The pathophysiology andpharmacology of hepcidinPiotr Ruchala and Elizabeta Nemeth

Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

Review

Inappropriate production of the iron-regulatory hor-mone hepcidin contributes to the pathogenesis of com-mon iron disorders. Absolute or relative deficiency ofhepcidin causes iron overload in hereditary hemochro-matosis and iron-loading anemias. Elevated hepcidincauses iron restriction in inflammatory conditionsincluding autoimmune disease, critical illness, somecancers, and chronic kidney disease. Multiple agentstargeting hepcidin and its regulators are under develop-ment as novel therapeutics for iron disorders. Thisreview summarizes hepcidin biology and discusses thecurrent landscape for hepcidin-targeting therapeuticstrategies.

Hepcidin regulates systemic iron homeostasisThe peptide hormone hepcidin is primarily produced inhepatocytes and it regulates plasma iron concentrations[1]. The molecular target of hepcidin is the cellular ironexporter ferroportin [2]. Ferroportin supplies iron intoplasma from duodenal enterocytes engaged in dietary ironabsorption, from macrophages of the spleen and liver thatrecycle old red blood cells, and from hepatocytes involved iniron storage (Figure 1) [3]. Hepcidin is the ligand forferroportin and their interaction results in rapid ubiquiti-nation of ferroportin and endocytosis and degradation ofthe ligand–receptor complex [4]. Loss of ferroportin fromcell membranes decreases the delivery of iron into plasma.If hepcidin is chronically elevated, persistent hypoferremiacan lead to the development of iron-restricted anemia.Conversely, chronic hepcidin deficiency results in excessiveiron absorption, increased levels of non-transferrin-boundiron in circulation, and the development of iron overload.Because of its critical role in iron homeostasis and thepathogenesis of iron disorders, hepcidin has emerged as apromising drug target. Here we review the biology ofhepcidin and highlight various pharmacological strategiesfocused on antagonizing or agonizing hepcidin.

Hepcidin is synthesized as a preprohormone that iscleaved intracellularly and secreted as a mature 25-aapeptide [5]. The production of hepcidin appears to beregulated predominantly at the transcriptional level.

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Corresponding author: Nemeth, E. (enemeth@mednet.ucla.edu).Keywords: iron disorders; anemia; iron overload; ferroportin.

The major stimuli regulating hepcidin production includeits substrate, iron, and the signals reflecting erythropoieticdemands for iron (Figure 1). Hepcidin is also an acute-phase reactant and is increased during inflammation. Anumber of other hepcidin regulators have also beendescribed, including growth hormones: hepatocyte growthfactor (HGF) and epidermal growth factor (EGF) [6], ster-oid hormones (estrogen [7], testosterone [8,9]), and meta-bolic pathways (starvation/gluconeogenesis [10]), but theirrole in hepcidin and iron homeostasis and pathobiology isless well understood.

Hepcidin regulation by iron

Hepcidin production increases in response to iron loading[11,12] and this prevents further absorption of dietary ironand the development of iron overload. Both plasma ironand liver iron stores regulate hepcidin transcription viadistinct but overlapping pathways [12,13]. These convergeonto the bone morphogenetic protein (BMP) pathway toincrease hepcidin transcription (Figure 2). Both serum ironand liver iron accumulation activate the BMP receptor andits Smad1/5/8 pathway [13], and increase hepcidin mRNAconcentrations in hepatocytes. The BMP co-receptor hemo-juvelin (HJV) is also required for this response [12].

It is not yet known how intracellular iron concentrationsare sensed. One of the mediators may be BMP6, whoseexpression increases with liver iron loading in mice [14].For extracellular iron, transferrin receptors 1 and 2 (TfR1and TfR2) are the likely sensors of holo-transferrin con-centrations, but multiple hypotheses have been proposedas to how this signal is transmitted. One line of evidencesuggests that HFE protein, an MHC class I molecule,shuttles between the two TfR receptors, depending onholo-Tf concentrations. At higher holo-Tf concentrations,HFE is displaced from TfR1 and associates with TfR2 [15].HFE and TfR2 in turn may interact with HJV [16], thuspotentiating BMP signaling. Other evidence suggests thatHFE and TfR2 regulate hepcidin independently [17], butthe details of this proposed mechanism are not yet known.Holo-Tf binding to TfR2 by itself stabilizes the receptorbecause TfR2 is redirected to a recycling pathway [18].

