Hepcidin Expression Research Articles

Effect of Radiotherapy on HB Level and Nutritional Status of Tumor Patients and Its Relationship with Hepcidin and EPO

We studied the expression features of Hepcidin (HEP), Erythropoietin (EPO), Albumin (ALB) and Prealbumin (PA), and the pathogenesis of anemia in tumor patients caused by radiotherapy. We tested the hemoglobin (HB) concentration in 446 cancer patients who received radiotherapy, and measured the serum HEP, EPO, ALB and PA n 92 cases to explore the pathogenesis of anemia caused by radiotherapy. The results were: (1) The HB level before and after radiotherapy in 446 patients were 119.85±19.87g/L and 116.32±24.25g/L (<i>t</i>=2.5211, <i>P</i><0.05). The cases of anemia before and after radiotherapy were 146 cases and 175 cases (<i>χ²</i>=4.09, <i>P</i><0.05). (2) The HB level before and after radiotherapy in 92 patients and in 20 cases of control group were respectively 127.33±17.12, 119.41±17.05 and 132.80±11.80g/L, the difference was statistically significant (<i>F</i>=11.70, <i>P</i><0.01). The anemia cases were 14 and 27, respectively, which were also statistically significant (<i>χ²</i>=8.68, <i>P</i><0.01). (3) The HEP level before and after radiotherapy in 92 patients and in 20 cases of control group were 3.38±2.48, 2.37±1.37 and 3.22±1.10mg/mL respectively, the difference was statistically significant (<i>F</i>=3.88, <i>P</i><0.05). (4) EPO level before and after radiotherapy in 92 patients and in 20 controls were 5.05±3.50, 5.75±1.99 and 3.81±1.15mg/mL respectively, the difference was statistically significant (<i>F</i>=9.18, <i>P</i><0.01). (5) The levels of ALB and PA before radiotherapy in 92 patients were higher than those after radiotherapy. (6) HEP was negatively correlated with HB before radiotherapy (<i>r</i>=-0.2938, <i>t</i>=2.9089, <i>P</i><0.01), but HEP was not correlated with HB after radiotherapy (<i>r</i>=- 0.0111, <i>P</i>>0.05). (7) There were positive correlation between ALB and HB before and after radiotherapy (all <i>P</i><0.01). (8) There were positive correlation between PA and HB before and after radiotherapy (all <i>P</i><0.01). Therefore, Radiotherapy could cause anemia and deteriorate nutritional status in cancer patients. Anemia negatively feedback inhibited the expression of HEP, while EPO was highly compensatory.

Hepcidin: Structure, Functions And Role In Hematological Disorders

Hepcidin is an essential iron regulatory protein encoded by human antimicrobial peptide gene. Structurally, hepcidin has three parts; the main structural framework, disulphide linkages and N-terminal region. Ferroportin, that allows the entry of iron into the blood, is degraded by hepcidin and inhibits this process. Transferrin receptor 1 and 2 play an important role along with hemochromatosis protein in sensing iron and increasing hepcidin expression. The role of hepcidin is instrumental in iron deficiency anemia. Iron-refractory iron deficiency anemia an autosomal recessive is associated with TMPRSS6 mutation that leads to increased concentration of serum hepcidin despite iron deficiency. In anemia of chronic disorder, hepcidin blocks the entry of iron from macrophages and duodenal enterocytes into blood, thus inhibiting the growth of microorganisms. In β-thalassemia major it has two effects; inhibitory and stimulatory. The inhibitory response is due to ineffective erythropoiesis and stimulatory response is due to multiple transfusions. In hereditary hemochromatosis type II hepcidin production is low due to gene mutation that leads to iron overload. This narrative review is compiled after relevant literature search from electronic databases. The key words used for searching relevant literature include iron, iron deficiency, iron deficiency anemia, iron metabolism, hepcidin, transferrin, causes of iron deficiency anemia and laboratory diagnosis of iron deficiency anemia. Reference hematology books were also consulted.

Improving greater amberjack (Seriola dumerili) defenses against monogenean parasite Neobenedenia girellae infection through functional dietary additives

The main objective of this study was to determine the effect of two feeding additives (cMOS: Actigen®, Alltech Inc., USA) and a phytogenic specifically designed for reducing ectoparasite incidence, combining garlic and labiatae herbal extracts (HERB; Delacon, Austria) on growth performance, stress and immune response of greater amberjack (Seriola dumerili) before and after an experimental parasitization with the skin ectoparasite Neobenedenia girellae, a monogenean with high incidence on Seriola cultured in sea cages. Two parallel trials were conducted in order to determine the effect of 70 days of functional diets supplementation (0.2% cMOS and 200 ppm HERB) on greater amberjack: a) Growth performance and stress resistance and b) disease resistance against N. girellae. Additionally, in order to evaluate the effects of the dietary supplements pre- and post- parasitization challenge against N. girellae, the expression of some immune-related genes was evaluated. The relative expression of Il-1β, piscidin, hepcidin, IgT, tnf-α, muc-2, cathelicidin, complement c3, cd8, and caspase-3 was determined in skin. The activity of protease and lysozyme in serum and skin mucus were also evaluated, as well as parasite load and parasite total size.The use of both functional additives did not affect fish growth performance. However, the dietary supplementation of HERB significantly (p<0.05) reduced circulating cortisol levels after the stress challenge, whereas dietary cMOS significantly (p<0.05) reduced the parasite load and total parasite size. When comparing pre and post parasitization challenge, skin mucus protease activity was significantly (p<0.05) higher in all the treatments before parasitization, while the use of both functional additives increased skin mucus lysozyme activity after challenge. From the eleven immune genes studied, only piscidin was significantly (p<0.05) up-regulated by HERB supplementation before challenge, whereas after parasitization, muc-2 and piscidin were significantly (p<0.05) up-regulated by HERB and tnf-α, il1-β, hep, c3, cd8 and casp3 were up-regulated by both functional additives. Moreover, hep, IgT, cath, c3 and cd8 expressions were negatively correlated with the parasite load.In general, greater amberjack supplementation with cMOS and HERB at the dose used in the present study was effective for reducing stress and for increasing the skin associated lymphoid tissue (SALT) immune response against N. girellae during the infection process.

