WO2018107241A1 - Treatment of iron disorders - Google Patents

Abstract

Treatment of a disease of iron metabolism including the step of administering a peptide effective for inhibiting the binding of hepcidin to α2- macroglobulin, to an individual requiring said treatment.

Description

Treatment of iron disorders

Field of the invention

The invention relates to diseases of iron metabolism including anemia, especially to anemia of chronic disease, to hepcidin, and to the synthesis and design of peptides contemplated for therapeutic use as competitive inhibitors.

Background of the invention

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Iron is an essential transition metal for life [1 ,2]. In most organisms, a highly coordinated system controls the regulation and cellular and systemic metabolism of iron [1 .2]. Hepcidin, the 'hormone of iron metabolism' appears to be a major regulator of systemic iron homeostasis [3].

The dysregulation of hepcidin is etiologically involved in many diseases of iron metabolism. Pathologically low hepcidin induces iron overload (e.g., in hereditary hemochromatosis types 1 -3), while pathologically high hepcidin typically induces iron- limited anemia [1 ], with the anemia of chronic disease being of particular concern [4].

Hepcidin is believed to exert its effects, principally through binding to the iron release transporter, ferroportin [3]. Ferroportin acts to release cellular iron into the circulation and results in the absorption of iron from the gut. By binding to ferroportin, hepcidin down-regulates ferroportin levels by causing its cellular internalisation and degradation [7]. This decrease in ferroportin prevents (1 ) iron being released from cellular stores {e.g., the liver) into the blood; and (2) iron uptake from the gut into the blood [7]. Hence, pathologically high hepcidin levels, as occurs in the anemia of chronic disease, leads to a debilitating anemia (as ferroportin expression is reduced) [3]. Considering its role in cellular iron release, ferroportin has been suggested as a target for the development of inhibitors for treatment of diseases of iron metabolism, whereby it has been hypothesised that inhibition of ferroportin I - hepcidin binding might enable treatment of these diseases [5]. In one study, the hepcidin pharmacophore at the ferroportin binding site was established [5]. However, the hepcidin analogs utilized in that study to define the pharmacophore were observed not to inhibit hepcidin binding to ferroportin in a ferroportin-GFP assay when hepcidin and each analog were co-incubated [5]. The results strongly suggested that these peptide analogs would not be useful for treatment of diseases of iron metabolism. This is because they do not have sufficient affinity to inhibit hepcidin- mediated ferroportin internalisation and degradation [5].

Hepcidin has also been shown to bind to other molecules including oc-2 macroglobulin [6], and the latter has been proposed as a target for screening for inhibitors for treatment of diseases of iron metabolism by increasing urinary excretion of hepcidin arising from competitive inhibition of hepcidin binding with -2 macroglobulin [8].

WO201 0/065815, WO2013/086143, WO2014/071015 and WO2016/1 09363 (all by The Regents of University of California) and WO2014/145561 and WO201 5/200916 (by Protagonist Therapeutics Inc) each propose peptides having hepcidin activity, especially activity for binding to ferroportin to cause degradation or internalisation of same, for treatment of iron related disorders.

There remains a need to treat disease of iron metabolism, particularly those diseases arising from or associated with excessive hepcidin production or activity, and for pharmaceutical compositions for same. There remains a need to treat anemia, in particular the anemia of chronic disease and for pharmaceutical compositions for same.

Summary of the invention

The invention seeks to address one or more of the above mentioned needs or limitations and in one embodiment provides peptides and methods of use of same for antagonising the binding of hepcidin to oc2 macroglobulin, or for increasing excretion of hepcidin from the body, or for increasing ferroportin -mediated export of Fe ions from a cell, or for improving red blood cell production, or for treating a disease of iron metabolism, especially a disease associated with excessive hepcidin production, expression or activity, and in particular, for treating anemia of chronic disease.

The peptides utilised in the methods of the invention for treatment of diseases associated with excessive hepcidin production or activity inhibit the binding of hepcidin to cc2 macroglobulin, and otherwise do not have hepcidin activity in the presence of natural or endogenous hepcidin. That is, they do not bind to ferroportin to cause internalisation or degradation of ferroportin that would otherwise prevent ferroportin - mediated export of Fe ions from inside a cell in the presence of natural or endogenous hepcidin because they do not inhibit hepcidin from binding to ferroportin the presence of natural or endogenous hepcidin. Thus, in one embodiment, there is provided a method of treating a disease associated with excessive hepcidin production or activity including providing an individual in need of said treatment with a peptide that: (i) inhibits the binding of hepcidin to a2 macroglobulin; and (ii) that does not have hepcidin activity, for example, does not prevent ferroportin from export of Fe ions from a cell, in the presence of natural or endogenous hepcidin.

In another embodiment, there is provided a method of treating anemia of chronic disease including providing an individual in need of said treatment with a peptide that: (i) inhibits the binding of hepcidin to oc2 macroglobulin; and (ii) that does not inhibit the binding of hepcidin to ferroportin. In these embodiments, the peptide has an amino acid sequence with at least 90% or greater amino acid homology to the sequence shown in SEQ ID No:1 , provided that at position 4, the residue is not Phe, and at position 6, the residue is not lie:

SEQ ID No: 1 AspThrHisPheProlleCysllePhe

In another embodiment, there is provided a method of treating a disease associated with excessive hepcidin production or activity, preferably for treating anemia of chronic disease, including providing in an individual in need of said treatment a peptide that inhibits the binding of hepcidin to oc2 macroglobulin, wherein the peptide does not have hepcidin activity, or does not inhibit the binding of hepcidin to ferroportin, and wherein the peptide includes or consists of an amino acid sequence according to Formula 1 A:

(SEQ ID No: 2) XiX₂X₃X₄X5X6CysllePhe or Formula 1 B:

(SEQ ID No:3) PhelleCysX₆X₅X4X3X2Xi wherein

Xi s Asp, Ala, Gly, Ser or Thr

x₂ s Thr, Ala or Gly

X3 is His, Asp or Glu

X₄ is Phe, Leu, Nle, Lys, lie or Val

X₅ is Pro, Ala or Gly

ΧΘ is lie, Lys, Arg, or His

In the above described embodiments where the peptide includes an amino acid sequence according to Formula 1 A, the peptide may consist of about 25 amino acids. For example, the peptide may have the sequence:

(SEQ ID No: 4)

XiX₂X₃X₄X5X6CysllePheCysCysGlyCysCysBBXXCysGlyXCysCysBThr wherein:

Xi to X₆ are as described above;

B is a positively charged residue, for example His, Asn, Arg or Lys

X is any amino acid residue.