Although the details of the iron-sensing circuitry remainto be determined, it has been shown that mutations in Hfe,TfR2, Hjv, Bmp6, BMP receptors Alk2 and Alk3, andSmad4 all impair hepcidin regulation by iron in mice[12,19,20]. HFE, TfR2 and HJV mutations have also beendescribed in humans and result in hepcidin levels that areinappropriately low for the degree of iron overload

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Erythropoie�c signal

Spleen

Bonemarrow

RBC

Liver

Duodenum

Hepcidin

Hepcidin

Iron signal

Hepcidin

Fpn

Fpn

Fpn

Inflamma�on

PlasmaFe-Tf

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Figure 1. The role of hepcidin in iron metabolism. Hepcidin–ferroportin interaction

determines the flow of iron into plasma. Hepcidin concentrations are in turn

regulated by iron, erythropoietic activity, and inflammation. Reproduced with

permission from Goodnough L.T. et al (2010) Detection, evaluation, and

management of iron-restricted erythropoiesis. Blood 116, 4754–4761. � The

American Society of Hematology.

Review Trends in Pharmacological Sciences March 2014, Vol. 35, No. 3

observed in these patients [21–23]. Hepcidin production isfurther modulated by the transmembrane serine proteaseTMPRSS6, also known as matriptase-2 [24,25], and byneogenin, a multifunctional transmembrane receptor[26]. Although the mechanism is still controversial, ithas been proposed that these proteins act by post-transla-tionally regulating the levels of membrane-associated HJV[27,28]. The specific involvement of these proteins in ironsensing is also uncertain.

BMPRTfR1

Fe-Tf

TfR2

HFE

Fe-Tf

MT2

BMPs

Smad1/5/8pathway

HJVNeo

Hepcidintranscrip�on

(A)

?

Figure 2. Regulation of hepcidin transcription. (A) Hepcidin regulation by extracellular iron

regulation of hepcidin expression. BMP pathway signaling is further modulated by hemoj

via the following mechanism. Binding of holotransferrin (Fe-Tf) to transferrin receptor 1 (T

The TfR2 protein is stabilized by binding of Fe-Tf. The HFE–TfR2 complex may promote BM

each other. Additional proteins [TMPRSS6/matriptase-2 (MT2) and neogenin] modulate

regulation by inflammation. It has been shown that IL-6 and other cytokines (e.g., oncosta

been proposed that activin B, acting via BMP receptors and the Smad1/5/8 pathway, stim

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Hepcidin regulation by erythropoietic signals

Hepcidin is suppressed in conditions associated withincreased erythropoietic activity, presumably to makemore iron available for hemoglobin synthesis. Hemor-rhage, hemolysis, and injections of erythropoietin, a hor-mone that promotes red blood cell production, all result ina rapid decrease in hepcidin [29]. In anemias with ineffec-tive erythropoiesis, hepcidin levels are chronically sup-pressed, and this is thought to be the cause of ironoverload in nontransfused patients [30]. It is not knownwhether the same pathways mediate both physiologicaland pathological suppression of hepcidin in response toincreased erythropoietic activity, but these likely involvesecretion of a hepcidin suppressor from bone marrowerythroid precursors [31].

Hepcidin regulation by inflammation

Hepcidin is rapidly increased by inflammatory and infec-tious stimuli via the IL-6 pathway [32], although otherpathways, including the BMP pathway, may also contri-bute (Figure 2) [33,34]. Inflammatory regulation of hepci-din may have evolved as a host defense mechanism to slowthe growth of microorganisms by sequestering iron fromthem. Although the role of hepcidin in infections remains tobe demonstrated, it is thought that hepcidin increases ininflammatory conditions contribute to the development ofiron-restricted anemia [1].