Effects of long-term ethanol ingestion on hepatic iron metabolism in two mouse strains.

The mechanisms responsible for dysregulation of iron metabolism in response to ethanol ingestion are poorly understood. Relatively brief ethanol exposures in rodents are associated with reduced hepatic hepcidin expression without increases in hepatic iron content. This study evaluated the effects of long-term ethanol treatment on hepatic iron metabolism in two mouse strains. Ethanol was administered in the drinking water to C57BL/6 and BALB/c mice for up to 11months. Hepatic histology and iron concentrations (HIC) were assessed, along with expression of relevant genes and proteins by real-time RT-PCR and western blot, respectively. The livers of ethanol-consuming mice of both strains showed mild steatosis without inflammation or fibrosis. Stainable hepatocyte iron was modestly increased in both strains ingesting ethanol, although hepatic iron concentrations were significantly higher only in C57BL/6 mice. Long-term ethanol did not affect hepcidin mRNA (Hamp1 or Hamp2) in either strain, nor was the expression of several oxidative stress-responsive genes (glutamate cysteine ligase, gamma-glutamyl transpeptidase, heme oxygenase-1 and growth differentiation factor 15) altered in response to ethanol, suggesting that oxidative stress and suppression of hepcidin expression in short-term ethanol feeding models may be transient phenomena that resolve as mice adapt to ethanol exposure. This murine model of chronic ethanol ingestion demonstrates modest increases in hepatic iron without changes in hepcidin expression, markers of oxidative stress or significant histologic liver injury. Further investigations are needed to characterize the mechanisms of dysregulated iron metabolism resulting from chronic ethanol ingestion.

The protective effect of the cardiac thioredoxin system on the heart in the case of iron overload in mice

BackgroundIron, which is essential for many vital biological processes, causes significant clinical pathologies in the case of its deficiency or excess. Cardiovascular protective pathways are activated by iron therapy. However, determining the appropriate iron concentration is essential to protect heart tissue from iron-induced oxidative stress. The thioredoxin system is one of the antioxidant systems that protect cells against oxidative stress. Moreover, it allows the binding of many transcription factors for apoptosis, myocardial protection, the stimulation of cell proliferation, and angiogenesis processes, especially the regulation of the cardiovascular system. This study’s goal was to understand how iron overload affects the gene and protein levels of the thioredoxin system in the mouse heart. MethodsBALB/c mice were randomly separated into two groups. The iron overload group was administered with intraperitoneal injections of an iron-dextran solution twice a week for three weeks. In parallel, the control group was intraperitoneally given Dextran 5 solution. The total iron content, the total GSH level, the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and thioredoxin reductase 1 (TXNRD1) activity were demonstrated spectroscopically. Changes in the iron metabolism marker genes and thioredoxin system genes were examined by qPCR. The quantitative protein expression of TXNRD1 and thioredoxin-interacting protein (TXNIP) was examined by western blotting. ResultsThe iron content of the heart increased in the iron overload group. The expression of hepcidin (Hamp) and ferroportin (Fpn) increased with iron overload. However, decreased expression was observed for ferritin (Fth). No changes were revealed in the GSH level and GSH/GSSG ratio. The gene expression of thioredoxin 1 (Txn1), Txnrd1, and Txnip did not change. TXNRD1 activity and protein expression increased significantly, while the protein expression of TXNIP decreased significantly. ConclusionIn the case of iron overload, the cardiac thioredoxin system is affected by the protein level rather than the gene level. The amount and duration of iron overload used in this study may be considered as a starting point for further studies to determine appropriate conditions for the iron therapy of cardiovascular diseases.

Retraction to: Minocycline ameliorates osteoporosis induced by ovariectomy (OVX) and iron accumulation via iron chelation, bone metabolism regulation and inhibition of oxidative stress.

