WO2018234538A1 - Hepcidin antagonist or agonist for use in the treatment of dysregulation of mo and/or mn metabolism - Google Patents

Abstract

Hepcidin antagonist or agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism There is a continued need to develop novel drugs and therapies for treating diseases associated to Mo and/or Mn systemic or cellular metabolism alterations or during which a better control of Mo or Mn systemic or cellular metabolism could improve the prognosis and/or quality of life. The inventors of the present invention have shown that, surprisingly, iron (Fe), manganese (Mn) and molybdenum (Mo) metabolisms share regulatory mechanisms involving the hepcidin/ferroportin axis. The present invention relates to a hepcidin antagonist or agonist for use in the treatment of dysregulation of molybdenum and/or manganese systemic or cellular metabolism, and related diseases.

Description

Hepcidin antagonist or agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism

Field of the invention

The present invention relates to a hepcidin antagonist or agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism.

Background of the invention

Molybdenum (Mo) is an essential trace element for eukaryotes, activities of some enzymes being Mo dependent, including sulfite oxidase, mitochondrial amidoxime reducing component, xanthine oxidoreductase and aldehyde oxidase (Schwarz, Mendel et al. 2009) 24. The association of Mo with these enzymes is carried out via the MoCo (Molybdenum Cofactor), synthetized by cells that incorporate Mo. Mo deficiencies are mostly rare genetic diseases linked to mutations in genes involved in the synthesis of MoCo and, leading to fatal neurological disease (Schwarz,

Mendel et al. 2009) 24. Excess of Mo are mainly related to occupational activities or contaminated nutriments. It has been reported in duck that spleen Mo accumulation decreases antioxidant capacity, favors cell apoptosis and increases Heat Shock proteins and TNFa in spleen (Cao,

Zhang et al. 2016) 8° (Zhang, Luo et al. 2016) 29. Similar data were found in chickens fed with high

Mo diet (Yang, Cui et al. 2011) 28. In humans, excess of Mo has been associated to purine metabolism modulation, joint symptoms and/or to the development of gout suggesting potential implication of Mo in this disease (Avakian, Nargizian et al. 1978 , Walravens, Moure-Eraso et al. 1979²⁷, Vyskocil and Viau 1999²⁶), xanthine oxidase being the Mo dependent enzyme involved in the uric acid production. In addition, it has been reported that Mo may interact with the surface of collagen fibers and could initiate the formation of microcrystals (Harris, Reiber et al. 2005)¹¹. Moreover, it is noteworthy that during HFE-hereditary hemochromatosis hyperferritinemia level, reflecting iron stores, was recently found associated to the risk of hyperuricemia (Flais, Bardou-Jacquet et al. 2017)⁹. Finally, it has been suggested that Molybdenum accumulation was correlated with C-parathyroid hormone (C-PTH) levels and may contribute to dialysis related arthritis (Hosokawa and Yoshida 1994) 12.

Manganese (Mn) is an essential component of metalloenzymes including Mn superoxide dismutase that is involved in the control of oxidative stress. If Mn deficiency is not well characterized in humans, excess of Mn may induce motor coordination, memory deficit and psychiatric disorders ( Pfalzer, A. C. and A. B. Bowman 2017) 18 , as found in patients exposed to environmental contamination and those exhibiting liver failure during chronic liver diseases (Rivera-Mancia, Rios et al. 2011) 22. Hfe-/- mice exposed to Mn demonstrate an alteration of spatial memory (Alsulimani, Ye et al. 2015)¹ and of emotional behavior in behavior tests.

Thus, there is a continued need to develop novel drugs and therapies for treating diseases associated to Mo and/or Mn systemic or cellular metabolism alterations or during which a better control of Mo or Mn systemic or cellular metabolism could improve the prognosis and/or quality of life.

The inventors of the present invention have discovered that, surprisingly, iron (Fe), manganese (Mn) and molybdenum (Mo) metabolisms share regulatory mechanisms involving the hepcidin/ferroportin axis. Summary of the invention

Hepatic hepcidin is known as being the key hormone in iron homeostasis; it is able to decrease plasma iron (Fe) levels by blocking iron absorption in the duodenum and iron release from macrophages thus targeting the two entrance gates for iron in the circulation. Several stimuli have been shown to be involved in hepcidin regulation including iron, hypoxia, erythropoietic demand and inflammation.

The present invention relies on the discovery that, surprisingly, iron (Fe), molybdenum (Mo) and manganese (Mn) share regulatory mechanisms involving the hepcidin/ferroportin axis which mean that treatments controlling the hepcidin/ferroportin axis activity represent a way to counteract Mo or Mn systemic or cellular metabolism alterations. Thus, the present invention relates to a hepcidin antagonist or agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism, and related diseases. For instance, the hepcidin agonist can be used in the prevention and/or treatment of osteoporosis.