Preferably the peptide may have the sequence:

(SEQ ID No:5)

X₁X₂X₃X4X₅X₆CysllePheCysCysGlyCysCysHisArgSerLysCysGlyMetCysCysLysTrir wherein X-, to X₆ are as described above

The peptide may be linear or cyclic. In some embodiments the peptides of the invention bind to oc2 macroglobulin with a binding affinity sufficient to inhibit the binding of hepcidin to oc2 macroglobulin..

Generally, the peptides of the invention are designed not to provide hepcidin hormonal activity in the presence of natural or endogenous hepcidin. As such, they do not generally prevent ferroportin from exporting Fe ions from a cell, for example by causing the degradation or internalisation of ferroportin that is otherwise seen when hepcidin binds to ferroportin. In this context, the peptides of the invention may be referred to as "null hepcidin analogues".

In one embodiment, a null hepcidin analog may mimic a hepcidin function or activity (such as oc2 macroglobulin-binding) although as mentioned above, a null hepcidin analog does not prevent ferroportin from export Fe ions from a cell in the presence of natural or endogenous hepcidin.

In one embodiment, there is provided a method of treating anemia of chronic disease comprising providing a peptide which does not bind to ferroportin in the presence of natural or endogenous hepcidin, wherein the peptide is selected from the group consisting of D1 A (SEQ ID No: 6), T2A (SEQ ID No: 7), H3D (SEQ ID No: 8), F4Nle (SEQ ID No: 9), F4K (SEQ ID No: 10), P5A (SEQ ID No: 1 1 ), I6K (SEQ ID No: 12), D1 A/H3D (SEQ ID No: 13), D1 A/F4K (SEQ ID No: 14), I6D (SEQ ID No: 15), C7G (SEQ ID No: 1 6) or cyclic Hep9 or CHH1 as described herein. Generally, a natural or endogenous hepcidin has the amino acid sequence shown in SEQ ID No: 17.

In some embodiments, the present invention provides compositions and medicaments which comprise at least one peptide, which may be isolated, synthesized and/or purified, comprising, consisting essentially, or consisting of Formula 1 A or 1 B as set forth herein.

In some embodiments, the present invention provides method of manufacturing medicaments for the treatment of diseases of iron metabolism, particularly disease that arise from or are associated with excessive hepcidin production, expression or activity, which comprise at least one peptide, which may be isolated and/or purified, comprising, consisting essentially or consisting of Formula 1 A or 1 B as set forth herein. Also, provided are methods of treating a disease of iron metabolism in a subject, such as a mammalian subject, preferably a human subject, which comprises administering at least one peptide, which may be isolated and/or purified, comprising, consisting essentially or consisting of Formula 1 A or 1 B, as set forth herein, or a composition comprising said at least one peptide to the subject. In some embodiments, the peptide is administered in a therapeutically effective amount. In some embodiments, the therapeutically effective amount is an effective daily dose administered as a single daily dose or as divided daily doses. The peptides of the present invention can also be administered at a variety of doses. In some embodiments the dose is given as a weekly dose, e.g. from 1 -10,000 g/kg/dose. In some embodiments, the daily dose is about 1 -1 ,000, preferably about 10-500 g/kg/day. Dosages can vary according to the type of formulation of peptidyl drug administered as well as the route of administration. One skilled in the art can adjust the dosage by changing the route of administration or formulation, so that the dosage administered would result in a similar pharmacokinetic or biological profile as would result from the preferred dosage ranges described herein. In some embodiments, the composition to be administered is formulated for oral, pulmonary or mucosal administration.

Some embodiments include any dosage with any route of administration which results in an effective pharmacokinetic and pharmacodynamic profile that decreases serum hepcidin levels and increases serum iron levels. Some preferred doses include those that result in a desired reduction in serum hepcidin. Administration of the peptidyl or protein formulations of the present invention includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is considered to be administering the drug to the patient.

In some embodiments, the present invention provides methods of binding an a2 macroglobulin, which comprises contacting the a2 macroglobulin with at least one peptide or composition as disclosed herein. In some embodiments, the present invention provides kits comprising at least one peptide or composition as disclosed herein packaged together with a reagent, a device, instructional material, or a combination thereof.

In some embodiments, the present invention provides complexes which comprise at least one peptide as disclosed herein bound to an cx2 microglobulin, preferably a human a2 macroglobulin, or an antibody, such as an antibody which specifically binds a peptide as disclosed herein, or a combination thereof.

In some embodiments, the present invention provides the use of at least one peptide, which may be isolated and/or purified, comprising, consisting essentially or consisting of Formula 1 A or 1 B as set forth herein or a composition comprising, consisting essentially of, or consisting of said at least one peptide for the manufacture of a medicament for treating a disease of iron metabolism and/or increasing the amount of extracellular iron in a subject in need thereof, wherein the medicament is prepared to be administered at an effective daily dose, as a single daily dose, or as divided daily doses. In some embodiments, the dose is about 1 -1 ,000, preferably about 1 0-500 pg/kg/day. In some embodiments, the medicament is formulated for subcutaneous injection or oral, pulmonary or mucosal administration.

Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

Brief description of the drawings

Figure 1 . Sequestration and transport of hepcidin in the circulation by native and activated a2-macroglobulin (a2M). (1 ) Hepcidin binds to native and activated a2M. (2) Hepcidin-binding to high Mr (725 kDa) a2M or its activated form largely prevents urinary excretion of low Mr hepcidin (2.8 kDa). (3) Hepcidin binding to native and activated a2M is labile and in the presence of its target, ferroportin (Fpn1 ), hepcidin can dissociate from α2Μ. Release of hepcidin and binding to Fpn1 prevents cellular iron release leading to increased iron storage.

Figure 2. Null hepcidin analogues compete for the hepcidin bound to a-2 macroglobulin and induce its urinary excretion. This is an innovative therapy for the anaemia of chronic disease, where there is inappropriate and excessive hepcidin production induced by a chronic disease (e.g., cancer, etc.). This excessive hepcidin prevents iron release from tissue stores, e.g., the liver and also decreases iron uptake from enterocytes in the gut. Iron is required for red blood cell production and a lack of iron release from tissues leads to a debilitating anaemia which exacerbates the underlying disease.