OBJECTIVE To investigate the effect and mechanism of minocycline on iron accumulation-related postmenopausal osteoporosis. METHODS The present study established a rat model of ovariectomy (OVX), gave rats ferric ammonium citrate (FAC) and treated them with minocycline, then examined the severity of osteoporosis and iron metabolism in rats. To further explore the mechanism, osteoblasts were treated with FAC and minocycline, then their effects on cell viability, apoptosis, alkaline phosphatase (ALP) activity, bone metabolism proteins, iron metabolism proteins, and oxidative stress in osteoblasts were measured. RESULTS In the animal study, OVX significantly decreased the serum estradiol level. Both OVX and FAC significantly increased the serum ferritin and tibial iron level, which was significantly decreased by minocycline (P < 0.05). Minocycline significantly increased the ratio of BV/TV, Tb.Th and Tb.N (P < 0.05), and the levels of BALP, BGP and CTX, but decreased the levels of TRAP and ratio of RANKL/OPG (P < 0.05 compared to OVX+FAC group). In the cell study, minocycline significantly decreased the cellular iron accumulation and induced cell death and apoptosis (P < 0.05). Minocycline significantly increased the ALP activity, the expression of Collagen I, Osteocalcin and OPG (P < 0.05). Minocycline significantly decreased the expression of Ferritin and hepcidin, and increased the expression of FPN) (P < 0.05). It also significantly decreased the cellular MD) and protein carbonyl level and RO) intensity, but increased the levels of SO) and GP) (P < 0.05). CONCLUSION Minocycline ameliorated osteoporosis induced by OVX and iron accumulation. The mechanism may involve iron chelation, regulation of bone and iron metabolism, and inhibition of oxidative stress.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication.

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

Ziltivekimab for Treatment of Anemia of Inflammation in Patients on Hemodialysis: Results from a Phase 1/2 Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

Patients with CKD who are on hemodialysis are hyporesponsive to erythropoiesis-stimulating agents (ESAs) because of anemia of inflammation. Interleukin-6 (IL-6) induced hepcidin expression is a key mediator of such inflammation. This phase 1/2, placebo-controlled trial assessed effects of ziltivekimab, a novel anti-IL-6 ligand antibody, in patients on hemodialysis with rs855791, a single nucleotide polymorphism of the TMPRSS6 gene that is hypothesized to heighten susceptibility to IL-6-mediated inflammatory effects. After a screening period documenting stable ESA and iron dosing, we randomized 61 patients with elevated IL-6 (≥4 pg/ml) to receive placebo or ziltivekimab (doses of 2, 6, or 20 mg), administered intravenously every 2 weeks for 12 weeks during hemodialysis. ESA dose adjustments were allowed after 4 weeks. We analyzed safety and effects on inflammation, iron metabolism, serum albumin, and anti-drug antibodies. No patient experienced dose-limiting toxicity. Four patients (two each in the 6- and 20-mg cohorts) died of a treatment-emergent adverse event. Compared with patients receiving placebo, those receiving ziltivekimab experienced significantly greater reductions of high-sensitivity C-reactive protein, serum amyloid A, and fibrinogen from baseline to end of treatment. Median ESA usage decreased by 15,000, 15,000, or 33,000 IU/wk per patient in the 2-, 6-, and 20-mg ziltivekimab cohorts, respectively, compared with no change in the placebo group. We also noted significant dose responses for decreased ESA resistance index and increased serum iron, total iron binding capacity, transferrin saturation, and serum albumin. Ziltivekimab significantly improved markers of inflammation, reduced ESA requirements, and increased serum albumin in patients on hemodialysis with inflammation and hyporesponsiveness to ESA therapy. Study to Assess the Safety, Pharmacokinetics, and Pharmacodynamics of Multiple Doses of COR-001, NCT02868229.

The HIF-PHI BAY 85-3934 (Molidustat) Improves Anemia and Is Associated With Reduced Levels of Circulating FGF23 in a CKD Mouse Model.

Fibroblast growth factor-23 (FGF23) is a critical factor in chronic kidney disease (CKD), with elevated levels causing alterations in mineral metabolism and increased odds for mortality. Patients with CKD develop anemia as the kidneys progressively lose the ability to produce erythropoietin (EPO). Anemia is a potent driver of FGF23 secretion; therefore, a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) currently in clinical trials to elevate endogenous EPO to resolve anemia was tested for effects on iron utilization and FGF23-related parameters in a CKD mouse model. Mice were fed either a casein control diet or an adenine-containing diet to induce CKD. The CKD mice had markedly elevated iFGF23 and blood urea nitrogen (BUN), hyperphosphatemia, and anemia. Cohorts of mice were then treated with a patient-equivalent dose of BAY 85-3934 (BAY; Molidustat), which elevated EPO and completely resolved aberrant complete blood counts (CBCs) in the CKD mice. iFGF23 was elevated in vehicle-treated CKD mice (120-fold), whereas circulating iFGF23 was significantly attenuated (>60%) in the BAY-treated CKD mice. The BAY-treated mice with CKD also had reduced BUN, but there was no effect on renal vitamin D metabolic enzyme expression. Consistent with increased EPO, bone marrow Erfe, Transferrin receptor (Tfrc), and EpoR mRNAs were increased in BAY-treated CKD mice, and in vitro hypoxic marrow cultures increased FGF23 with direct EPO treatment. Liver Bmp-6 and hepcidin expression were downregulated in all BAY-treated groups. Femur trabecular parameters and cortical porosity were not worsened with BAY administration. In vitro, differentiated osteocyte-like cells exposed to an iron chelator to simulate iron depletion/hypoxia increased FGF23; repletion with holo-transferrin completely suppressed FGF23 and normalized Tfrc1. Collectively, these results support that resolving anemia using a HIF-PHI during CKD was associated with lower BUN and reduced FGF23, potentially through direct restoration of iron utilization, thus providing modifiable outcomes beyond improving anemia for this patient population. © 2021 American Society for Bone and Mineral Research (ASBMR).