Another object of the invention is a method for treating dysregulation of cellular Mo and/or Mn metabolism comprising administering to a subject in need thereof a therapeutically effective amount of a hepcidin antagonist or agonist as disclosed above.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat the dysregulation.

Detailed description

The systemic iron metabolism is tightly controlled through the hepcidin/ferroportin axis. Hepcidin (encoded by the Hamp gene), a small peptide secreted in plasma by hepatocytes, interacts with the iron exporter ferroportin, a protein localized on the cell membranes of enterocytes and macrophages that are the main providers of iron for plasma (Brissot and Loreal 2016)⁶. Hepcidin limits the expression and activity of the ferroportin protein, the iron exporter, and thus reduces the iron egress from these cells toward plasma (Nemeth, Turtle et al. 2004)¹⁵. Therefore, the modulation of the expression and secretion of hepcidin controls the iron distribution. The main signals regulating hepcidin are those related iron status, inflammation and anemia/erythropoiesis (Ganz 2011¹⁰, Brissot and Loreal 2016⁶).

During secondary iron overload, the iron excess induces an increase of hepatic hepcidin expression which favors iron sequestration in macrophages (Pigeon, Ilyin et al. 2001)¹⁹ especially in spleen in order to limit toxic effect of iron. Conversely, iron overload consecutive to genetic hemochromatosis (GH) related to p.Cys282Tyr mutation in HFE gene is characterized by a loss of this adaptive mechanism, with an abnormally low level of hepcidin despite an iron repletion state (Nicolas, Bennoun et al. 2001¹⁶, Gehrke, Kulaksiz et al. 2003³⁰, Bardou-Jacquet,

Philip et al. 2014 3^J 1¹). Similar findings are involved in the genesis of rare hemochromatosis linked to mutations in hemojuvelin (HJV) (Papanikolaou, Samuels et al. 2004) 17 or transferrin receptor 2 (TFR2) (Camaschella, Roetto et al. 2000)⁷ or hepcidin (HAMP) genes (Roetto, Papanikolaou et al. 2003)²³.

A lot of potential bridges between iron metabolism and non-iron metals being suggested, the inventors investigated the impact of iron overload conditions on metals for which metabolic alterations could modulate the expression of a large panel of diseases. Iron, manganese, copper, zinc and molybdenum concentrations were determined using ICP-MS in liver and spleen of Hfe- /-, Hjv7- and Bmp67- mice vs their wild-type controls, and carbonyl iron overloaded C57BL/6 and iron dextran overloaded mice vs their controls. Hepatic Hepcidin mRNA levels were determined by RT-qPCR. The inventors surprisingly found that Spleen Mo and Mn concentrations parallel the modulation of spleen Fe concentrations that is dependent of hepcidin/ferroportin axis, meaning an increase in concentration during secondary iron overloads and a decrease in concentration during genetic iron overloads related to hepcidin deficiency. It is therefore possible to modulate Mo and Mn cellular or plasmatic concentrations by controlling the hepcidin/ferroportin axis activity. Thus, the present invention relates to hepcidin antagonist or agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism. According to the invention, a "hepcidin antagonist" refers to a compound which is an inhibitor of the hepcidin expression or hepcidin activity.

According to the invention, a "hepcidin agonist" refers to a compound which replaces hepcidin activity or stimulate its endogenous production. As used herein, the terms "treating" or treatment" relates to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

According to the invention, a "metabolism" refers to systemic or cellular metabolism. In a preferred embodiment the metabolism is cellular metabolism.

According to the present invention, the hepcidin antagonist is used in the treatment of Mo and/or Mn cellular excess or systemic deficiencies. Blocking the hepcidin expression or activity allows to decrease the Mo and/or Mn concentration in every tissue and cells expressing ferroportin, including macrophages, enterocytes, and hepatocytes as well as in tumor cells expressing ferroportin and to increase the Mo and/or Mn concentration in plasma.

According to the present invention, the hepcidin agonist is used in the treatment of Mo and/or Mn cellular deficiencies or systemic excess. Stimulating the hepcidin expression or activity allows increase the Mo and/or Mn concentration in cells and to decrease the Mo and/or Mn concentration in plasma. Hepcidin Antagonists

Inhibitors of the hepcidin expression

One object of the present invention relates to a hepcidin antagonist which is an inhibitor of the hepcidin expression.