Figure 3. Null hepcidin analogue competition assay in vitro. The peptides D1 A and I6K effectively compete with hepcidin for binding to a2-macroglobulin (a2M). Hence, this indicates that these peptides can displace hepcidin from a2M more effectively, leading to the urinary excretion of low molecular weight hepcidin. This is beneficial in the anaemia of chronic disease where there is excessive quantities of hepcidin produced. Densitometry is mean + SD (3 expts).

Figure 4. I6K given i.v increases serum Fe & attenuates hepcidin activity. In vivo study in mice showing that I6K at 100-fold excess to hepcidin significantly (p<0.001 ) increases serum Fe and totally prevents hepcidin's ability to decrease serum Fe levels. The increase in serum Fe above the control is probably due to I6K competing with hepcidin on a2M (Figs. 2 & 3). Result is mean + SEM (n=5~6).

Figure 5. I6K given i.v. dose-dependently increases serum Fe in vivo in mice. I6K dose-dependently (at 1 -, 10- and 100-fold excess of hepcidin) increases serum Fe and was well tolerated. Thus, these new data in vivo show the great potential of our analogues as they increase serum Fe to fight the anaemia of chronic disease which affects millions. Result is mean + SEM (n=5~6).

Figure 6 Hepcidin and its analogues, structure and activity correlations. Detailed description of the embodiments

A particularly important finding of the invention is that certain peptides that had previously been considered not useful for treatment of diseases of iron metabolism, because they do not competitively inhibit binding of hepcidin with ferroportin and therefore, do not have hepcidin activity for degradation of ferroportin in the presence of natural or endogenous hepcidin, have been found to inhibit the binding of oc-2 macroglobulin to hepcidin.

This inhibition is important because the binding of oc 2 macroglobulin to hepcidin enables hepcidin to function as a hormone and increases the half-life of hepcidin in the body. That is, in the absence of hepcidin binding to high molecular weight oc-2 macroglobulin protein (725 kDa), the low molecular weight hepcidin hormone (2kDa) is excreted by the kidney into the urine.

In light of this finding the inventors have realised that these peptides could be used to ostensibly elute hepcidin from oc2 macroglobulin, thereby enabling excretion of hepcidin via the kidney. The result is to reduce the amount of hepcidin in individuals having pathologically excessive hepcidin production, expression or activity.

The present invention provides peptides which are useful in the study and treatment of diseases of iron metabolism, and in particular, diseases associated with, or arising from, excessive hepcidin activity or production. Such a disease may be one 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.

In one embodiment the disease is an anemia including anemia of chronic disease, anemia of inflammation, anemia of infection, hypochromic microcytic anemia, iron- deficiency anemia, iron-refractory iron deficiency anemia, anemia of chronic kidney disease, iron deficiency of obesity, congenital dyserythropoietic anemia, or other anemias.

In another embodiment, the disease may be a condition, for example a benign or malignant tumor that overproduces hepcidin, or induces its over-production, conditions with hepcidin excess, Friedreich ataxia, gracile syndrome, Hallervorden-Spatz disease, Wilson's disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer, hepatitis, cirrhosis of liver, pica, chronic renal failure, insulin resistance, diabetes, atherosclerosis, neurodegenerative disorders, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

In a particularly preferred embodiment of the present invention, the disease of iron metabolism is anemia of chronic disease. Anemia of chronic disease is a condition well known in the art, affecting more than 80% of hospitalised patients suffering from infections, malignancy and inflammation (NEJM 2005;352;1 01 1 ). This disorder is associated with excessive levels of hepcidin, as a response to chronic diseases. Excessive hepcidin promotes liver iron storage, leading to inadequate iron release for red blood cell production, causing a severe anemia.

The peptides of the present invention may be made using methods known in the art including chemical synthesis (solid-phase, solution phase, or a combination of both), biosynthesis, or in vitro synthesis using recombinant DNA methods,. See e.g. Kelly & Winkler (1990) Genetic Engineering Principles and Methods, vol. 12, J. K. Setlow ed., Plenum Press, NY, pp. 1 -1 9; Merrifield (1964) J Amer Chem Soc 85:2149; Houghten (1985) PNAS USA 82:51 31 -5135; and Stewart & Young (1 984) Solid Phase Peptide Synthesis, 2ed. Pierce, Rockford, IL, which are herein incorporated by reference. The peptides of the present invention may be purified using protein purification techniques known in the art such as reverse phase high- performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, precipitation, filtration, size exclusion, or electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem. 12(16):31 21 - 31 26; and Scopes (1 982) Protein Purification, Springer- Verlag, NY, which are herein incorporated by reference.

Alternatively, the peptides of the present invention may be made by recombinant DNA techniques known in the art. Thus, polynucleotides that encode the polypeptides of the present invention are contemplated herein. In preferred embodiments, the polynucleotides are isolated. As used herein "isolated polynucleotides" refers to polynucleotides that are in an environment different from that in which the polynucleotide naturally occurs. In some embodiments, the peptides of the present invention are substantially purified. As used herein, a "substantially purified" compound refers to a compound that is at least about 60% free, preferably about 75% free, and most preferably about 90% free from other macromolecular components with which the compound may otherwise be associated, or a compound that is at least about 60%> free, preferably about 75% free, and most preferably about 90% free from other peptide components as measured by HPLC with detection at 214 nm.

The peptides of the present invention bind a2 macroglobulin, preferably human oc2 macroglobulin. Preferred peptides of the present invention specifically bind human oc2 macroglobulin. 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. According to the invention there is provided a method for treating a disease of iron metabolism, especially anemia of chronic disease including the step of administering a peptide effective for inhibiting the binding of hepcidin to a2- macroglobulin, to an individual requiring said treatment, wherein the peptide has an amino acid sequence according to Formula 1 :

XiX₂X₃X₄X5X6CysllePhe

wherein

Xi is Asp, Ala, Gly, Ser or Thr

X₂ is Thr, Ala or Gly

X₃ is His, Asp or Glu

X₄ is Phe, Leu, Nle, Lys, lie or Val

X₅ is Pro, Ala or Gly X& is lie, Lys, Arg, or His wherein the peptide does not have hepcidin activity in the presence of

endogenous or natural hepcidin.

Typically the peptide has a binding affinity similar to that of hepdicin for a-2 macroglobulin in a competitive binding assay described herein.

In one embodiment, Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Phe, X₅ is Pro, and X₆ is lie. Preferably, Xi is Ala.

