEG3-K(Palmitoyl)-NH₂ 23

22 22 Ida-THFPrMiF-PEG3-K(Palmitoyl)-NH₂ 46 (366, 48)

23 23 Ida-THFPrCrF-PEG5-K(Palmitoyl)-NH₂ 21

24 24 Ida-THFPRCrF-PEG3-K(Palmitoyl)-NH₂ 31

25 25 Ida-THFPrCRF-PEG3-K(Palmitoyl)-NH₂ 77

26 26 S ar-THFPRCRF-PEG3 -K(Palmitoyl)-NH₂ 189

27 27 Hoo-THFPRCRF-PEG3-K(Palmitoyl)-NH₂ 52

28 28 Ida-THFPrCrY-PEG3-K(Palmitoyl)-NH₂ 97

29 29 Ida-THFPrCrf-PEG3-K(Palmitoyl)-NH₂ 137

30 30 Ida-THFPrCrK-PEG3-K(Palmitoyl)-NH₂ 343

C+-Ahx-DTHFPr-C+-rF-PEG3-K(Palmitoyl)-

31 36 121

NH₂

32 37 DTHFPrSrF-PEG2-K(Palmitoyl)-NH2 321

Ac-C+-DTHFPr-C+-rF-PEG2-K(Palmitoyl)-

33 38 40

NH2

34 39 Ac-C+-DTHFPrC+-rF-PEG3-K(Palmitoyl)-NH₂ 343 (373)

Isovaleric-C+DTHFPiC+rF-PEG3-K(Palm)-

35 40 549

NH2

36 41 Ac-C+DTHFPiC+rF-PEG3-0(Palm)-NH₂ 189

37 42 Ac-C+DTHFPrC+iF-PEG5-K(Palm)-NH₂ 311

Isovaleric-C+DTHF-Aib-rC+rF-PEG3-0(Palm)-

38 43 482

NH₂

Ida-THFPrC(Me)rF-PEG3(16atoms)-

39 44 800

K(Palmitoyl)-NH₂

Ida-THFPrCrF-PEG3-K(Palmitoyl)_NH₂

40 45 11

Disulfide Dimer values in parenthesis indicate a second measurement

each value represents an average with n=3 Further, the average IC₅₀ has been determined for several Comparative Compounds:

Table 5: Average IC₅₀ of several Comparative Compounds according to Ferroportin Internalization Assay

In the in vivo studies the Comparative Compound No. 1 turned out to be less effective than Example Compound No. 12.

Example 3 Biological in vivo Studies in a Mouse Model

Hbb^th3/+ mice (Yang et al., 1995) were bred at Jackson Laboratory, and all experiments were performed at Bayer HealthCare. Mice received ad libitum access to water and chow that contained 35 ppm of iron. Hepcidin mimetic peptide test compounds were prepared in 10% ethanol, 30% polyethylenglycol-400, and 60% water. For serum iron regulation studies, the test compounds according to Example Compounds 12 and 13 were injected subcutaneously, and after 24h blood was collected and serum prepared using serum separator tubes (Sarstedt, Numbrecht, Germany). For chronic β-thalassemia studies, animals were dosed subcutaneously twice weekly for 6 weeks with Example Compound 12 (3mg/kg, SC). Five μΐ tail vein blood were collected once weekly, 1 day before next dose, and analyzed by flow cytometry as described below. Necropsy was performed 24h after the last dose. Blood, spleen, liver, and kidney were collected. Organ weights were recorded, and complete blood counts were measured using an XT-2000iV hematology analyzer (Sysmex, Kobe, Japan), serum iron was measured as described below, and part of the organs were fixed for histopathology or snap frozen for total organ iron analysis as described (Killilea and Ames, 2008; Killilea et al., 2003; McColl et al., 2008).

Serum iron and transferrin saturation assay

Serum iron and total iron binding capacity (TIBC) were measured using a commercial kit in a microtiter plate format modified from the manufacturer's instructions (Pointe Scientific Inc., Canton, MI). In brief, 125 μΐ assay buffer were combined with 25 μΐ sample. Baseline absorbance was recorded at 560 nm using a plate reader (SpectraMax 360plus; Molecular Devices, Sunnyvale, CA). Iron color reagent was added and absorbance recorded for 20 min. Iron levels were calculated using mean absorbance from both readings following the manufacturer's instructions.

Total iron binding capacity was determined by measuring unsaturated iron binding capacity (UIBC) using the same kit. In brief, excess iron was added to the serum sample to bind any unsaturated transferrin and unbound iron was measured as described above. Transferrin saturation was calculated as the percentage of serum iron over the sum of total iron binding capacity and serum iron.