In one embodiment, the compound according to the invention can be an erythropoiesis- stimulating agent like erythropoietin (see Ashby et al., Haematologica, 2010, 95(3), 505-8) or an erythroferone inducer.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of hepcidin gene expression for use in the present invention. Hepcidin gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that hepcidin gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of hepcidin gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of hepcidin mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of hepcidin gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing hepcidin. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication- deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991.

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double- stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In a preferred embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.

Inhibitors of the hepcidin activity Another aspect of the present invention relates to a hepcidin antagonist which is an inhibitor of the hepcidin activity.

By "hepcidin activity", it is herein meant the ability of the Hepcidin to limit the expression and activity of the ferroportin protein, the iron exporter, and thus reducing the Fe/Mn/Mo egress from these cells toward plasma By "inhibitor of the hepcidin activity", it is herein referred to a compound which is capable of reducing or suppressing the activity of hepcidin. In view of the teaching of the present disclosure, particularly of the examples, it falls within the ability of the skilled person to assess whether a compound is an inhibitor of the hepcidin activity.

In a particular embodiment, the present invention relates to a compound which is an inhibitor of the hepcidin activity for use in the treatment of diseases associated to Mo and/or Mn metabolism wherein said compound is an anti-hepcidin antibody which neutralizes hepcidin (see for example Cooke et al., Blood, 2013, 122(17); Sasu et al., Blood, 2010, 115(17), 3616-24; US8629250; WO2009058797; WO2010017070; WO2009139822; WO2014152006) or an anti-hepcidin antibody fragment which neutralizes hepcidin.

Antibodies directed against hepcidin can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against hepcidin can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-hepcidin single chain antibodies. Hepcidin activity inhibitors useful in practicing the present invention also include anti-hepcidin antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to hepcidin.

Humanized anti-hepcidin antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of hepcidin are selected.

In still another embodiment, hepcidin activity inhibitors may be selected from aptamers. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then, for this invention, neutralizing aptamers of hepcidin are selected. In a particular embodiment, the compound according to the invention is an anti-hepcidin spiegelmer® like lexaptepid pegol (NOX-H94) (see Schwoebel et al., Blood, 2013, 121(12), 2311-2315; WO2012055573; WO2010124874) which is produced by NOXXON.

In a further embodiment, the compound according to the invention is an anticalin® that binds to hepcidin like PRS-080 (see Hohlbaum et al., Am J Hematol., 2013, 5(88), E41; WO2012022742) which is produced by PIERIS AG. General methods for producing anticalins are for example described in W09916873.

Hepcidin agonist Hepcidin and its variants

In one embodiment, the compound according to the invention can be the hepcidin peptide or one of its active variants.

Hepcidin mimics

One object of the present invention relates to a hepcidin agonist which replaces hepcidin activity.

In one embodiment, the compound according to the invention can be mini-hepcidins. Mini- hepcidins are peptide-based hepcidin agonists which are designed based on the region of hepcidin that interacts with ferroportin. Mutagenesis studies and biomolecular modeling indicated that the first 9 amino acids of the hepcidin N-terminus were important for hepcidin activity. Synthetic N-terminal peptides were further engineered to increase their bioavailability. These small modified mini-hepcidin peptides show bioactivity in vivo as determined by their ability to induce hypoferremia in mice and prevent iron accumulation in hepcidin-deficient mice. Example of suitable mini-hepcidin may be PR65, PR73 or mHS17.

Stimulators of hepcidin production

Another aspect of the present invention relates to a hepcidin agonist which stimulates its endogenous production.

In one embodiment, the compound according to the invention can be TMPRSS6 inhibitors (see Fung, E., & Nemeth, E. (2013). Manipulation of the hepcidin pathway for therapeutic purposes. Haematologica, 98(11), 1667-1676).

TMPRSS6 is a negative regulator for hepcidin expression in both mouse and human models. Thus, targeting of Tmprss6 is a possible approach for the treatment of dysregulation of Mo and/or Mn metabolism.