The Role of Erythroferrone Hormone as Erythroid Regulator of Hepcidin and Iron Metabolism during Thalassemia and in Iron Deficiency Anemia- A Short Review

Erythroferrone (ERFE) is a hormone produced by erythroblasts in the bone marrow in response to erythropoietin controlling iron storage release through its actions on hepcidin, which acts on hepatocytes to suppress expression of the hormone hepcidin. Erythroferrone now considered is one of potential clinical biomarkers for assessing erythropoiesis activity in patients with blood disorders regarding to iron imbalance. Since discovery of in 2014 by Dr. Leon Kautz and colleagues and till now no more enough studies in Erythroferrone among human, most studies are conducted in animals. In this review we briefly address the Role of Erythroferrone hormone as erythroid regulator of hepcidin and iron metabolism during thalassemia and in iron deficiency anemia. Studies in this review were identified through a search using the following electronic databases: PubMed, Academia, Scopus, Google Scholar, and another open database source. Conclusion: Most of studies concluded that, in people with thalassemia and iron deficiency anemia, erythroferrone levels in the blood are higher than in people without thalassemia and iron deficiency anemia. Knowing the mechanisms of erythroferrone as erythroid regulator of hepcidin and iron metabolism during thalassemia and in iron deficiency anemia important in the diagnosis and treatment for both conditions. The erythroferrone hormone may act as potential factor in physiological hepcidin suppressor in cases with iron deficiency anemia and thalassemia disease and play a key role in treatment process among those patients in status of iron deficiency or iron overload. However, till now few studies of the function of ERFE in humans because is recently discovered and remains to be investigated and most studies are conducted among animals.

Myricetin reduces cytotoxicity by suppressing hepcidin expression in MES23.5 cells.

Multiple studies implicate iron accumulation in the substantia nigra in the degeneration of dopaminergic neurons in Parkinson's disease. Indeed, slowing of iron accumulation in cells has been identified as the key point for delaying and treating Parkinson's disease. Myricetin reportedly plays an important role in anti-oxidation, anti-apoptosis, anti-inflammation, and iron chelation. However, the mechanism underlying its neuroprotection remains unclear. In the present study, MES23.5 cells were treated with 1 × 10-6 M myricetin for 1 hour, followed by co-treatment with 400 nM rotenone for 24 hours to establish an in vitro cell model of Parkinson's disease. Our results revealed that myricetin alleviated rotenone-induced decreases in cell viability, suppressed the production of intracellular reactive oxygen species, and restored mitochondrial transmembrane potential. In addition, myricetin significantly suppressed rotenone-induced hepcidin gene transcription and partly relieved rotenone-induced inhibition of ferroportin 1 mRNA and protein levels. Furthermore, myricetin inhibited rotenone-induced phosphorylation of STAT3 and SMAD1 in MES23.5 cells. These findings suggest that myricetin protected rotenone-treated MES23.5 cells by potently inhibiting hepcidin expression to prevent iron accumulation, and this effect was mediated by alteration of STAT3 and SMAD1 signaling pathways.

Global loss of Tfr2 with concomitant induced iron deficiency greatly ameliorates the phenotype of a murine thalassemia intermedia model.

β-thalassemias result from mutations in β-globin, causing ineffective erythropoiesis and secondary iron overload due to inappropriately low levels of the iron regulatory hormone hepcidin. Mutations in transferrin receptor 2 (TFR2) lead to hereditary hemochromatosis (HH) as a result of inappropriately increased iron uptake from the diet, also due to improperly regulated hepcidin. TFR2 is also thought to be required for efficient erythropoiesis through its interaction with the erythropoietin receptor in erythroid progenitors. Transmembrane serine protease 6 (TMPRSS6), a membrane serine protease expressed selectively in the liver, participates in regulating hepcidin production in response to iron stores by cleaving hemojuvelin (HJV). We have previously demonstrated that inhibiting TMPRSS6 expression with a hepatocyte-specific siRNA formulation, induces hepcidin, mitigates anemia, and reduces iron overload in murine models of β-thalassemia intermedia and HH. Here, we demonstrate that Tmprss6 siRNA treatment of double mutant Tfr2Y245X/Y245X HH Hbbth3/+ thalassemic mice induces hepcidin and diminishes tissue and serum iron levels. Importantly, treated double mutant animals produce more mature red blood cells and have a nearly 50% increase in hemoglobin compared to untreated β-thalassemic mice. Furthermore, we also show that treatment of Tfr2Y245X/Y245X HH mice leads to increased hepcidin expression and reduced total body iron burden. These data indicate that siRNA suppression of Tmprss6, in conjunction with the targeting of TFR2, may be superior to inhibiting Tmprss6 alone in the treatment of the anemia and secondary iron loading in β-thalassemia intermedia and may be useful as a method of suppressing the primary iron overload in TFR2-related (type 3) hereditary hemochromatosis.