In another embodiment, Xi is Asp, X2 is Ala or Gly, X3 is His, X₄ is Phe, X5 is Pro, and e is lie. Preferably X2 is Ala. In a further embodiment, Xi is Asp, X2 is Thr, X3 is Asp or Glu, X₄ is Phe, X5 is

Pro, and X& is lie. Preferably X3 is Asp.

In yet a further embodiment, Xi is Asp, X2 is Thr, X3 is His, X₄ is Leu, lie or Val, X₅ is Pro, and X₆ is lie. Preferably X₄ is Nle or Leu.

In still a further embodiment, Xi is Asp, X2 is Thr, X₃ is His, X₄ is Phe, X5 is Ala or Gly, and X₆ is lie. Preferably X₅ is Ala.

In still a further embodiment, Xi is Asp, X2 is Thr, X₃ is His, X₄ is Phe, X5 is Pro, and X₆ is Lys, Arg or His. Preferably X₆ is Lys.

In still a further embodiment, wherein X^ is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is Asp or Glu, X₄ is Phe, X5 is Pro, and Xe is lie. Preferably Xi is Ala and X3 is Asp. In still a further embodiment, Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X is Lys, Arg or His, X5 is Pro, and X& is lie. Preferalby Xi is Ala and X₄ is Lys.

The peptide may be linear or cyclic, preferably linear.

In one embodiment, the peptide may contain an alkyl group at the C-terminal residue. A peptide as described above may be provided for use in the method of the invention in the form of a pharmaceutical composition including the peptide and an excipient, diluent or carrier.

In further embodiments there are provided peptides and uses of same for treatment of iron disorders, particularly disorders such as anemia of chronic disease. These peptides do not have hepcidin activity in the presence of natural or endogenous hepcidin. These peptides inhibit the binding of hepcidin to 2- macroglobulin. In one embodiment, there is provided a peptide for inhibiting the binding of hepcidin to a2- macroglobulin, wherein the peptide has an amino acid sequence according to Formula 1 :

XiX₂X₃X₄X5X6CysllePhe

wherein

Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is Asp or Glu, X₄ is Phe, X₅ is Pro, and X₆ is lie. Preferably Xi is Ala and X₃ is Asp.

In another embodiment, the peptide has an amino acid sequence according to

Formula 1 C:

XiX₂X₃X₄X5X6CysllePheCysCysGlyCysCysBBXXCysGlyXCysCysBThr wherein

Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is Asp or Glu, X is Phe, X₅ is Pro, and is lie. Preferably Xi is Ala and X₃ is Asp.

B is a positively charged residue, for example His, Asn, Arg or Lys

X is any amino acid residue.

Preferably the peptide of Formula 1 C may have the sequence:

X₁X₂X3X4X₅X₆CysllePheCysCysGlyCysCysHisArgSerLysCysGlyMetCysCysLysThr where Xi is Ala and X₃ is Asp

In another embodiment there is provided a peptide for inhibiting the binding of hepcidin to oc2- macroglobulin, wherein the peptide has an amino acid sequence according to Formula 1 :

XiX₂X₃X₄X₅X₆CysllePhe

wherein Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Lys, Arg or His, X5 is Pro, and X₆ is lie. Preferably Xi is Ala and X₄ is Lys.

In another embodiment, the peptide has an amino acid sequence according to Formula 1 C: XiX₂X₃X₄X5X6CysllePheCysCysGlyCysCysBBXXCysGlyXCysCysBThr wherein

Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Lys, Arg or His, X5 is Pro, and X₆ is lie. Preferably Xi is Ala and X₄ is Lys.

B is a positively charged residue, for example His, Asn, Arg or Lys

X is any amino acid residue.

Preferably the peptide of Formula 1 C may have the sequence:

X₁X₂X3X4X₅X₆CysllePrieCysCysGlyCysCysHisArgSerLysCysGlyMetCysCysLysThr where Xi is Ala and X₄ is Lys.

In another embodiment there is provided a peptide having an amino acid sequence according to Formula 1 C:

XiX₂X₃X₄X₅X₆CysllePheCysCysGlyCysCysBBXXCysGlyXCysCysBThr wherein:

Xi is Asp, Ala, Gly, Ser or Thr

X₂ is Thr, Ala or Gly

X₃ is His, Asp or Glu

X is Phe, Leu, Nle, Lys, lie or Val

X₅ is Pro, Ala or Gly

X₆ is lie, Lys, Arg, or His

B is a positively charged residue, for example His, Asn, Arg or Lys

X is any amino acid residue. wherein the peptide does not have hepcidin activity in the presence of natural or endogenous hepcidin.

Typically the peptide has a binding affinity similar to that of hepdicin for a-2 microglobulin in a competitive binding assay described herein. Preferably the peptide of Formula 1 C may have the sequence:

XiX₂X₃X₄X₅X₆CysllePheCysCysGlyCysCysHisArgSerLysCysGlyMetCysCysLysThr

In one embodiment of Formula 1 C, Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Phe, X₅ is Pro, and X₆ is lie. Preferably, Xi is Ala. In another embodiment of Formula 1 C, Xi is Asp, X2 is Ala or Gly, X3 is His, X₄ is

Phe, X₅ is Pro, and X₆ is lie. Preferably X₂ is Ala.

In a further embodiment of Formula 1 C, Xi is Asp, X₂ is Thr, X₃ is Asp or Glu, X₄ is Phe, X5 is Pro, and X& is lie. Preferably X3 is Asp.

In yet a further embodiment of Formula 1 C, Xi is Asp, X2 is Thr, X3 is His, X₄ is Leu, lie or Val, X5 is Pro, and XQ is lie. Preferably X₄ is Nle or Leu.

In still a further embodiment of Formula 1 C, Xi is Asp, X2 is Thr, X3 is His, X₄ is Phe, X₅ is Ala or Gly, and X₆ is lie. Preferably X₅ is Ala.

In still a further embodiment of Formula 1 C, Xi is Asp, X₂ is Thr, X₃ is His, X₄ is Phe, X₅ is Pro, and X₆ is Lys, Arg or His. Preferably X₆ is Lys. In still a further embodiment of Formula 1 C, wherein Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is Asp or Glu, X is Phe, X₅ is Pro, and X₆ is lie. Preferably Xi is Ala and X₃ is Asp.

In still a further embodiment of Formula 1 C, Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Lys, Arg or His, X5 is Pro, and X& is lie. Preferalby Xi is Ala and X₄ is Lys.

In certain embodiments, the present invention includes pharmaceutical compositions comprising one or more null hepcidin analogues of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutically acceptable carrier, diluent or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in "Remington's Pharmaceutical Sciences", 1 7th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985. For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used.