Two studies harnessed RNA-based technologies to decrease the levels of Tmprss6 and demonstrated their effectiveness in increasing hepcidin transcription and reverting iron overload in mouse models. Guo et al. utilized anti-sense oligonucleotides (ASOs) against Tmprss6 mRNA. ASOs are single-stranded nucleic acids containing modified bases that protect ASOs from degradation by exonucleases. These ASOs trigger the degradation of their target mRNA based on the RNaseH mechanism. Schmidt et al. used small interfering RNA (siRNA) against Tmprss6.61 Unlike ASOs, siRNAs are double- stranded nucleic acids designed to inhibit the expression of its target genes through the RNA interference pathway. Tmprss6 siRNA was packaged in lipid nanoparticles that promotes their delivery to the liver, the main site of TMPRSS6 expression and activity. Both studies evaluated the effect of Tmprss6 knockdown in HFE-/- mice and thalassemic th3/+ mice after six weeks. ASOs were injected twice a week (100 mg/kg/wk) and siRNA every two weeks (1 mg/kg). The effects of the two approaches on endogenous hepcidin expression, iron and hematologic parameters were remarkably similar. With both approaches, hepcidin mRNA increased 2-3 fold in HFE-/- and th3/+ mice compared to control injections.60, 61 In HFE-/- mice, serum iron and liver iron concentrations were reduced compared to the vehicle control group, and spleen iron was increased. A mild reduction in hemoglobin was also observed indicating some iron restriction. In the mouse model of thalassemia intermedia (th3/+), 6-week treatment decreased liver iron but also improved anemia and ineffective erythropoiesis, with reduced spleen size and improved maturation of erythroid precursors, reproducing the effect of transgenic hepcidin overexpression in th3+/- mice.

In another embodiment, the compound according to the invention can be BMP (Bone Morphogenetic Protein) agonists, for instance BMP6 or the isoflavone genistein (see Fung, E., & Nemeth, E. (2013). Manipulation of the hepcidin pathway for therapeutic purposes. Haematologica, 98(11), 1667-1676).

There are nearly 20 bone morphogenetic proteins (BMPs) expressed in mammals, and among these, BMP2, BMP4, BMP5, BMP6, BMP7, and BMP9 can induce Hamp (the gene encoding Hepcidin) expression. A small-scale chemical screen in zebrafish embryos identified the isoflavone genistein as an enhancer of hepcidin transcription. In a cellular system in vitro, genistein promoted hepcidin expression via both Stat3 and BMP-dependent pathways, but not via estrogen receptors, known targets of genistein.

Ferroportin antagonist Since hepcidin inhibits ferroportin thus preventing iron and other metals such as Mo and Mn from being exported from the cells, a ferroportin antagonist would therefore have the same effect as a hepcidin agonist. As such, hepcidin agonist in the meaning of the present invention may also include ferroportin antagonist.

Ferroportin antagonist may be antagonists well known in the art such as antibodies, aptamers, small inhibitory RNAs (siRNAs), ribozymes, antisense oligonucleotides... (see above the "hepcidin antagonist" part). Administration

It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The hepcidin antagonist or agonist according to the invention can be administered by any suitable route of administration. For example, the antagonist or agonist according to the invention can be administered by oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous, intra- articular and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

The antagonists or agonist of the present invention, together with one or more conventional adjuvants, carriers, or diluents may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredients commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral uses. Formulations containing about one (1) milligram of active ingredient or, more broadly, about 0.01 to about one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms. Applications according to the invention

The present invention relates to hepcidin antagonist or agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism. In particular: One aspect of the invention is the use of hepcidin antagonist in the treatment of systemic Mn deficiencies or cellular Mn excess. These alterations may promote or favor the development of lesions.

Mn systemic deficiencies can be detected by measuring the level of Mn, especially in whole blood, plasma, erythrocytes, urine and hair (See for instance: Goule et al. Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair: Reference values - Forensic Science International 153 (2005) 39-44). Quantification in tissues, including, liver, bones, etc. are also possible.

Example of diseases or disorders associated with Mn systemic deficiencies may include SLC39A8 deficiency (See for instance: J Inherit Metab Dis. 2017 Mar;40(2):261-269.Epub 2016 Dec 19).

Another aspect of the invention is the use of hepcidin antagonist in the treatment of systemic Mo deficiencies or cellular Mo excess.

Mo systemic deficiencies can be detected by measuring the level of Mo in whole blood, plasma, erythrocytes, urine and hair (See Goule et al. cited above) as well as in tissues. Another aspect of the invention is the use of hepcidin agonist in the treatment of systemic Mn excess or cellular Mn deficiency.

Systemic iron Mn excess can be detected by measuring the level of Mn in whole blood, plasma, erythrocytes, urine and hair and tissues (See Goule et al. cited above). Localised Mn excess can be detected by MRI, especially in brain. Example of diseases or disorders associated with systemic Mn excess may be motor coordination, memory deficit and psychiatric disorders such as manganism and diseases associated to manganism, hepatic encephalopathy, neuroinflammation, childhood developmental disorders, neurodegenerative diseases and mental disorders (schizophrenia or psychosis-related phenotype) (See for instance: Curr Opin Pediatr. 2016 Apr;28(2):243-9). Another aspect of the invention is the use of hepcidin agonist in the treatment of systemic Mo excess or cellular Mo deficiency.