Relation of hepcidin gene expression in blood mononuclear cells with iron overload severity among β-thalassemia major patients

Iron overload is the main cause of morbidity and mortality in β-thalassemia major patients, and cardiac iron overload is the most common reason for death in these transfusion-dependent patients. Hepcidin, a liver-derived peptide hormone, plays a key role in plasma iron levels regulation by controlling two main stages, digestive iron absorption in enterocytes, and iron recycling in macrophages. Although hepcidin is mainly secreted from hepatocytes in the liver, it is also synthesized from mononuclear cells consisting of monocytes and lymphocytes. Binding of this molecule to ferroportin, a specific cellular exporter of iron, leads to degradation of the ligand-receptor complex, which reduces the iron overload by lowering the amounts of iron released into the plasma. Likewise, the same mechanism has been proved to be true for lymphocyte-drived hepcidin.The expression levels of hepcidin mRNA were evaluated using quantitative real time PCR (qRT-PCR) in 50 β-thalassemia major patients, as well as 25 healthy volunteers as the group of control. There was a significantly positive correlation between the cardiac iron concentration, showed by higher T2 values, and hepcidin levels in the patients (p = 0.028; r = 0.311). However, hepcidin expression levels did not significantly correlate with ferritin and liver iron concentrations.Hepcidin can act as a beneficial marker to determine iron overload degrees, particularly in the heart, in β-thalassemia major patients and be used as a logical therapeutic agent for treatment of β-thalassemia disorders.

BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues

BackgroundThe bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6. MethodsBMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics. ResultsN-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour. ConclusionsIn BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS. General significanceThis study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders.

DISC-a, the First in a Novel Class of Potent and Selective Matriptase-2 Inhibitors for the Treatment of Hematologic Disorders Characterized By Low Hepcidin

Hepcidin is known as the master regulator of systemic iron homeostasis with reduction in synthesis leading to the development of iron overload. Hepcidin gene expression is negatively modulated by matriptase-2 (MT-2), a liver-specific type II transmembrane serine protease. MT-2 cleaves hemojuvelin (HJV), leading to the extracellular release of soluble HJV fragments and suppression of hepcidin expression. Loss-of-function of MT-2 leads to increased hepcidin expression, as has been established by human genetics (Finberg et al., 2008) and genetic mouse models (Du et al., 2008). Therefore, inhibition of MT-2 represents a potential therapeutic strategy for diseases caused by inappropriately low hepcidin leading to iron overload or where therapeutic iron restriction may be used to control excessive erythrocytosis. Here we describe the characteristics of DISC-A, a potent (low nM Ki) small molecule MT-2 inhibitor for treatment of low hepcidin disorders, with a favorable pharmacokinetics profile in rats (Clp 6.4 ml/min/kg, and t ½ 4.6 hr) and monkeys (Clp 8.1 ml/min/kg, and t ½ 2.8 hr) and drug-like properties. DISC-A inhibits proteolytic activity of MT-2 expressed on the surface of transfected HEK293 cells and prevents shedding of MT-2 from the membrane (autocleavage). In addition, in MT-2 and HJV co-transfected HEK293A cells, DISC-A shows a dose dependent inhibition of HJV cleavage. The efficacy of DISC-A was evaluated in a rat model of low hepcidin. In this model, when Sprague-Dawley rats who are fed a standard iron diet (45 ppm) reach 8 - 9 weeks of age, they are administered erythropoietin (EPO) at 30 IU/animal/day for 4-consecutive days, before dosing with DISC-A. Under the conditions of the model, the increased erythropoiesis leads to increased iron utilization and consequently suppressed hepcidin levels. We determine hepcidin changes by measuring the changes in the expression of liver HAMP (the gene that encodes hepcidin) mRNA expression. Circulating soluble HJV is assayed as a direct measure of MT-2 activity. In this model, a single subcutaneous administration of DISC-A at 20 mg/kg resulted in a 50% reduction in soluble HJV, 14-fold increase in liver HAMP expression and &amp;gt;50% reduction in serum iron and transferrin saturation (TSAT) at 2, 4, 6, and 8 hours. The pharmacokinetics/pharmacodynamics response was robust. In summary, we have identified DISC-A, a novel, potent inhibitor of MT-2. We have demonstrated that DISC-A inhibits MT-2 proteolytic activity, prevents cleavage of HJV, and modulates hepcidin gene expression and iron homeostasis in vitro and in vivo. The favorable pharmacokinetics suggest compounds from these chemical series have the potential for clinical therapeutic benefit. Disclosures Hong: Disc Medicine: Current Employment, Current equity holder in private company. Babu:Aurigene Discovery Technologies: Current Employment. Blaustein:Disc Medicine: Current Employment, Current equity holder in private company. Nguyen:Disc Medicine: Current Employment, Current equity holder in private company. Rao:Aurigene Discovery Technologies: Current Employment. Savage:Disc Medicine: Current Employment, Current equity holder in private company. MacDonald:Disc Medicine: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Beconi:Disc Medicine: Current Employment, Current equity holder in private company. Venkatraman:Disc Medicine: Current Employment, Current equity holder in private company.