Hepcidin and iron disordersHepcidin deficiency in iron overload disorders

Hepcidin deficiency is the pathogenic cause of iron overloadin most forms of hereditary hemochromatosis. Hepcidininsufficiency results from deleterious mutations in thegenes encoding hepcidin regulators (HFE, TfR2, and

IL-6

IL6R

Stat3pathway

Hepcidintranscrip�on

BMPR

ac�vin B

Smad1/5/8pathway

IL-6-likecytokines

(B)

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. The bone morphogenetic protein (BMP)–Smad pathway is central to transcriptional

uvelin (HJV), a BMP co-receptor. It is proposed that sensing of holotransferrin occurs

fR1) displaces HFE from its complex with TfR1 and promotes its interaction with TfR2.

P signaling by forming a complex with HJV, or even acting independently of HJV or

the cleavage of membrane HJV and thus alter hepcidin transcription. (B) Hepcidin

tin M, IL-22) regulate hepcidin expression by activating the Stat3 pathway. It has also

ulates hepcidin expression during inflammation.

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HJV) or hepcidin itself [1]. In all of these cases, dietary ironis hyperabsorbed, resulting in deposition of excess iron inthe liver and other parenchyma. The degree of hepcidindeficiency correlates with the severity of iron overload:mutations in HJV or hepcidin, which are associated withabsolute hepcidin deficiency, cause juvenile hemochroma-tosis, whereas mutations in HFE and TfR2, for which thehepcidin response to iron loading is partially preserved[12,21], result in a less severe adult form of the disease. Arare form of hemochromatosis is also caused by mutationsin the hepcidin receptor ferroportin, which lead to ferro-portin resistance to hepcidin-induced endocytosis [35,36].Hereditary hemochromatosis patients are currently trea-ted by bleeding. Each 1 ml of blood removed eliminates1 mg of iron from the body. As new red blood cells are made,excess iron from other organs is mobilized and used forerythropoiesis. Although this treatment is effective,repeated phlebotomy is inconvenient for many patientsand may be difficult for those with poor venous access orcoexisting medical conditions. Furthermore, once iron-depleted, patients with hereditary hemochromatosisexperience a dramatic decrease in hepcidin [21] and thisfurther accelerates iron absorption and the need for furtherphlebotomy.

As mentioned before, hepcidin deficiency also contri-butes to iron loading in non-transfused patients sufferingfrom anemias with ineffective erythropoiesis, includingb-thalassemia and congenital dyserythropoietic ane-mias. In these patients, despite iron loading, hepcidinremains insufficient because of the suppressive effect ofexcess erythroid activity [30]. In transfused patients,hepcidin levels decrease towards the end of the transfu-sion cycle [37] suggesting that relative hepcidin defi-ciency could contribute to iron loading even in thesepatients. Currently, iron overload in thalassemia is trea-ted by iron chelation, which can have significant sideeffects [38].

Hepcidin excess in iron-restrictive disorders

It is thought that increased hepcidin and associated hypo-ferremia contribute to the development of anemia indiverse human disorders [1]. The causes of increases inhepcidin include high levels of inflammatory cytokines,decreased clearance of hepcidin, and mutations in thenegative regulators of hepcidin. IL-6 and other cytokinescause high hepcidin levels in autoimmune disorders, infec-tions, and some cancers. In chronic kidney disease (CKD),apart from the common presence of inflammation,decreased clearance of hepcidin in the kidneys may alsocontribute to the development of anemia [39]. Finally,mutations in protease TMPRSS6, a hepcidin suppressor,lead to the development of iron-refractory iron deficiencyanemia (IRIDA) [25].

The degree of contribution of hepcidin excess to anemiaof inflammation remains to be determined. Although hep-cidin knockouts develop milder anemia in mouse models ofinflammation [40], decreases in hemoglobin are notentirely prevented, suggesting that other mechanisms alsocontribute. These may include direct effects of cytokinessuch as interferon-g and IL-6 on hematopoietic precursordifferentiation and erythrocyte maturation [41,42].