Mo excess can be detected by measuring the level of Mo in whole blood, plasma, urine and hair as well as in tissues (See Goule et al. cited above). Example of diseases or disorders potentially associated with Mo excess may be joint symptoms and diseases; gout; the formation of intra- articular microcrystals and dialysis related arthritis. Mo could also modulate immunity and therefore play a role in autoimmune diseases.

Another aspect of the invention is the use of hepcidin agonist in the prevention and/or treatment of osteoporosis. Without being bound by theory, the inventors surprisingly discovered that hepcidin can directly or indirectly, through an alteration of Mo and or Mn metabolisms, play a role in the development of osteoporosis (see Example 2).

Brief Description of the figures

Figure 1. Correlation between spleen iron and molybdenum concentrations, between spleen iron and manganese concentrations, and their graphic representation: in carbonyl iron overloaded mice (A and B) and dextran iron overloaded mice (C and D). Correlations were studied using the Spearman test with correlation coefficient (Rho) and p-value (p). Carbonyl iron overloaded mice include mice with iron added to normal diet at different concentrations (0,5% - 1,5% - 3%) and control mice (C). Dextran iron overloaded mice include mice with one subcutaneously single injection of iron dextran at different concentrations (0,25 - 0,5 - 1 g/kg) and control mice (C).

Figure 2. Bmp6-/- mice, Hjv-/- mice and control mice (WT) : iron (Fe), copper (Cu), manganese (Mn), zinc (Zn) and molybdenum (Mo) concentrations in liver (upper panel) and spleen (lower panel). Median is represented by a horizontal line within the boxes, 25th and 75th percentiles are respectively represented by the lower and upper lines of boxes, 10th and 90th percentiles are represented by horizontal lines located on either side boxes. Statistically significant differences are presented as *p<0,05 ; **p<0,01 and ***p<0,001. Figure 3. Correlation between spleen iron and molybdenum concentrations (A and C), between spleen iron and manganese (B and D) concentrations, and their graphic representation : in Hfe +/+ and Hfe -/- mice (A and B) and in Bmp6 KO, Hjv KO and control mice (WT) (C and D). Statistically significant correlations are represented by correlation coefficient (Rho) and p-value (p). Non statistically significant correlations are represented by ns.

Figure 4. Determination of iron, manganese and molybdenum concentrations in bones.

A- Quantification in 12 months old Hfe -/- mice (H12M) and their controls (C12M). B - Quantification in iron dextran model (6 months old mice that received one injection of iron dextran 4 months earlier). Controls received either physiological serum or dextran alone. Figure 5. Evaluation of bone structure.

A - Iron dextran injected mice and the two control groups. Bone trabeculae evaluated by micro CT and BV/TV, were similar between iron dextran injected mice and the two control groups; B - Hfe -/- mice 6 months (H6M) or 12 months old (H12M) and their respective controls (C6M & C12M). Hfe -/- 6 mice presented osteoporosis with altered bone trabeculae and reduced BV/TV.

Example 1

MATERIAL AND METHODS

Animals

Animals were maintained in UMS Biosit animal facilities in Rennes for Hfe related and secondary iron overload models and in Toulouse for Hjv and BMP6 knock out models. Experimental protocols were approved respectively by the local animal ethic committees of Rennes (b-2007-OL-02 and 03) and Midi-Pyrenees Animal Ethics Committee (for. Animals were given free access to tap water and standard laboratory mouse chow diet. Hfe-/- (n=8) and Hfe+/+ (n=6) C57BL/6 male mice of 12 months, receiving normal diet and tap water, were studied. Animals were maintained in, in compliance with french law and regulations. Mice were anaesthetized and blood, obtained from a trans-diaphragmatic intracardiac puncture, was sampled in sodium heparin tubes suitable for trace elements analysis. Mice were sacrificed, and livers and spleens were dissected and weighed. Liver and spleen samples were quickly frozen in liquid nitrogen, and then stored at -80°C to perform trace elements quantification and niRNA extraction. Liver and spleen samples were also fixed in 4% buffered-formaldehyde for histological studies.

Experimentally iron overloaded C57BL/6 male mice (Janvier Lab, Le Genest-Saint-Isle, France) were also included for this study by using liver and spleen samples obtained from previous studies and stored at -80°C in the Rennes Experimental Iron Biobank (REIB). Thus, C57BL/6 male mice were iron loaded using carbonyl iron or iron dextran as previously reported. Briefly : i) carbonyl iron was added to normal diet at different concentrations (0,5% (n=6) - 1,5% (n=6) - 3%(n=6) of 5 weeks mice during 4 months and controls (n=5) received normal diet, or ii) six weeks male received one subcutaneously single injection of iron dextran (0,25 g/kg n=5; 0.5g/kg (n=6) ; lg/kg; n=6 ) whereas controls (n=6) received one subcutaneously single injection of dextran only and animals were studied 2 months later.