Ambient fine particulate matter aggravates atherosclerosis in apolipoprotein E knockout mice by iron overload via the hepcidin-ferroportin axis

AimsExposure to fine particulate matter (PM2.5) is correlated to atherosclerosis, but the mechanism remains largely undefined. Iron overload is a significant contributor to atherosclerosis, and iron homeostasis is highly regulated by the hepcidin-ferroportin (FPN) axis. Here we aimed to investigate the association between iron overload and PM2.5-induced atherosclerotic mice. Main methodsApolipoprotein E knockout (ApoE−/−) mice were randomly assigned to filtered air (FA group) or PM2.5 (PM2.5 group) for 3-month inhalation. Daily PM2.5 mass concentrations, serum levels of ferritin, iron, pro-atherosclerotic cytokines and lipid profiles, atherosclerotic lesion areas, hepcidin, FPN and iron depositions in atherosclerotic lesions, hepcidin, FPN mRNA and protein expressions in the aorta were detected, respectively. Key findingsThe daily average concentration of atmospheric PM2.5 was 68.2 ± 21.8 μg/m3. Serum levels of ferritin, iron, VEGF, MCP-1, IL-6, TNF-α, TC and LDL-C, atherosclerotic lesion areas, hepcidin and iron depositions in atherosclerotic lesions, hepcidin mRNA and protein expressions in the PM2.5 group were observably higher than those in the FA group. Nevertheless, FPN deposition in atherosclerotic lesions, FPN mRNA and protein expressions in the aorta of the PM2.5 group were markedly lower than those of the FA group. SignificancePM2.5 inhalation could exacerbate the formation and development of atherosclerosis in ApoE−/− mice, the potential mechanisms may be partly associated with iron overload via the hepcidin-FPN axis, as well as iron-triggered systemic inflammation and hyperlipidemia.

DISC-0974, a Novel, First-in-Class, Anti-Hemojuvelin Monoclonal Antibody Is Predicted to be Efficacious at Low Doses in Humans As Determined By Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling in Non-Clinical Studies

DISC-0974 is a humanized monoclonal anti-HJV antibody with high binding affinity for human, rat and cynomolgus monkey HJV (100 pM, 240 pM and 110 pM Kd, respectively) [Kovac 2016]. DISC-0974 inhibits the interaction between HJV and the key hepcidin regulatory ligands, bone morphogenetic proteins (BMPs), leading to decreased SMAD phosphorylation (SMAD-P) and reduced hepcidin expression [Kovak 2016]. Elevated hepcidin is a hallmark of and plays a critical role in the pathogenesis of anemia of inflammation (and other forms of anemia) by reducing transferrin saturation and erythroid iron availability, leading to failure of erythropoiesis and decreased hemoglobin synthesis. DISC-0974 is being developed to reduce hepcidin synthesis and treat anemia in these patients. A critical early signal of therapeutic effect is the effect of DISC-0974 on transferrin saturation (TSAT). TSAT has therefore been used as the primary pharmacodynamic marker in a series of non-clinical studies to determine the relationship between drug exposure and pharmacological activity. In non-human primates (NHP) DISC-0974 elicits a dose-dependent reduction in hepcidin levels leading to increases in serum iron and consequently TSAT. PK/PD responses were examined over 60 days for a single 6 mg/kg intravenous dose of DISC-0974 in three male NHPs and in repeat dose studies occurring at 14 day intervals administered 3 times (vehicle, 0.6 and 3 mg/kg) or two times (60 mg/kg). In these studies, the serum iron response saturated in an exposure-dependent manner over the 0.6-60 mg/kg dose range. The onset of iron elevation lagged compared to the DISC-0974 maximal plasma concentrations by 1- 4 days post dose and persisted beyond what could be predicted by DISC-0974 plasma concentration alone. The observed PD responses were well-described by a delayed response PK/PD model of serum iron which was fit globally to all studies. We will present single and multiple dose simulations showing that low monthly doses of DISC-0974 are sufficient to elicit a biologically significant and controlled PD modulation (serum iron, TSAT, hepcidin) over a sustained period-of-time (Figure 1). DISC-0974 is under development for the treatment of anemia of inflammation associated with high hepcidin. Disclosures Blaustein: Disc Medicine:Current Employment, Current equity holder in private company.King:Disc Medicine:Current Employment, Current equity holder in private company.Nguyen:Disc Medicine:Current Employment, Current equity holder in private company.Savage:Disc Medicine:Current Employment, Current equity holder in private company.Beconi:Disc Medicine:Current Employment, Current equity holder in private company.

Hepcidin Peptidomimetics - Oral Efficacy in Pre-Clinical Disease Model of Iron Overload