Manipulation of the hepcidin pathway for therapeuticpurposesBecause hepcidin deficiency or excess plays importantroles in the pathogenesis of various iron disorders, hepci-din agonists and antagonists may be potentially useful inclinical practice. Genetic studies in animal models haveprovided initial proof of the principle that hepcidin could bean effective therapeutic target. For example, overexpres-sion of hepcidin in Hfe�/� mice, a model of the mostcommon form of human hereditary hemochromatosis, pre-vented the liver iron overload normally seen in thesemutants [43]. Similarly, in mouse models of b-thalassemiaintermedia, moderate transgenic hepcidin expressiondecreased iron loading in the liver, diminished splenome-galy, and improved anemia [44]. In the case of anemia ofinflammation, hepcidin knockout mice demonstratedmilder anemia with faster recovery than wild type mice[40].

Hepcidin agonists

Compounds that can mimic the function of hepcidin orpotentiate its endogenous synthesis may be able to preventsystemic accumulation of iron. The development of bothtypes of therapeutics is in progress (Table 1). Such com-pounds may provide additional treatment options forpatients who do not respond well to standard treatmentregimens, such as hereditary hemochromatosis patientswith limited tolerance to phlebotomy and b-thalassemiapatients who suffer from side effects when undergoing ironchelation therapy.

Hepcidin mimics. The hepcidin peptide itself does nothave desirable pharmacological properties. The hormonehas a short half-life in circulation (several minutes) [45].Moreover, synthesis of the bioactive peptide is difficult andexpensive, mostly because of complicated disulfide bondconnectivity (four -S–S- bridges) that diminishes the yieldof the correctly folded product. In addition, recently studiesaimed at developing more stable cyclic full-length hepcidinanalogs were unsuccessful, because the cyclic analogsshowed no biological activity despite increased stabilityin human serum [46]. Hepcidin mimetics may thus be moreviable therapeutic candidates.

Minihepcidins are peptide-based hepcidin agonists thatwere rationally designed based on the region of hepcidinthat interacts with ferroportin [47]. Mutagenesis and trun-cation studies established that a 9-aa N-terminal fragmentof hepcidin (DTHFPICIF) is crucial for its hormonal activ-ity [47]. This particular fragment was further engineered:unnatural amino acids (N-substituted and b-homo aminoacids) were introduced to increase resistance to proteolysis,and fatty acids were conjugated to prolong the half-life incirculation. This yielded analogs that are at least as potentas full-length hepcidin and have a longer duration of action[48]. One such analog, the minihepcidin PR65, was testedin hepcidin knockout mice, a model of severe hemochro-matosis. A 2-week treatment prevented the development ofiron overload in non-overloaded hepcidin knockout mice[48]. Treatment of mice with pre-existing iron overload wasless effective but still led to partial redistribution of ironfrom the liver to the spleen within 2 weeks [48]. At high

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Table 1. Principles of hepcidin-targeting therapeutic approaches

Therapeutic

approach

Targeted disease Mode of action Agents

Hepcidin

agonists

Iron overload (hereditary

hemochromatosis and iron-loading

anemias)

Hepcidin mimics Minihepcidins [47]

Stimulators of hepcidin production Gene silencing of TMPRSS6 [50,51]

BMP pathway agonists [52]

Hepcidin

antagonists

Iron-restricted anemias (anemia of

inflammation, anemia of chronic kidney

disease, anemia of cancer, IRIDA)

Suppressors of hepcidin production BMP pathway inhibitors [54,56,74]

Anti-inflammatory agents [60–62]

Erythropoiesis-stimulating agents [65]

Gene silencing of hepcidin and its regulators

[66]a

Hepcidin peptide neutralizing binders Anti-hepcidin antibodies [67]b

Anticalins [68]

Spiegelmers [69]

Agents interfering with hepcidin–

ferroportin interaction

Anti-ferroportin antibodies [71]

Thiol modifiers [72]

ahttp://ir.isispharm.com/phoenix.zhtml?c=222170&p=irol-newsArticle&ID=1828284&highlight=

bhttp://www.clinicaltrials.gov/ct2/show/NCT01340976

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doses, PR65 caused profound iron restriction and anemia,indicating that minihepcidin therapy will likely requiretitration to effect to avoid excessive hypoferremia and ironrestriction. Collectively, these data strongly suggest thatminihepcidins may be useful for the prevention of ironoverload or as an auxiliary to phlebotomy or chelation forthe treatment of existing iron overload. Development of thenext generation of minihepcidins is under way (P. Ruchala,unpublished data).