Hjv7- mice on a 129S6/SvEvTac background (Huang, Pinkus et al. 2005) 13 were bred to

Bmp6tmlRob mice (Bmp67-) on an outbred CD1 background (Solloway, Dudley et al. 1998) 25. Experiments were done on 12 wild-type (WT), Bmp67-, Hjv-/-, littermates of the F2 progeny.

Trace elements quantification

All samples were handled with care in order to avoid environmental contamination during their manipulation.

Liver and spleen samples, stored at -80°C, were desiccated at 120°C for 15 hours in an oven. Thereafter, dried samples were weighed and mineralized according to the following protocol: in teflon tubes, nitric acid solution (Fisher Chemical - Optima Grade®) was added to dried samples and then teflon tubes were placed in a MARS6® (CEM) microwave with a temperature maintained at 180°C. Solutions were preserved at 4°C until metals quantification.

Iron (56Fe), manganese (55Mn), copper (63Cu), zinc (66Zn), and molybdenum (95Mo) were quantified by ICP-MS (Inductively Coupled Plasma Mass Spectrometry), on a X-Series II from Thermo Scientific® equipped with collision cell technology (Platform EM2, University of Rennes 1 / Rennes Hospital). The source of plasma was argon (Messer®) with a purity >99.999%. The collision/reaction cell used was pressurized with a mixture of helium (93%) and hydrogen (7%) (Messer®). Ultrapure water was obtained from Millipore Direct-Q® 3 water station. Nitric acid solution was suprapur, at 69% (Fisher Chemical - Optima Grade®). The internal standard used was rhodium (Fisher Scientific®). Calibration ranges preparation was carried out using a multi-element calibrator solution (SCP Science® Plasma Cal). Calibration and verification of instrument performance were realized using multi-element solutions, respectively tune F and tune A (Thermo®). Certified reference materials were obtained from NCS (bovine liver ZC71001). Quantification of hepatic hepcidin 1 mRNA level

Expression level of hepcidin 1 mRNA transcripts was determined in the liver by quantitative RT- quantitative PCR. Total mouse liver RNAs were isolated using SV Total RNA Isolation System (Promega®) for carbonyl iron overloaded mice and the Nucleospin® 8 RNA (Macherey-Nagel) for dextran iron overloaded mice and Hfe-/- and Hfe+/- mice. Then mRNAs were reverse transcribed with the M-MLV reverse transcriptase (Promega®). Specific primers have been used to amplify hepcidin 1 (Hepcl), HPRT for carbonyl iron overloaded mice, and TBP for dextran iron overloaded mice and Hfe-/- and Hfe+/- mice. Quantitative real-time PCR assays were using the qPCR MasterMix Plus for SYBR® Green I (Eurogentec®) and the system StepOne Plus (Real-Time PCR System - Applied Biosystems®). All results were analysed by StepOne Software v2.1 (Applied Biosystem®). For each cDNA sample, the difference between the threshold cycle for Hepcl amplification and the threshold cycle for HPRT or TBP was calculated. This allowed normalization of the amount of target to the endogenous reference, HPRT or TBP.

Statistical analysis

Results were expressed as mean +/- SD. Non parametric Kruskall-Wallis test, followed, when appropriate, by a pair- wise comparison using nonparametric Mann-Whitney test were performed. Correlations were studied using the Spearman test. A p<0.05 was considered significant. RESULTS

1 - Impact of secondary iron overload on liver and spleen metal concentrations.

Carbonyl iron overloaded mice exhibited, as expected (Pigeon, Turlin et al. 1999 20 , Pigeon,

Legrand et al. 2001 21 ), a dose dependent and strong increase of liver and spleen Fe concentrations reaching 10.47 and 2.9 folds respectively in 3% carbonyl iron overloaded animals, compared to controls. Whereas no significant modulation of the concentration of other metals studied was found in liver, a dose-dependent increase of Mo, found in spleen, reaching 4 folds of control values in 3% carbonyl iron overloaded animals, compared to controls as well as a slight increase (35%) of spleen Mn concentration was observed.

In order to avoid a potential impact of iron content in diet on the digestive absorption of other metals, we investigated the effect of a secondary iron overload related to parenteral iron-dextran administration. As expected, in this model (Pigeon, Ilyin et al. 2001¹⁹, Pigeon, Legrand et al. 200120 ), we also found a dose-dependent increase of liver and spleen iron concentration reaching 35.5 and 3.4 folds respectively in in animals injected with lg/kg iron-dextran, compared to controls. In addition, in the spleen, a dose-dependent increase of both Mo and Mn (3.5 and 2.6 fold respectively) was observed similarly to carbonyl iron overload model.