The results are shown in Figure 1A and IB.

Therefrom it is apparent, that in animals treated subcutaneously with Example Compound 12 (see Fig. 1A) and with Example Compound 13 (see Fig. IB) serum iron levels in serum collected after 24h were significantly reduced, measured by colorimetric assay. The results of the chronic β-thalassemia studies are shown in Figures 2A to 2E.

Therefrom it is apparent, that in complete blood counts performed at the end of the 6 week study showed reduced reticulocyte numbers, increased RBC counts, hemoglobin, and hematocrit, which indicates a more effective erythropoiesis (see Fig. 2A)

Average spleen size was significantly reduced in the treated group suggesting a reduction in extramedullary hematopoiesis (see Fig. 2B). Spleen iron was increased, liver iron unchanged, and kidney iron reduced (see Fig. 2D). In comparison, Casu and co-workers reported reduction in spleen, liver, and kidney iron following a 6 week study with test compound M004, however, they collected the organs for iron measurement 3.5 days after the final dose (Casu et al., 2016). At that timepoint, their compound M004 was likely no longer active. In the present studies, organs were collected 1 day after the last dose, when serum iron was still reduced (see Fig 2E). Ferroportin was likely still downregulated in spleen and liver which are rich in macrophages and macrophage-like cells, possibly accounting for the iron increase in these organs at this particular timepoint. In the kidney, macrophages may not be as prominent, and therefore kidney organ iron was reduced in the present studies (see Fig 2D).

Histopathology

Part of spleen and liver were fixed in neutral-buffered formalin, paraffin sections were prepared and hematoxylin-eosin stained. Sections were analyzed by a pathologist in a blinded fashion and findings were graded semiquantitatively from 0-5.

There was significant reduction in three pathologic parameters: Not only extramedullary hematopoiesis in the spleen, but also reduction in liver glycogen, and reduction in liver vacuolation (one tailed non-parametric Mann- Whitney, *p<0.05, or **p<0.01) (see Fig. 2C).

Flow cytometry

During the chronic 6 week study, cells were stained for flow cytometry analysis in two steps modified from procedures previously described (Alt et al., 2009; Casu et al., 2016). In brief, cells were stained with directly conjugated antibodies for Terl l9, CD44, and CD71 (BD Biosciences, San Jose, CA) for 20 min on ice and washed. Then cells were stained for reactive oxygen species with 2 μΜ CM-H2DCFDA for 30 min at room temperature according to the manufacturer's instructions (ThermoFisher, Eugene, OR), washed, and stained for with 50nM DAPI. Samples were measured with an LSRII (BD Biosciences, San Jose, CA). Data were analyzed with FCS Express (De Novo Software, Los Angeles, CA).

The results are shown in Figure 2F.

A significant improvement in the percentage of mature CD447CD71^", Terl l9⁺ red blood cells (RBCs) and a reduction in reactive oxygen species (ROS) (see Figures 2F) has been demonstrated.

In summary, the in vivo mouse model has shown, that treatment with Example Compound 12 ameliorated β-thalassemia.

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Jordan, J.B., Poppe, L., Haniu, M., Arvedson, T., Syed, R., Li, V., Kohno, H., Kim, H., Schnier, P.D., Harvey, T.S., et al. (2009). Hepcidin revisited, disulfide connectivity, dynamics, and structure. J. Biol. Chem. 284, 24155-24167.

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Killilea, D.W., Atamna, H., Liao, C, and Ames, B.N. (2003). Iron Accumulation During Cellular Senescence in Human Fibroblasts In vitro. Antioxid. Redox Signal. 5, 507-516.

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Park, C.H., Valore, E.V., Waring, A.J., and Ganz, T. (2001). Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J. Biol. Chem. 276, 7806-7810. Powell, L.W., Seckington, R.C., and Deugnier, Y. (2016). Haemochromatosis. The Lancet 388, 706-716.

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Rivera, S., Nemeth, E., Gabayan, V., Lopez, M., Farshidi, D., Ganz, T. (2005). Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin- containing organs. Blood 106, 2196-2199.

Santini, V., Girelli, D., Sanna, A., Martinelli, N., Duca, L., Campostrini, N., Cortelezzi, A., Corbella, M., Bosi, A., Reda, G., et al. (2011). Hepcidin Levels and Their Determinants in Different Types of Myelodysplastic Syndromes. PLoS ONE 6, e23109.