Stimulators of hepcidin production. One approach forincrease hepcidin production is to antagonize TMPRSS6, anegative regulator of hepcidin. Homozygous inactivation ofTmprss6 in thalassemic th3/+ mice increased hepcidinlevels, ameliorated iron overload, and improved ineffectiveerythropoiesis [49]. Subsequently, targeting of Tmprss6with RNA-based therapeutics proved to be a promisingapproach. Both antisense oligonucleotides (ASOs) and siR-NAs against Tmprss6 were successfully used in mousemodels of iron overload [50,51]. Hfe�/� mice treated for 6weeks showed increases in hepcidin mRNA and decreases inserum and liver iron concentrations. A small reduction inhemoglobin was also noted, indicating mild iron restriction.Treatment of thalassemic th3/+ mice with ASOs and siRNAagainst Tmprss6 reproduced the effect of transgenic hepci-din overexpression in the th3+/� mouse model [44] andimproved anemia while decreasing iron overload [50,51].

Other approaches for increasing hepcidin productionmay include administration of BMP6 and its agonists[52]. However, clinical application of these agents in irondisorders will require extensive further research becausethe BMP pathway plays a role in bone morphogenesis,cell growth, differentiation, apoptosis, angiogenesis, andcancer.

Hepcidin antagonistsElevated hepcidin concentrations are associated with var-ious pathologies: anemia of inflammation, chronic kidneydisease, some cancers, and iron-refractory iron deficiencyanemia. These conditions are usually treated with erythro-poiesis-stimulating agents (ESAs) with or without high-dose intravenous iron. However, the effectiveness of these

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therapies is thought to be impaired by high hepcidin.Hepcidin-mediated iron restriction likely contributes toerythropoietin resistance, and high hepcidin may decreasemobilization of iron from macrophages, which process IViron preparations. Considering the side effects reported forESAs when used at high doses [53], introduction of hepci-din antagonists may offer a therapeutic advantage.

Antagonism of hepcidin activity can be achieved bydecreasing hepcidin production, neutralizing hepcidin pep-tide, blocking hepcidin–ferroportin binding, preventingferroportin endocytosis, or promoting ferroportin synthesis(Table 1).

Suppressing hepcidin production by targeting the

pathways regulating hepcidin transcription

BMP pathway. Because the BMP–Smad pathway is cru-cial for regulation of hepcidin transcription (Figure 2), it isnot surprising that agents interfering with the BMP path-way are effective at decreasing hepcidin production. How-ever, questions remain about what other processes may beaffected in vivo by these agents given the pleiotropic role ofthe BMP pathway in various biological responses.

It was recently reported that sequestration of BMPs byheparin decreases hepcidin expression in hepatic cell linesand in mice [54]. In patients undergoing low-molecular-weight heparin therapy to prevent deep vein thrombosis,serum hepcidin concentrations decreased by �80% within2–5 days after the start of the treatment. This was asso-ciated with increased serum iron and transferrin satura-tion. Because heparin is a known anticoagulant, novel non-anticoagulant heparins were also developed and tested [55]and showed potent activity in vitro and in vivo in mice,even in the presence of an inflammatory stimulus. Notably,heparin itself is an anti-inflammatory agent [54], whichmay be a contributory factor in its anti-hepcidin activity.

HJV, a BMP co-receptor essential for hepcidin expres-sion, is another molecular target that can be exploited tointerfere with hepcidin production. Membrane-linked HJVand its soluble form (sHJV) have opposing effects on hep-cidin expression, and sHJV decreases Smad signaling andhepcidin levels [56]. Soluble HJV-Fc fusion protein(sHJV.Fc) ameliorated anemia of inflammation (AI) in a

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rat model [57] in which AI was induced with group Astreptococcal peptidoglycan–polysaccharide (PG-APS).Four-week therapy resulted in increased hemoglobinand serum iron, although hepcidin mRNA had not signifi-cantly decreased by this point. Notably, in vitro studiessuggested that sHJV.Fc acts as a broad-spectrum BMPantagonist [58], and it remains to be determined whetherthis therapy will have significant off-target effects in vivo.