Analyzing relationships between spleen iron concentration and spleen molybdenum or manganese concentrations, we found that the Mo and Mn levels parallels the iron level in carbonyl iron supplemented model and in iron dextran model (Figure 1).

Accordingly to mechanisms regulating the expression of hepatic hepcidin mRNA during secondary iron overload, the hepatic iron excess was responsible of an hepcidin mRNA level increase in both models, 7.1 and 5.2 folds respectively.

2 - Impact of iron overload related to hepcidin deficiency in mouse genetic hemochromatosis models on liver and spleen metal concentrations.

Spleen Mo and Mn levels being increased during iron overload inducing hepcidin expression increase, we analysed the impact of iron overload related to hepcidin deficiencies. Mild (Hfe-/-) and severe Hjv-/- or Bmp6-/- mouse genetic hemochromatosis phenotype models of hepcidin deficiencies and iron overload phenotypes were investigated.

As expected, Hfe-/- mice presented a slight increase (3.9 fold) of liver iron concentration compared to wild- type animals and hepatic hepcidin mRNA level was only slightly (54%) decreased. In addition, there was no significant difference between Hfe-/- mice and wild type mice for hepatic copper, zinc, manganese and molybdenum concentrations. Spleen iron concentration were not different between Hfe-/- and control mice and Mn was the only metal that was slightly decreased (36%).

In mice presenting a strong hepcidin deficiency, meaning Hjv-/- or Bmp6-/- mice with 91% and 99%), we found a strong increase of hepatic iron concentration compared to control mice in Bmp6-/- (20,1 fold) and Hjv-/- (14,3 fold) mice (Figure 2). In addition, spleen iron concentration was significantly decreased in Bmp6-/- (65%) and Hjv-/- (56%) compared to control mice (Figure 2). Moreover, spleen Mo concentration was significantly decreased in the two knock-out mice models (52% and 56% in Bmp6-/- and Hjv-/- mice respectively). We also found a spleen manganese concentration decrease in Bmp6-/- mice (26%) but not in Hjv-/- mice. In addition, in the two knock-out models, spleen copper level was significantly higher whereas liver copper concentration was lower compared to control mice. Finally liver Mn concentration was increased in Bmp6— /- mice. It is noteworthy that in spleen Fe and Mo concentrations were strongly correlated in the three genetic models (Figure 3) whereas Mn and iron concentrations in spleen were correlated in Bmp6-/- and HJV7- mice and not in Hfe -/- mice. 3 - Relationships between hepcidin expression and iron and metals in liver and spleen

Spleen iron concentration being strongly regulated by hepcidin levels, we hypothesized that hepatic hepcidin expression levels could be related to Mo concentration in spleen. Thus, we found that spleen Mo levels were correlated to hepatic mRNA hepcidin levels in all our models but Hfe-/- ; In addition, spleen Mn level was correlated to hepatic hepcidin mRNA in Hjv7- model and iron dextran models.

In summary our data demonstrate that spleen Mo and Mn concentrations parallel the modulation of spleen iron concentrations in iron overloads.

Example 2: The respective role of iron excess, hepcidin level and their interactions with manganese and molybdenum in the development of osteoporosis during iron overload situations were evaluated.

The following was used:

the Hfe^'A mice model that induces an iron overload due to hepcidin-deficiency mimicking hemochromatosis found in Humans (with wild type mice as control). The analyses were performed both at 6 months and 12 months of age.

the iron dextran injected animal model that reproduce an iron overload model with adapted regulation of hepcidin level considering the iron status increase. Animals were injected with iron dextran at 2 months of age to cause iron overload and the analysis were performed at 6 months of age. Control groups included animals injected with saline serum and animals injected with dextran alone.

1 - Impact of iron overload on iron, molybdenum and manganese concentrations in bone

In iron-dextran overloaded mice (Figure 4), the bone iron concentration was strongly and significantly increased in iron injected animals compared to control groups. Bone concentrations of manganese and molybdenum were significantly increased in the iron dextran group compared to control mice (p = 0.007 for manganese and p = 0.035 for molybdenum). Bone concentration of manganese was increased in the iron dextran group compared to the dextran group (p = 0.03), whereas molybdenum concentration was not.

In Hfe^'A mice (Figure 4) the bone iron concentration was increased in Hfe^'A animals at 12 months compared to the control group. 2 - Relationships between the concentrations of the three metals in liver, spleen and bone.