Suitable pH-buffering agents may, e.g., be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine or acetate (e.g. as sodium acetate), or mixtures thereof. The term further encompasses any carrier agents listed in the US Pharmacopeia for use in animals, including humans. It is to be understood that the inclusion of a null hepcidin analogue of the invention (i.e., one or more null hepcidin analogue peptide monomers of the invention or one or more null hepcidin analogue peptide dimers of the present invention) in a pharmaceutical composition also encompasses inclusion of a pharmaceutically acceptable salt or solvate of a null hepcidin analogue of the invention. In particular embodiments, the pharmaceutical compositions further comprise one or more pharmaceutically acceptable carrier, excipient, or vehicle.

In certain embodiments, the invention provides a pharmaceutical composition comprising a null hepcidin analogue, or a pharmaceutically acceptable salt or solvate thereof, for treating a variety of conditions, diseases, or disorders as disclosed herein or elsewhere (see, e.g., Methods of Treatment, herein).

In particular embodiments, the invention provides a pharmaceutical composition comprising a null hepcidin analogue peptide monomer, or a pharmaceutically acceptable salt or solvate thereof, for treating a variety of conditions, diseases, or disorders as disclosed herein elsewhere (see, e.g., Methods of Treatment, herein). In particular embodiments, the invention provides a pharmaceutical composition comprising a null hepcidin analogue peptide dimer, or a pharmaceutically acceptable salt or solvate thereof, for treating a variety of conditions, diseases, or disorders as disclosed herein elsewhere (see, e.g., Methods of Treatment, herein). The null hepcidin analogues of the present invention may be formulated as pharmaceutical compositions which are suited for administration with or without storage, and which typically comprise a therapeutically effective amount of at least one null hepcidin analogue of the invention, together with a pharmaceutically acceptable carrier, excipient or vehicle. In some embodiments, the null hepcidin analogue pharmaceutical compositions of the invention are in unit dosage form. In such forms, the composition is divided into unit doses containing appropriate quantities of the active component or components. The unit dosage form may be presented as a packaged preparation, the package containing discrete quantities of the preparation, for example, packaged tablets, capsules or powders in vials or ampoules. The unit dosage form may also be, e.g., a capsule, cachet or tablet in itself, or it may be an appropriate number of any of these packaged forms. A unit dosage form may also be provided in single-dose injectable form, for example in the form of a pen device containing a liquid-phase (typically aqueous) composition. Compositions may be formulated for any suitable route and means of administration, e.g., any one of the routes and means of administration disclosed herein.

In particular embodiments, the null hepcidin analogue, or the pharmaceutical composition comprising a null hepcidin analogue, is suspended in a sustained-release matrix. A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. One embodiment of a biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid). In certain embodiments, the compositions are administered enterally or parenterally. In particular embodiments, the compositions are administered orally, intracisternally, intravaginally, intraperitoneally, intrarectally, topically (as by powders, ointments, drops, suppository, or transdermal patch, including delivery intravitreally, intranasally, and via inhalation) or buccally. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal and intraarticular injection and infusion. Accordingly, in certain embodiments, the compositions are formulated for delivery by any of these routes of administration.

In certain embodiments, pharmaceutical compositions for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, beta-cyclodextrin, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

Injectable depot forms include those made by forming microencapsule matrices of the null hepcidin analogue in one or more biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), poly(anhydrides), and (poly)glycols, such as PEG. Depending upon the ratio of peptide to polymer and the nature of the particular polymer employed, the rate of release of the null hepcidin analogue can be controlled. Depot injectable formulations are also prepared by entrapping the null hepcidin analogue in liposomes or microemulsions compatible with body tissues.

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

Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye. Compositions for topical lung administration, including those for inhalation and intranasal, may involve solutions and suspensions in aqueous and nonaqueous formulations and can be prepared as a dry powder which may be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredient may be finely divided form may be used in admixture with a larger-sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 micrometers in diameter. Suitable inert carriers include sugars such as lactose.

Alternatively, the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied propellant medium and indeed the total composition may be such that the active ingredient does not dissolve therein to any substantial extent. The pressurized composition may also contain a surface active agent, such as a liquid or solid non-ionic surface active agent or may be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.

A further form of topical administration is to the eye. A null hepcidin analogue of the invention may be delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the null hepcidin analogue is maintained in contact with the ocular surface for a sufficient time period to allow the null hepcidin analogue to penetrate the corneal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera. The pharmaceutically acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an encapsulating material. Alternatively, the null hepcidin analogues of the invention may be injected directly into the vitreous and aqueous humour.

Compositions for rectal or vaginal administration include suppositories which may be prepared by mixing the null hepcidin analogues of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at room temperature but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active compound.

Null hepcidin analogues of the present invention may also be administered in liposomes or other lipid-based carriers. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a null hepcidin analogue of the present invention, stabilizers, preservatives, excipients, and the like. In certain embodiments, the lipids comprise phospholipids, including the phosphatidyl cholines (lecithins) and serines, both natural and synthetic. Methods to form liposomes are known in the art.

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

In some aspects, the invention provides a pharmaceutical composition for oral delivery. Compositions and null hepcidin analogues of the instant invention may be prepared for oral administration according to any of the methods, techniques, and/or delivery vehicles described herein. Further, one having skill in the art will appreciate that the null hepcidin analogues of the instant invention may be modified or integrated into a system or delivery vehicle that is not disclosed herein, yet is well known in the art and compatible for use in oral delivery of peptides.

In certain embodiments, formulations for oral administration may comprise adjuvants (e.g. resorcinols and/or nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to artificially increase the permeability of the intestinal walls, and/or enzymatic inhibitors (e.g. pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) or trasylol) to inhibit enzymatic degradation. In certain embodiments, the null hepcidin analogue of a solid-type dosage form for oral administration can be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffmose, maltitol, dextran, starches, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, or glyceride. These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrators, binders, thickeners, buffering agents, pH adjusting agents, sweetening agents, flavoring agents or perfuming agents.