Sebastiani, G., Wilkinson, N., and Pantopoulos, K. (2016). Pharmacological Targeting of the Hepcidin/Ferroportin Axis. Front. Pharmacol. 7, 160.

Temraz, S., Santini, V., Musallam, K., and Taher, A. (2014). Iron overload and chelation therapy in myelodysplastic syndromes. Crit. Rev. Oncol. Hematol. 91, 64-73.

Walker, V.J., and Agarwal, A. (2016). Targeting Iron Homeostasis in Acute Kidney Injury. Semin. Nephrol. 36, 62-70.

Walter, P.B., Harmatz, P., and Vichinsky, E. (2009). Iron Metabolism and Iron Chelation in Sickle Cell Disease. Acta Haematol. 122, 174-183.

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Zeng, C, Chen, Q., Zhang, K., Chen, Q., Song, S., and Fang, X. (2015). Hepatic Hepcidin Protects against Polymicrobial Sepsis in Mice by Regulating Host Iron Status: Anesthesiology 122, 374-386. SEQUENCE LISTING

SEQ ID 1 Ida-THFPrCrF-PEG3-K-NH₂

SEQ ID 2 Ida-THFPrCrF-PEG3-K(Ac)-NH₂

SEQ ID 3 Ac-GTHFPRCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 4 THFPRCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 5 DTHFPR-M-RF-PEG2-K(Palmitoyl)-NH₂

SEQ ID 6 DTHFPR-Nle-RF-PEG2-K(Palmitoyl)-NH₂

SEQ ID 7 DTHFPRSRF-PEG2-K(Palmitoyl)-NH₂

! SEQ ID 8 Ac-C+-DTHFPR-C+-RF-PEG2-K(Palmitoyl)-NH₂

SEQ ID 9 Ac-DTHFPRCRF-PEG3-K(Octanoyl)-NH₂

SEQ ID 10 Hoo-DTHFPRCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 11 DTHFPRCRF-PEG3-R-K(Palmitoyl)-NH₂

SEQ ID 12 Ida-THFPrCrF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 13 Ida-THFPRCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 14 Ida-THFPrCrF-PEG3-k(Palmitoyl)-NH₂

SEQ ID 15 Ida-THFPRCRF-PEG3-0(Palmitoyl)-NH₂

SEQ ID 16 Ida-THF-Oic-rCiF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 17 Ida-THFPr-hC-rF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 18 Ida-THFPrCrF-PEG3-K(Dodecanoyl)-NH₂

SEQ ID 19 Ida-THF-Pip-rCrF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 20 Ida-THFPrCiF-PEG3-E( 1 - Aminohexadecane)-NH₂

SEQ ID 21 Ida-TH-Dip-PrCrF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 22 Ida-THFPrMrF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 23 Ida-THFPrCrF-PEG5-K(Palmitoyl)-NH₂

SEQ ID 24 Ida-THFPRCrF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 25 Ida-THFPrCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 26 Sar-THFPRCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 27 Hoo-THFPRCRF-PEG3-K(Palmitoyl)-NH₂

SEQ ID 28 Ida-THFPrCrY-PEG3-K(Palmitoyl)-NH₂

SEQ ID 29 Ida-THFPrCrf-PEG3-K(Palmitoyl)-NH₂

SEQ ID 30 Ida-THFPrCrK-PEG3-K(Palmitoyl)-NH₂

SEQ ID 31 Ida-TH-Dip-BHomoPro-RCR-BHomoPhe-PEG3-Palm

SEQ ID 32 DTHFPICIF-OH

SEQ ID 33 DTHFPICIF-NH₂

SEQ ID 34 Ida-THFPRCRF-OH

SEQ ID 35 Ac-C+-DTHFPR-C+-RF-NH₂ SEQ ID 36

SEQ ID 37

SEQ ID 38

SEQ ID 39

SEQ ID 40

SEQ ID 41

SEQ ID 42

SEQ ID 43

SEQ ID 44

SEQ ID 45

Claims

1. A peptide having the following formula (I)

X0 - X12 - X13 - XI - Thr - His - X2 - X3 - X4 - XI 1 - X5 - X6 - X7 - [X8(- X9)] - X10

(I) or a derivative or analogue or pharmaceutically acceptable salts or solvates thereof, wherein

X0 is Gly, 2,2-dimethylglycine (Aib), sarcosine (Sar), acetyl, C₃-Ci₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), pyroglutamate, Q-C6 alkyl, Ci-C₆ dialkyl or X0 is absent;