In Hfe^{~ '} mice, the molybdenum concentration in bone was significantly correlated with its concentration in the spleen (Rho = + 0.566, p <0.001).

In iron-dextan iron overload model, the iron concentration in bone was correlated with i) the hepatic (Rho = + 0.686, p <0.001) and splenic (Rho = + 0.674, p <0.001) concentrations, ii) the manganese bone concentrations (Rho = + 0.580, p = 0.001) and molybdenum bone concentration (Rho = + 0.486, p = 0.007). Bone concentration of manganese correlated with its splenic concentration (Rho = + 0.495, p = 0.01), but not with its hepatic concentration.

3 - Hepcidin deficient animals, but not secondary iron overloaded mice, develops osteoporosis · Bone Morphology :

There was no significant difference for bone microarchitecture parameters between iron- dextran mice and the two control groups.

In Hfe^'A mice, there was a significant decrease in bone trabecular volume (BV / TV) demonstrating osteoporosis in 12-month-old Hfe^'A mice (p = 0.022). This difference was not significant at 6 months (Figure 5). The thickness of bone trabeculae (Tb.Th) was significantly decreased at 6 months and 12 months in Hfe^'A mice compared to control mice of the same age. Finally, there was a significant increase in the Tb.Pf index, reflecting the decrease in connectivity between the bone trabeculae, at 12 months in Hfe^'A mice compared to control mice of the same age. All these differences demonstrate that Hfe^_/" developed osteoporosis.

• microarchitecture study by microCT :

3D reconstructions of the trabecular and cortical bone of the tibias, performed with the MicroCT Skyscan showed a decrease of bone trabeculae number (Tb.N), beginning in Hfe^'A mice of 6 months and becoming significant at 12 months compared to control mice of the same age,. In addition, there was a decrease of the thickness of the trabeculae (Tb.Th) at 12 months in Hfe^_/" mice.

There was no difference between iron- dextran, Dextran and control mice.

• Relationships between tissues metal concentration and histomorphometry.

In the Hfe^'A mice group, the BV / TV, was correlated with the molybdenum bone concentration (rho = -0.571, p <0.01). The molybdenum bone concentration was also correlated with Tb.N (rho = -0.696, p <0.001) and trabecular spacing Tb.Sp ; rho = + 0.680, p <0.001). The splenic molybdenum concentration was correlated with Tb.Pf (rho = + 0.505, p <0.05), Tb.N (rho = -0.8, p <0.001) and Tb.Sp (rho = + 0.794). , p <0.001). Finally, the splenic concentration of manganese correlated with Tb.N (rho = 0.626, p <0.001) and Tb.Pf (rho = + 0.613, p <0.001).

There was no significant correlation in the group of secondary iron overload mice. Summary

In iron-dextran iron overload, despite the strong increase of the bone iron concentration, no osteoporosis was detected. Both Mn and Mo concentrations were also significantly increased in bone.

In hepcidin deficient animals (Hfe^'A), the bone iron concentration was also increased, but much lower than in iron-dextran animals. However, osteoporosis was found in 12 month old mice. There was a lack of elevation of molybdenum and manganese concentration in bones.

The relative decrease of the concentration of Mo and Mn, with respect to the iron concentration increase in Hfe^'A mice, could limit the mechanisms of defense involved in the protection against mechanisms leading to organ lesions, in particular oxidative stress.

Without being bound by theory, this results show that hepcidin can directly or indirectly, through an alteration of Mo and or Mn metabolisms, play a role in the development of osteoporosis. Therefore, the hepcidin agonist of the present invention can be used in the prevention and/or treatment of osteoporosis.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

1. A hepcidin antagonist for use in the treatment of dysregulation of Mo and/or Mn metabolism.

2. The hepcidin antagonist according to claim 1, for use in the treatment of Mo and/or Mn cellular excess.

3. The hepcidin antagonist according to claim 1, for use in the treatment of Mo and/or systemic deficiencies.

4. A hepcidin agonist for use in the treatment of dysregulation of Mo and/or Mn metabolism.

5. The hepcidin agonist according to claim 4, for use in the treatment of Mo and/or Mn cellular deficiencies.

6. The hepcidin agonist according to claim 4, for use in the treatment of Mo and/or Mn systemic excess.

7. The hepcidin antagonist for use according to any of the claims 1 to 3 or the hepcidin agonist for use according to any of the claims 4 to 6, wherein the dysregulation is Mo dysregulation.

8. The hepcidin antagonist for use according to any of the claims 1 to 3 or the hepcidin agonist for use according to any of the claims 4 to 6, wherein the dysregulation is Mn dysregulation.

9. A hepcidin agonist for use in the prevention and/or treatment of osteoporosis.