In particular embodiments, oral dosage forms or unit doses compatible for use with the null hepcidin analogues of the present invention may include a mixture of null hepcidin analogue and nondrug components or excipients, as well as other non- reusable materials that may be considered either as an ingredient or packaging. Oral compositions may include at least one of a liquid, a solid, and a semi-solid dosage forms. In some embodiments, an oral dosage form is provided comprising an effective amount of null hepcidin analogue, wherein the dosage form comprises at least one of a pill, a tablet, a capsule, a gel, a paste, a drink, a syrup, ointment, and suppository. In some instances, an oral dosage form is provided that isdesigned and configured to achieve delayed release of the null hepcidin analogue in the subject's small intestine and/or colon. In one embodiment, an oral pharmaceutical composition comprising a null hepcidin analogue of the present invention comprises an enteric coating that is designed to delay release of the null hepcidin analogue in the small intestine. In at least some embodiments, a pharmaceutical composition is provided which comprises a null hepcidin analogue of the present invention and a protease inhibitor, such as aprotinin, in a delayed release pharmaceutical formulation. In some instances, pharmaceutical compositions of the instant invention comprise an enteric coat that is soluble in gastric juice at a pH of about 5.0 or higher. In at least one embodiment, a pharmaceutical composition is provided comprising an enteric coating comprising a polymer having dissociable carboxylic groups, such as derivatives of cellulose, including hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate and cellulose acetate trimellitate and similar derivatives of cellulose and other carbohydrate polymers. In one embodiment, a pharmaceutical composition comprising a null hepcidin analogue of the present invention is provided in an enteric coating, the enteric coating being designed to protect and release the pharmaceutical composition in a controlled manner within the subject's lower gastrointestinal system, and to avoid systemic side effects. In addition to enteric coatings, the null hepcidin analogues of the instant invention may be encapsulated, coated, engaged or otherwise associated within any compatible oral drug delivery system or component. For example, in some embodiments a null hepcidin analogue of the present invention is provided in a lipid carrier system comprising at least one of polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems. To overcome peptide degradation in the small intestine, some embodiments of the present invention comprise a hydrogel polymer carrier system in which a null hepcidin analogue of the present invention is contained, whereby the hydrogel polymer protects the null hepcidin analogue from proteolysis in the small intestine and/or colon. The null hepcidin analogues of the present invention may further be formulated for compatible use with a carrier system that is designed to increase the dissolution kinetics and enhance intestinal absorption of the peptide. These methods include the use of liposomes, micelles and nanoparticles to increase Gl tract permeation of peptides.

Various bioresponsive systems may also be combined with one or more null hepcidin analogue of the present invention to provide a pharmaceutical agent for oral delivery. In some embodiments, a null hepcidin analogue of the instant invention is used in combination with a bioresponsive system, such as hydrogels and mucoadhesive polymers with hydrogen bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, Eudragit®, chitosan and alginate) to provide a therapeutic agent for oral administration. Other embodiments include a method for optimizing or prolonging drug residence time for a null hepcidin analogue disclosed herein, wherein the surface of the null hepcidin analogue surface is modified to comprise mucoadhesive properties through hydrogen bonds, polymers with linked mucins or/and hydrophobic interactions. These modified peptide molecules may demonstrate increase drug residence time within the subject, in accordance with a desired feature of the invention. Moreover, targeted mucoadhesive systems may specifically bind to receptors at the enterocytes and M-cell surfaces, thereby further increasing the uptake of particles containing the null hepcidin analogue.

Other embodiments comprise a method for oral delivery of a null hepcidin analogue of the present invention, wherein the null hepcidin analogue is provided to a subject in combination with permeation enhancers that promote the transport of the peptides across the intestinal mucosa by increasing paracellular or transcellular permeation. For example, in one embodiment, a permeation enhancer is combined with a null hepcidin analogue, wherein the permeation enhancer comprises at least one of a long-chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent. In one embodiment, a permeation enhancer comprising sodium N-[hydroxybenzoyl)amino] caprylate is used to form a weak noncovalent association with the null hepcidin analogue of the instant invention, wherein the permeation enhancer favors membrane transport and further dissociation once reaching the blood circulation.

In another embodiment, a null hepcidin analogue of the present invention is conjugated to oligoarginine, thereby increasing cellular penetration of the peptide into various cell types. Further, in at least one embodiment, a noncovalent bond is provided between a peptide inhibitor of the present invention and a permeation enhancer selected from the group consisting of a cyclodextrin (CD) and a dendrimers, wherein the permeation enhancer reduces peptide aggregation and increasing stability and solubility for the null hepcidin analogue molecule. Other embodiments of the invention provide a method for treating a subject with a null hepcidin analogue of the present invention having an increased half-life. In one aspect, the present invention provides a null hepcidin analogue having a half-life of at least several hours to one day in vitro or in vivo (e.g., when administered to a human subject) sufficient for daily (q.d.) or twice daily (b.i.d.) dosing of a therapeutically effective amount. In another embodiment, the null hepcidin analogue has a half-life of three days or longer sufficient for weekly (q.w.) dosing of a therapeutically effective amount. Further, in another embodiment, the null hepcidin analogue has a half-life of eight days or longer sufficient for bi-weekly (b.i.w.) or monthly dosing of a therapeutically effective amount. In another embodiment, the null hepcidin analogue is derivatized or modified such that is has a longer half-life as compared to the underivatized or unmodified null hepcidin analogue.

In another embodiment, the null hepcidin analogue contains one or more chemical modifications to increase serum half-life. When used in at least one of the treatments or delivery systems described herein, a null hepcidin analogue of the present invention may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form. The total daily usage of the null hepcidin analogues and compositions 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 null hepcidin analogue employed; e) the duration of the treatment; f) drugs used in combination or coincidental with the specific null hepcidin analogue employed, and like factors well known in the medical arts. In particular embodiments, the total daily dose of the null hepcidin analogues of the invention to be administered to a human or other mammal host 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. In certain embodiments, a dosage of a null hepcidin analogue of the present invention is in the range from about 0.0001 to about 1 00 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.

In various embodiments, a null hepcidin analogue 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. Such regular null hepcidin analogue administration regimens of the invention may, in certain circumstances such as, e.g., during chronic long-term administration, be advantageously interrupted for a period of time so that the medicated subject reduces the level of or stops taking the medication, often referred to as taking a "drug holiday." Drug holidays are useful for, e.g., maintaining or regaining sensitivity to a drug especially during long-term chronic treatment, or to reduce unwanted side-effects of long-term chronic treatment of the subject with the drug. The timing of a drug holiday depends on the timing of the regular dosing regimen and the purpose for taking the drug holiday (e.g., to regain drug sensitivity and/or to reduce unwanted side effects of continuous, long- term administration). In some embodiments, the drug holiday may be a reduction in the dosage of the drug (e.g. to below the therapeutically effective amount for a certain interval of time). In other embodiments, administration of the drug is stopped for a certain interval of time before administration is started again using the same or a different dosing regimen (e.g. at a lower or higher dose and/or frequency of administration). A drug holiday of the invention may thus be selected from a wide range of time-periods and dosage regimens. An exemplary drug holiday is two or more days, one or more weeks, or one or more months, up to about 24 months of drug holiday. So, for example, a regular daily dosing regimen with a peptide, a peptide analogue, or a dimer of the invention may, for example, be interrupted by a drug holiday of a week, or two weeks, or four weeks, after which time the preceding, regular dosage regimen (e.g. a daily or a weekly dosing regimen) is resumed. A variety of other drug holiday regimens are envisioned to be useful for administering the null hepcidin analogues of the invention.