Hepcidin peptidomimetics that are orally stable and systemically active will mark a paradigm change in management of blood disorders that exhibit aberrant iron homeostasis (e.g. hereditary hemochromatosis) and in conditions that can be influenced by modulating stressed iron homeostasis (e.g. polycythemia vera). Hepcidin modulates the iron exporter membrane protein ferroportin and is the master regulator of iron homeostasis in the body. Orally bioavailable "Minihepcidins" have been previously shown to be efficacious in lowering serum iron in mice when dosed peroral (PO) (Preza GC et. al., Journal of Clinical Investigation 2011). Here we describe hepcidin mimetic peptides that are metabolically stable in the gastrointestinal tract, systemically absorbed when delivered orally, and pharmacodynamically active in reducing serum iron parameters in pre-clinical models. Further, we also demonstrate improvement in disease parameters in a mouse model for hereditary hemochromatosis. The oral peptides, PN20076 and PN20089, have EC50 of 16.5 nM and 1.39 nM respectively in cell based ferroportin internalization assay (Table 1). In comparison EC50 was 67.8 nM for Hepcidin and 6.12 nM for PTG-300. (PTG-300 is an injectable hepcidin mimetic currently in Phase 2 clinical studies for polycythemia vera and hereditary hemochromatosis.) Oral stability of the peptides was evaluated in a panel of assays, including in vitro matrices simulating the gastric and intestinal conditions, and ex vivo matrices of serum/plasma from different species. Table 1 shows data for peptides PN20018, PN20076 and PN20089. PN20076 demonstrated extended stability in gastric and intestinal conditions, and degradation half-life of &amp;gt;24 hr in mouse plasma and 14.8 hr in rat serum. Based on their stability and potency data from the above battery of screening assays, the peptides were selected for in vivo evaluation in healthy mice to characterize their pharmacodynamic (PD) and pharmacokinetic (PK) properties. PN20076 and PN20089 showed equivalent PD response of reduction in serum iron concentration in wild type mice. After two successive PO doses of PN20076 or PN20089 approximately 24 hr apart, serum iron concentration was reduced from ~30 µM to ~10 µM (group averages), i.e. ~66% reduction, at 4.5 hr post-second dose for both peptides (Fig. 1). At 4.5 hr post-dose, the serum concentration of PN20076 was ~262 nM. PN20076 was further evaluated for its effect in lowering iron overload in a mouse model for hemochromatosis (HFE2-/- with homozygous deletion of hemojuvelin, a positive regulator of hepcidin expression). This mouse model is marked by hyper-absorption of dietary iron, higher transferrin saturation and deposition of excessive iron in liver, all manifestations of aberrant iron homeostasis caused by the genetic disruptions of the hepcidin-iron pathway. Liver iron accumulation was significantly prevented in groups treated with PN20076 once daily (QD) by PO administration for over two weeks, as compared to vehicle treated controls (Fig. 2). The reduction in non-heme iron concentration in liver homogenates (measured using a colorimetric iron assay) was statistically significant in the female group treated with PN20076. We have described orally stable and systemically active hepcidin mimetic peptides and demonstrated oral activity in preventing liver iron overload in hemochromatosis mice. The effective reduction of iron absorption from the diet and the steady state lowering of transferrin-saturation can potentially prevent tissue iron toxicity in hereditary hemochromatosis. Similarly, the sustained reduction of systemic iron levels with an oral hepcidin mimetic to control stressed iron homeostasis should reduce excessive erythrocytosis, a hallmark of polycythemia vera and other congenital and acquired erythropoietic disorders. Disclosures Bourne: Protagonist Therapeutics: Current Employment, Other: shareholder. Zhang:Protagonist Therapeutics: Current Employment, Other: shareholder. Frederick:Protagonist Therapeutics: Current Employment, Other: shareholder. Tran:Protagonist Therapeutics: Current Employment, Other: shareholder. Vengalam:Protagonist Therapeutics: Current Employment, Current equity holder in private company. McMahon:Protagonist Therapeutics: Current Employment, Other: shareholder. Huie:Protagonist Therapeutics: Current Employment, Other: shareholder. Ledet:Protagonist Therapeutics: Current Employment, Other: shareholder. Zhao:Protagonist Therapeutics: Current Employment, Other: shareholder. Tovera:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Lee:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yang:Protagonist Therapeutics: Current Employment, Other: shareholder. Dion:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yuan:Protagonist Therapeutics: Current Employment, Other: shareholder. Zemede:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Nguyen:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Masjedizadeh:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Cheng:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Mattheakis:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Liu:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Smythe:Protagonist Therapeutics: Current Employment, Other: shareholder.

DISC-0974, a Novel, First-in-Class, Anti-Hemojuvelin Monoclonal Antibody Decreases Hepcidin and Increases Transferrin Saturation in a Non-Human Primate Model of Cytokine (IL-6) Induced Hypoferremia