A small-molecule inhibitor of the BMP type I receptorwas another effective suppressor of hepcidin. LDN-193189,a derivative of dorsomorphin, which specifically antago-nizes the kinase activity of BMP receptor isotypes ALK2,ALK3, and ALK6, effectively reversed anemia in the ratmodel of AI caused by PG-APS [57]. In a rat model ofanemia of chronic kidney disease caused by adenine treat-ment, 5 weeks of LDN-193189 injections decreased hepci-din mRNA and increased serum iron and the hemoglobincontent of reticulocytes, although no change in hemoglobinwas noted [59].

Inflammatory pathway. Inflammation strongly induceshepcidin expression via IL-6–Stat3 and possibly otherpathways (Figure 2). As expected, neutralizing monoclonalantibodies against IL-6 or IL-6 receptors decreased hepci-din synthesis in animal models and humans with inflam-matory conditions. For example, anti-IL6 mAb decreasedhepcidin and improved anemia in renal cell carcinoma,multiple myeloma, and Castleman disease [60,61]. Simi-larly, administration of anti-TNFa antibody decreasedhepcidin levels in patients with rheumatoid arthritis[62]. Drawbacks of the anti-cytokine regimens are usuallyrelated to an impaired host defense immunoresponse [63],which translates to an increased risk of infections.

Erythropoietic pathway. Although the mechanism ispoorly understood, increased erythropoietic activity sup-presses hepcidin, and potentiation of this pathway could beused in iron-restricted conditions associated with elevatedhepcidin. High doses of erythropoietin (EPO) can overcomethe resistance to erythropoietin seen in AI [64], and thismay be partly caused by hepcidin suppression. Similarly,prolyl hydroxylase inhibitors, which stabilize hypoxia-inducible factor and increase EPO synthesis, decreasedhepcidin and increased hemoglobin in CKD patients [65].However, administration of large doses of ESAs in patientswith CKD or cancer has been associated with rare butsevere adverse effects [53]. Thus, agents that specificallytarget hepcidin production would be needed to alleviatesafety concerns.

Gene silencing of hepcidin and its regulators. Genesilencing agents preferentially accumulate in the liver,and thus hepcidin and its regulators may be feasibletargets for this approach. Antisense oligonucleotides andsiRNAs for hepcidin (http://ir.isispharm.com/phoe-nix.zhtml?c=222170&p=irol-newsArticle&ID=1828284&-highlight=), TfR2, and HJV have been developed [66].Experiments in the PG-APS rat model of AI showed thatTfR2 siRNA administration decreased hepcidin mRNAand reversed anemia [66]. Considering that human muta-tions in hepcidin, HJV, and TfR2 are exclusively associated

with iron overload disorders so far, off-target effects couldbe minimal.

Neutralizing the hepcidin peptide

Neutralization of hepcidin bioactivity may be achieved bydirect binding or sequestration of the hormone in circula-tion. To this end, (i) monoclonal antibodies (mAbs), (ii)engineered proteins (anticalins), and (iii) RNA-based bin-ders (spiegelmers) have been investigated.

A fully human anti-hepcidin antibody (12B9m) wasevaluated in transgenic mice expressing human hepcidinusing a model of AI caused by heat-killed Brucella abor-tus. MAb-mediated neutralization of hepcidin increasedhemoglobin and reversed the erythropoietin resistanceobserved in this model [67]. Furthermore, the MAb sup-pressed hepcidin in cynomolgus monkeys and caused aprolonged dose-dependent increase in serum iron.Another fully humanized mAb against hepcidin(LY2787106) is currently in Phase I human trials forcancer-related anemia (http://www.clinicaltrials.gov/ct2/show/NCT01340976).

Anticalins are small proteins (�20 kDa) derived fromlipocalins, which are naturally occurring proteins thatcan bind diverse biological ligands. Target-specific antic-alins with high specificity and picomolar affinity can beengineered via site-directed random mutagenesis incombination with selection via phage display. Onesuch compound, the anticalin PRS-080, binds humanhepcidin with subnanomolar affinity. In nonhuman pri-mates (cynomolgus monkeys), PRS-080 administrationcaused effective mobilization of iron and hyperferremia[68].