Thus, the null hepcidin analogues may be delivered via an administration regime which comprises two or more administration phases separated by respective drug holiday phases. During each administration phase, the null hepcidin analogue is administered to the recipient subject in a therapeutically effective amount according to a pre-determined administration pattern. The administration pattern may comprise continuous administration of the drug to the recipient subject over the duration of the administration phase. Alternatively, the administration pattern may comprise administration of a plurality of doses of the null hepcidin analogue to the recipient subject, wherein said doses are spaced by dosing intervals.

A dosing pattern may comprise at least two doses per administration phase, at least five doses per administration phase, at least 1 0 doses per administration phase, at least 20 doses per administration phase, at least 30 doses per administration phase, or more.

Said dosing intervals may be regular dosing intervals, which may be as set out above, including once daily, twice daily, once every two, three, four, five or six days, once or twice weekly, once or twice monthly, or a regular and even less frequent dosing interval, depending on the particular dosage formulation, bioavailability, and pharmacokinetic profile of the null hepcidin analogue of the present invention.

An administration phase may have a duration of at least two days, at least a week, at least 2 weeks, at least 4 weeks, at least a month, at least 2 months, at least 3 months, at least 6 months, or more. Where an administration pattern comprises a plurality of doses, the duration of the following drug holiday phase is longer than the dosing interval used in that administration pattern. Where the dosing interval is irregular, the duration of the drug holiday phase may be greater than the mean interval between doses over the course of the administration phase. Alternatively the duration of the drug holiday may be longer than the longest interval between consecutive doses during the administration phase.

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

Within these constraints, a drug holiday phase may have a duration of at least two days, at least a week, at least 2 weeks, at least 4 weeks, at least a month, at least 2 months, at least 3 months, at least 6 months, or more, depending on the administration pattern during the previous administration phase.

An administration regime comprises at least 2 administration phases. Consecutive administration phases are separated by respective drug holiday phases. Thus the administration regime may comprise at least 3, at least 4, at least 5, at least 10, at least 1 5, at least 20, at least 25, or at least 30 administration phases, or more, each separated by respective drug holiday phases.

Consecutive administration phases may utilise the same administration pattern, although this may not always be desirable or necessary. However, if other drugs or active agents are administered in combination with a null hepcidin analogue of the invention, then typically the same combination of drugs or active agents is given in consecutive administration phases. In certain embodiments, the recipient subject is human.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Examples Example 1 - Screening assays for determining appropriate a2- macroqlobulin binders.

For competition assay experiment, biotinylated hepcidin (BioHepcidin) was incubated at a 4: 1 molar ratio of BioHepcidin:a2M/a2M-MA in the presence of unlabeled hepcidin or its analogues. In these studies, 27.6 pmol of a2M or a2M-MA were incubated with 1 1 0.4 pmol BioHepcidin in the presence of hepcidin or its analogue at 25, 50, 1 00 or 200 molar excess of BioHepcidin, for 1 h at 37 °C. Any unbound BioHepcidin was removed from the a2M*BioHepcidin/a2M-MA^»BioHepcidin complexes using Sephadex G-25 columns. Samples are then applied dropwise to a nitrocellulose membrane before biotin-streptavidin-HRP detection via chemiluminescence (Chemiluminescence Detection Module, Thermo Scientific). Example 2 - Screening assays for determining peptides that do not have other hepcidin activities

Assay to determine the activity of hepcidin analogues were performed in two stages. First, cells were pre-treated with culture media alone (DMEM + 10% fetal calf serum; control), or this medium containing DFO or FAC for 24 h/37°C (primary incubation). This medium was then removed and the cells rinsed with serum-free media. In the second stage, cells were incubated with serum-free DMEM or this medium containing DFO (100 μΜ) or FAC (250 pg/mL) in the presence or absence of either: hepcidin and hepcidin analogues (0.7 μΜ), for 6 h/37°C (secondary incubation). At the end of the secondary incubation, cells were lysed for assessment of Fpn1 expression by Western analysis. The activity of hepcidin analogues are measured in terms of Fpn1 expression relative to cell treated with hepcidin.

Example 3 - Screening assays for determining peptides that do not degrade/cause internalisation of Ferroportin. To measure serum iron levels in vivo, 100 μΙ_ 0.9% saline (Control) or this vehicle containing: Hepcidin (0.9 nmole), or hepcidin analogues at 0.9 nmole (1 x), 9 nmole (10x) or 90 nmole (100x) in the presence or absence of Hepcidin (0.9 nmole) were injected into the tail vein of mice. After 2 h, blood (600 μΙ_) was collected by cardiac puncture from anesthetized mice and placed in Capiject® Micro Collection tubes and serum isolated. Serum iron was measured using a Konelab Clinical Chemistry Analyzer (Thermo Scientific, Waltham, MA).

References

1 . Lawen A., Lane DJR. Antioxid. Redox Signal. 18, 2473-2507 (2013).