In diseases characterized by chronic inflammation, hepcidin levels are often elevated causing reduced transferrin-bound iron, reduced erythroid iron availability, diminished erythropoiesis and anemia despite the presence of adequate iron stores. Hemojuvelin (HJV) is a key selective regulator of hepcidin production in humans; patients with homozygous loss-of-function in the gene coding for HJV exhibit dramatically reduced hepcidin levels and a severe iron overload syndrome, juvenile hemochromatosis Type 2A. DISC-0974 is a humanized monoclonal anti-HJV antibody with high binding affinity for human, rat and cynomolgus monkey HJV (100 pM, 240 pM and 110 pM Kd, respectively) [Kovac 2016]. DISC-0974 inhibits the interaction between HJV and the key hepcidin regulatory ligands, bone morphogenetic proteins (BMPs), leading to decreased SMAD phosphorylation (SMAD-P) and reduced hepcidin expression [Kovak 2016]. The multiple-dose pharmacokinetics and pharmacodynamics (PK/PD) of DISC-0974 was first evaluated in healthy NHPs in which regulation of iron metabolism is primarily controlled by BMP/SMAD regulation of hepcidin synthesis. Dose levels were selected to modulate the pharmacodynamic response (transferrin saturation [TSAT]) but to be either below saturation (0.6 mg/kg), or to saturate the TSAT response (3 and 60 mg/kg). As expected from the mechanism of action, hepcidin concentrations decreased following DISC-0974 administration with return to baseline that was exposure dependent. The decrease in hepcidin was associated with increased TSAT, which DISC-0974 modulated in a dose-dependent manner. At 0.6 mg/kg, the TSAT response was maintained below saturation (~65%) after the first dose; saturation increased following subsequent doses, consistent with higher DISC-0974 serum concentrations. At the 3.0 and 60 mg/kg doses TSAT saturation (&amp;gt;85%) was achieved within 5 days post first dose and remained elevated through the end of the study, also consistent with the serum concentrations of DISC-0974. In vivo efficacy of DISC-0974 was evaluated in an NHP model of inflammation-induced (IL-6) high-hepcidin to establish whether inhibition of BMP/SMAD signaling could effectively control hepcidin increases generated by inflammatory signals. In this study, monkeys were challenged with 6×104 IU IL-6/kg on Day 1 and on Day 4. Groups of N=3 received 0 (vehicle), 0.6 or 6 mg/kg DISC-0974. On Day 11 all groups received a second challenge of 6×104 IU IL-6/kg. TSAT, hepcidin-25 and DISC-0974 concentrations were determined. DISC-0974 was effective in preventing IL-6-induced hepcidin increase in a dose-dependent manner in this model (Figure 1). In summary, in healthy NHPs, administration of DISC-0974 causes reduction in hepcidin levels leading to release of stored iron from macrophages of the reticuloendothelial system, making iron available for erythropoiesis as demonstrated by the dose-dependent modulation of TSAT. DISC-0974 is also effective in preventing hepcidin increases caused by IL-6, which is generally regarded at the classical cytokine stimulator of hepcidin synthesis. Given the similarity of the mechanism of hepcidin regulation between NHPs and humans, DISC-0974 is anticipated to provide a similar effect in the treatment of anemia of inflammation in humans by lowering cytokine-induced increases in hepcidin and improving iron availability for erythropoiesis. Figure Disclosures MacDonald: Disc Medicine: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Blaustein:Disc Medicine: Current Employment, Current equity holder in private company. Nguyen:Disc Medicine: Current Employment, Current equity holder in private company. King:Disc Medicine: Current Employment, Current equity holder in private company. Hong:Disc Medicine: Current Employment, Current equity holder in private company. Savage:Disc Medicine: Current Employment, Current equity holder in private company. Venkatraman:Disc Medicine: Current Employment, Current equity holder in private company. Beconi:Disc Medicine: Current Employment, Current equity holder in private company.

Characterization of INCB00928, a Potent and Selective ALK2 Inhibitor for the Treatment of Anemia

A significant population of patients with myelofibrosis (MF) develop anemia and either require red blood cell (RBC) transfusions or have an inadequate response to the currently available therapies and become transfusion-dependent. In patients with MF, elevated levels of serum hepcidin, a key iron regulatory hormone, is associated with increased dependence on RBC transfusions and reduced overall survival. Elevated hepcidin expression has also been observed to cause severe functional iron deficiency anemia and is central to the pathophysiology of anemia of chronic disease. Thus, to ensure proper maintenance of iron homeostasis, hepcidin levels are tightly regulated. Specifically, the production of hepcidin is controlled by the bone morphogenetic protein (BMP) type I receptor ACVR1, a gene that encodes the serine/threonine kinase ALK2. In preclinical models, knockdown or complete loss of ALK2 decreases hepcidin production resulting in elevated serum iron levels. In this study, we report characterization of INCB00928, a novel small molecule inhibitor of ALK2 for the treatment of anemia. INCB00928 was observed to have subnanomolar activity against ALK2 and selectivity over ALK1 and ALK3 in biochemical enzyme assays. In cell-based profiling studies, INCB00928 inhibited ALK2 potently and selectively over ALK1 and ALK3 as determined by the inhibition of ligand-induced SMAD pathway signaling. Importantly, in both an immortalized human liver cell line as well as primary human hepatocytes, INCB00928 inhibited BMP-induced production of hepcidin with nanomolar activity. INCB00928 was also observed to have suitable absorption, distribution, metabolism, and excretion properties to be dosed in in vivo rodent studies. In tumor- and inflammation-induced mouse models of anemia, INCB00928 improved RBC count, hemoglobin, and hematocrit levels while decreasing hepcidin levels in a dose-dependent manner. Additionally, consistent with the improved symptoms of anemia, pSMAD1/5 inhibition was observed in a dose-dependent manner in liver tissues collected from INCB00928-treated mice. In summary, INCB00928 is a potent, selective, and orally available small molecule inhibitor of ALK2, which significantly reduces the production of hepcidin in human liver cells, primary hepatocytes, and in rodent models of anemia. For the majority of patients with MF, the management of anemia remains an unmet need. The preclinical findings from this study suggest ALK2 kinase inhibition with INCB00928 may be a promising novel treatment to reduce the production of hepcidin and improve MF-related anemia in humans, thus warranting further investigation. Disclosures Chen: Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Stubbs:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Pusey:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Wen:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Collins:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Kapilashrami:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Rupar:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Thekkat:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Lin:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Bowman:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Yang:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Diamond:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Yeleswaram:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Kim:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Koblish:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Chen:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Wee:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company.