XI is Ala, Asn, Asp, Gin, Glu, Gly, 2,2-dimethylglycine (Aib), iminodiacetic acid

(Ida), orotic acid (OA), dihydroorotic acid (Hoo), sarcosine (Sar), or a group [6-aminohexanoic acid-Asp] ([Ahx-D]), or XI is absent;

X2 is Phe, substituted Phe, diphenylalanine (Dip), Trp, 1-napthylalanine (1-Nal), or

2-napthylalanine (2-Nal);

X3 is Pro, β-proline, a proline mimetic, a proline spacer, Gly-Gly, sarcosine (Sar), or 2,2-dimethylglycine (Aib);

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X7 is a PEG spacer, having the formula (la)

.0

or having the formula (lb)

(lb)

with n = 1 to 6,

or a group

XI -XT wherein X7 is a PEG spacer according to formula (la) or (lb) with n = 1 to 6 and

XT Arg (R);

X8 is Lys, or ornithine (Om);

X9 is a C₈ to C₂o fatty acid;

X10 is from 0 up to 15 additional amino acids, with an amide or carboxylic acid C- terminus;

XI 1 is Cys, Met, Ser, Cys-Me, N-Me-Cys or Penicillamine (Pen);

XI 2 is 1, 2 or 3 additional amino acids and/or spacer, wherein the spacer may be selected from 6-aminohexanoic acid (Ahx), 10-aminodecanoic acid, 11- undecanoic acid, 12-aminododecanoic acid, PEG1, β-alanine, and 4- aminobutyric acid, or XI 2 is absent

Cys residues, N-Me-Cys or Penicillamine (Pen), or X13 is absent;

2. A peptide according to claim 1, wherein

X0 is Gly, 2,2-dimethylglycine (Aib), sarcosine (Sar), acetyl, C₃-Ci fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), pyroglutamate, Cj-C₆ alkyl, Ci-C₆ dialkyl or X0 is absent; XI is Ala, Asn, Asp, Gin, Glu, Gly, 2,2-dimethylglycine (Aib), iminodiacetic acid (Ida), orotic acid (OA), dihydroorotic acid (Hoo), sarcosine (Sar), or XI is absent;

X4 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X5 is Arg, He, an arginine mimetic, or an isoleucine mimetic;

X7 is a PEG spacer, having the formula (la)

(la) or the formula (lb)

(lb)

with n = 1 to 6;

X8 is Lys, or ornithine (Orn);

X9 is a C₈ to C₂o fatty acid;

X10 is from 0 up to 15 additional amino acids, with an amide or carboxylic

terminus;

XI 1 is Cys, Met, Ser, N-Me-Cys or Penicillamine (Pen); X12 is 1, 2 or 3 additional amino acids and/or spacer, wherein the spacer may be selected from 6-aminohexanoic acid (Ahx), 10-aminodecanoic acid, 11-undecanoic acid and 12-aminododecanoic acid, PEG1, β-alanine and 4-aminobutyric acid, or X12 is absent

X13 is Cys which, when XI 1 is Cys, can optionally form a disulfide bond via the Cys residues, N-Me-Cys or Penicillamine (Pen), or X13 is absent.

3. The peptide according to claim 1 or 2 or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X7 is a PEG spacer as defined in formula (la) and (lb) with n = 2 to 5, preferably with n = 3 to 5, more preferably with n = 3, 4 or 5, even more preferably with n = 2 or 3;; such as PEG2, PEG3, PEG4 or PEG5 or as PEG2-acetic, PEG3-acetic, PEG4-acetic or PEG5-acetic.

4. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X9 is a fatty acid > C₈; preferably a fatty acid > Ci₂, preferably a fatty acid > Ci₄, preferably a Q₂ to Q₈ fatty acid, preferably a Ci₆ fatty acid such as palmitic acid (palmitoyl, Palm).

5. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

both X4 and X5 are Arg and are independently selected from L-Arg, D-Arg, homo- arginine, nor-arginine, an arginine mimetic,

or wherein

both X4 and X5 are He and are independently selected from L-Ile, D-Ile, homo- isoleucine, nor-isoleucine, an isoleucine mimetic;

preferably

X4 and X5 are both L-Arg, or

X4 and X5 are both D-Arg, or

one of X4 and X5 is L-Arg and the other is D-Arg, or

X4 and X5 are both L-Ile, or

X4 and X5 are both D-Ile, or one of X4 and X5 is L-Ile and the other is

6. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X8 is Lys.

7. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X8 is ornithine (Orn).

8. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

XI lis Cys, such as preferably L-Cys, D-Cys or homo-Cys.

9. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

XI lis Cys and

X13is Cys,

which together form a disulfide bond, forming a peptide of the following formula (II):

X0 - X12 - Cys - XI - Thr - His - X2 - X3 - X4 - Cys - X5 - X6 - X7 - [X8(- X9)]

- X10

(II)

10. The peptide according to any one of claims 1 to 7 or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

XI lis Met, or XI lis Ser, or

XI 1 is Cys-Me.

The peptide according to any one of claims 1 to 7 or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

XI lis Met, or

XI lis Ser.

The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X0 is acetyl, a C₃-C₁₆ fatty acid which can be branched or cyclic, orotic acid (OA), dihydroorotic acid (Hoo), or X0 is absent,

preferably

X0 is acetyl, isovaleric acid, orotic acid (OA), dihydroorotic acid (Hoo), or X0 is absent,

more preferably

X0 is acetyl, isovaleric acid, dihydroorotic acid (Hoo), or X0 is absent.

XI is Asp, iminodiacetic acid (Ida), Gly, Ahx-Asp or is absent,

preferably

XI is Asp, iminodiacetic acid (Ida), Ac-Gly, Me-Gly or is absent,

preferably

XI is Asp, Gly, iminodiacetic acid (Ida) or is absent,

preferably

XI is Asp or iminodiacetic acid (Ida).

14. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X2 is Phe, substituted Phe, diphenylalanine (Dip).

X3 is Pro, β-proline, a proline mimetic, Gly-Gly, sarcosine (Sar), 2,2-dimethylglycine (Aib), or a proline spacer which is selected from the group consisting of g- aminobutyric acid, morpholine-3-carboxylic acid, nipecotic acid (Nip), octahydroindole (Oic), piperidine-2-carboxylic acid (Pip).

16. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X6 is Phe, substituted Phe, diphenylalanine (Dip), Tyr.

17. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X2 is Phe,

X4 is Arg,

X5 is Arg, and

X6 is Phe.

18. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X2 is Phe,

X4 is Arg,

XI 1 is Cys or Met,

X5 is Arg,

X6 is Phe, and X7 is PEG3.

19. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X2 is Phe,

X3 is Pro,

X4 is Arg,

X5 is Arg, and

X6 is Phe.

20. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, wherein

X2 is Phe,

X3 is Pro,

X4 is Arg,

XI 1 is Cys or Met

X5 is Arg,

X6 is Phe, and.

X7 is PEG3.

21. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 3, SEQ ID 4, SEQ ID 7, SEQ ID 8, SEQ ID 10, SEQ ID 12, SEQ ID 13, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 22, SEQ ID 38 and SEQ ID 45.

22. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 12 and SEQ ID 13.

23. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 3, SEQ ID 4, SEQ ID 7, SEQ ID 8, and SEQ ID 10.

24. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 22, SEQ ID 38 and SEQ ID 45.

25. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ 8, SEQ ID 22, SEQ ID 38 and SEQ ID 45.

26. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 22.

27. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, which are selected from the group comprising peptides having the sequence ID SEQ ID 45.

28. The peptide according to any one of the preceding claims or a derivative or analogue or pharmaceutically acceptable salt or solvate thereof, being a homodimer according to the structural formula (IV)

The peptide or the derivative or analogue or pharmaceutically acceptable salt or solvate thereof according to any one of the preceding claims, which acts as a hepcidin mimetic and/or which binds ferroportin or induces ferroportin internalization and/or degradation, for the use in the prophylaxis and treatment of hepcidin- associated disorders.

The peptide or derivative, analogue or a pharmaceutically acceptable salt or solvate according to claim 28, wherein the hepcidin-associated disorders comprise disorders related with reduced hepcidin levels or reduced responsiveness to hepcidin; disorders related with increased serum iron levels, such as in particular iron overload, hemochromatosis; iron-loading anemias such as thalassemia; diseases being associated with ineffective erythropoiesis such as myelofibrosis, myelodysplastic syndrome, and sickle cell disease; diseases with augmented erythropoiesis such as polycythemia vera; reduction of iron levels in patients with chronic kidney diseases; reduction of iron levels in bacterial infections and polymicrobial sepsis.

A pharmaceutical composition comprising at least one peptide, derivative or analogue as defined in any one of the preceding claims or a pharmaceutically acceptable salt or solvate thereof, which may optionally comprise at least one pharmaceutically acceptable excipient and/or optionally at least one further active ingredient being active in the prophylaxis or treatment of the disorders or diseases as defined in claim 28 or 29.