2. Richardson DR, Lane DJR, Becker EM et al. Proc. Natl. Acad. Sci USA 107(24), 1 0775-10782 (201 0).

3. Gant T. Blood 1 17(17), 4425-4433 (201 1 ).

4. Gant T. Pediatr. Blood Cancer 46(5), 554-557 (2006).

5. Clark RJ et al. Chemistry & Biology 18 336-343 (201 1 ).

6. Peslova G. et al. Blood 1 13 (24) 6225-6236 (2009).

7. Nemeth E. et al. Science 306, 2090-2093 (2004).

8. Lane DJR, Richardson DR Future Med Chem 6(1 ) 1 -4 (2014.

Claims

1 . A method for treating a disease of iron metabolism including the step of

administering a peptide effective for inhibiting the binding of hepcidin to a2- rmacroglobulin, to an individual requiring said treatment, wherein the peptide has an amino acid sequence according to Formula 1 :

XiX₂X₃X₄X5 6CysllePhe

wherein

Xi is Asp, Ala, Gly, Ser or Thr

X₂ is Thr, Ala or Gly

X₃ is His, Asp or Glu

x₄ is Phe, Leu, Lys, Nle, lie or Val

X₅ is Pro, Ala or Gly

X₆ is lie, Lys, Arg, or His

2. The method of claim 1 wherein Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Phe, X5 is Pro, and Χε is lie.

3. The method of claim 2 wherein Xi is Ala.

4. The method of claim 1 wherein Xi is Asp, X₂ is Ala or Gly, X3 is His, X₄ is Phe, X5 is Pro, and X₆ is lie.

5. The method of claim 4 wherein X₂ is Ala. 6. The method of claim 1 wherein Xi is Asp, X2 is Thr, X3 is Asp or Glu, X₄ is Phe, X5 is Pro, and X& is lie.

7. The method of claim 6 wherein X3 is Asp.

8. The method of claim 1 wherein Xi is Asp, X₂ is Thr, X₃ is His, X₄ is Leu, lie or Val, X₅ is Pro, and X₆ is lie. 9. The method of claim 8 wherein X₄ is Nle or Leu.

10. The method of claim 1 wherein Xi is Asp, X2 is Thr, X3 is His, X₄ is Phe, X5 is Ala or Gly, and X₆ is lie.

1 1 . The method of claim 10 wherein X5 is Ala.

12. The method of claim 1 wherein Xi is Asp, X₂ is Thr, X3 is His, X₄ is Phe, X5 is Pro, and Xe is Lys, Arg or His.

13. The method of claim 12 wherein Xe is Lys. 14. The method of claim 1 wherein Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is Asp or Glu, X₄ is Phe, X5 is Pro, and XQ is lie.

15. The method of claim 14 wherein Xi is Ala and X3 is Asp.

16. The method of claim 1 wherein Xi is Ala, Gly, Ser or Thr, and X2 is Thr, X3 is His, X₄ is Lys, Arg or His, X₅ is Pro, and X₆ is lie. 17. The method of claim 16 wherein X-i is Ala and X₄ is Lys.

18. The method of any one of the preceding claims wherein the peptide is linear or cyclic.

19. The method of any one of the preceding claims wherein the peptide is contains an alkyl group at the C-terminal residue. 20. The method of any one of the preceding claims wherein the peptide is provided in the form of a pharmaceutical composition including the peptide and an excipient, diluent or carrier.

21 . The method of any one of the preceding claims wherein the disease is anemia of inflammation. 22. The method of any one of the preceding claims wherein the peptide is provided in an amount of about 10-500mg/kg individual.

23. The method of any one of the preceding claims wherein the peptide is provided daily.

24. The method of any one of the preceding claims wherein the peptide is

administered IV. The method of any one of the preceding claims wherein the peptide is provided once to the individual in the form of a sustained release formulation.

A peptide for inhibiting the binding of hepcidin to cc2- macroglobulin, wherein the peptide has an amino acid sequence according to Formula 1 :

XiX₂X₃X4X5X6CysllePhe

wherein

Xi is Ala, Gly, Ser or Thr, and X2 is Thr, X₃ is Asp or Glu, X₄ is Phe, X5 is Pro, and X6 is lie.

27. The peptide of claim 26 wherein Xi is Ala and X3 is Asp. 28. A peptide for inhibiting the binding of hepcidin to oc2- macroglobulin, wherein the peptide has an amino acid sequence according to Formula 1 :

XiX₂X₃X4X5X6CysllePhe

wherein

Xi is Ala, Gly, Ser or Thr, and X₂ is Thr, X₃ is His, X₄ is Lys, Arg or His, X₅ is Pro, and X₆ is lie.

29. The peptide of claim 28 wherein Xi is Ala and X₄ is Lys.

30. A peptide for inhibiting the binding of hepcidin to oc2- macroglobulin, wherein the peptide has an amino acid sequence:

AspThrHisPheProlleCysllePhe

wherein the peptide is a cyclic peptide, or wherein the peptide contains an alkyl group at the C-terminal residue.

31 . The peptide according to any one of claims 26 to 29 wherein the peptide contains about 25 amino acids.

32. The peptide according to any one of claims 26 or 29 wherein the peptide has the sequence

X₁X₂X₃X₄X₅X₆CysllePheCysCysGlyCysCysHisArgSerLysCysGlyMetCysCysLysThr

33. A peptide having an amino acid sequence according to Formula 1 C:

XiX2X₃X₄X₅X6CysllePheCysCysGlyCysCysBBXXCysGlyXCysCysBThr wherein:

Xi is Asp, Ala, Gly, Ser or Thr

X₂ is Thr, Ala or Gly

X₃ is His, Asp or Glu

X₄ is Phe, Leu, Nle, Lys, lie or Val

X₅ is Pro, Ala or Gly

X₆ is lie, Lys, Arg, or His

B is a positively charged residue, for example His, Asn, Arg or Lys

X is any amino acid residue. 34. The peptide of claim 33 wherein X-i is Ala, Gly, Ser or Thr, and X2 is Thr, X₃ is His, X₄ is Phe, X₅ is Pro, and X₆ is lie, preferably, Xi is Ala.

35. The peptide of claim 33 wherein Xi is Asp, X2 is Ala or Gly, X3 is His, X₄ is Phe, X₅ is Pro, and X₆ is lie, preferably X₂ is Ala.

36. The peptide of claim 33 wherein Xi is Asp, X₂ is Thr, X₃ is Asp or Glu, X₄ is Phe, X₅ is Pro, and X₆ is lie, preferably X₃ is Asp.

37. The peptide of claim 33 wherein X is Asp, X₂ is Thr, X₃ is His, X is Leu, lie or Val or Nle, X5 is Pro, and Χε is lie, preferably X₄ is Leu or Nle.

38. The peptide of claim 33 wherein Xi is Asp, X2 is Thr, X₃ is His, X₄ is Phe, X5 is Ala or Gly, and X₆ is lie, preferably X₅ is Ala. 39. The peptide of claim 33 wherein Xi is Asp, X2 is Thr, X₃ is His, X₄ is Phe, X5 is Pro, and X₆ is Lys, Arg or His, preferably X₆ is Lys.

40. A pharmaceutical composition including a peptide of any one of claims 26 to 39.