Spiegelmers are L-enantiomeric oligonucleotides thatwere engineered to bind pharmacologically relevant tar-gets with high affinity. Therefore, they are conceptually aform of aptamer. Because to their non-natural structure,which utilizes L-ribose instead of D-ribose in nucleotideunits, they are highly resistant to degradation bynucleases, stable in circulation, immunologically passive,and generally well tolerated. Spiegelmers for specific tar-gets are selected by screening of large combinatoriallibraries. NOX-H94 is a spiegelmer that neutralizeshuman hepcidin. In a Phase I human trial, NOX-H94dose-dependently increased serum iron and transferrinsaturation [69]. NOX-H94 is currently in Phase II clinicaltrials to examine its efficacy in patients with anemia ofcancer.

The high specificity of hepcidin binders for their target isthe main advantage of these approaches. The main chal-lenge for their efficacy is likely the high rate of hepcidinproduction, estimated at �12 mg/day per 75 kg for humans[70] and even higher during inflammation. Considering thedifference in molecular weight between hepcidin (only2.7 kDa) and its neutralizing binders, massive doses ofintravenously delivered therapeutic agents may berequired. Moreover, neutralization of hepcidin withlarge-molecular-weight binders will most likely impedeits natural clearance rate, resulting in further hepcidinaccumulation. However, even partial or intermittent neu-tralization of hepcidin activity may be sufficient to reachdesired therapeutic effects.

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Blocking hepcidin–ferroportin interaction

A humanized anti-ferroportin monoclonal antibody(LY2928057) was developed against an extracellular loopof ferroportin adjacent to the hepcidin-binding region [71].LY2928057 prevents hepcidin binding to the transporterwithout interfering with the iron efflux through ferropor-tin. LY2928057 increased serum iron in a dose-dependentmanner in cynomolgus monkeys, and clinical evaluation isunder way.

Mechanistically, hepcidin binding to ferroportin relieson the extracellular loop of ferroportin that contains thesulfhydryl Cys326 residue and surrounding hydrophobicresidues [47]. The thiol-reactive compound fursultiamineprevented hepcidin binding to ferroportin in vitro [72],although the short half-life of fursultiamine in circulationprevented any consistent effects in mice in vivo. Similaragents may be effective in preventing hepcidin–ferroportininteraction, provided that nonspecific effects on other pro-teins containing an active thiol are minimized.

Targeting ferroportin endocytosis or synthesis

Although no agents have been tested in preclinical modelsyet, any approaches that prevent ferroportin endocytosis orstimulate ferroportin synthesis would be expected toincrease iron delivery to plasma, thus circumventing theeffect of elevated hepcidin. Ferroportin endocytosis isdependent on ubiquitination of several intracellular lysineresidues [4]. Because of the fairly generic nature of endo-cytic pathways, this process may be difficult to targetunless E3 ubiquitin ligases and/or other endocytic compo-nents with high ferroportin substrate specificity are iden-tified.

Unrelated to the effect on systemic iron metabolism,agents that increase ferroportin synthesis may also bebeneficial in the treatment of some cancers. Ferroportinexpression was a strong and independent predictor ofprognosis in breast cancer, and higher ferroportin wasassociated with a favorable prognosis [73].

Concluding remarksThe hepcidin–ferroportin axis plays an important role inthe pathogenesis of iron disorders including iron over-load diseases and iron-restricted anemias. It is thereforenot surprising that within only a dozen years since thefirst publications on hepcidin, multiple hepcidin-target-ing strategies have been developed. Although mostagents have only been evaluated in preclinical studies,several have reached human clinical trials. It is still tooearly to try to assess which of the many approaches havethe best chance of success. Although the clinical effec-tiveness of hepcidin-targeted therapies remains to beestablished, the hope is that hepcidin agonists andantagonists will improve the treatment of patients withiron disorders, either alone or in combination with exist-ing therapies.

Disclaimer statementElizabeta Nemeth is a stockholder and consultant for IntrinsicLifeSciences, a biotech company developing hepcidin diagnostics, andMerganser Biotech, a biotech company developing hepcidin therapeutics.Piotr Ruchala is a stockholder and consultant for Merganser Biotech.

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