Hepcidin Regulation Research Articles

Preterm Piglets Born by Cesarean Section as a Suitable Animal Model for the Study of Iron Metabolism in Premature Infants.

Preterm infants are most at risk of iron deficiency. However, our knowledge of the regulation of iron homeostasis in preterm infants is poor. The main goal of our research was to develop and validate an animal model of human prematurity to assess iron status in preterm infants. We performed a cesarean section on sows on the 109th day of pregnancy, which corresponds to the last trimester of human pregnancy. Preterm piglets showed decreased body weight, red blood cell indices, plasma iron level and transferrin saturation. Interestingly, higher hepatic and splenic non-heme iron content and plasma and hepatic ferritin levels were found in premature piglets compared with term ones. In addition, premature piglets showed higher mRNA levels of iron-regulatory hormone hepcidin in the liver than term animals, which have not been reflected in higher plasma hepcidin-25 levels. We also showed changes in hepcidin regulators, including hepatic bone morphogenetic protein 6, plasma erythroferrone and growth differentiation factor 15 in preterm piglets. Consequently, no difference was observed in iron-exporter ferroportin levels in the spleen and liver. Overall, it seems that premature piglets show a pattern of iron metabolism characteristic of functional iron deficiency and iron accumulation in the tissue.

Hepcidin: A missing link at the interface of malaria and hypertension

Malaria and hypertension are seemingly unrelated communicable and non-communicable diseases, both highly pervasive in the global south. There is a debate about the potential connection between these two disorders beyond any obvious factors. The emerging evidence suggests an emerging genetic selection pressure for hypertension in malaria-endemic regions. Furthermore, the impact of the liver hormone hepcidin, which has been overlooked, is significant in the development of hypertension due to malaria. Malaria exposure leads to changes in iron metabolism through the regulation of hepcidin, resulting in iron deficiency and, consequently, the development of hypertension.

Determining the expression of vitamin D receptor, regulator of iron metabolism hepcidin and cathelicidin antimicrobial peptide genes for development of future diagnostic and therapeutic options in patients with inflammatory bowel disease.

Patients with inflammatory bowel diseases (IBD) frequently experience malnutrition and develop nutrients deficiencies such as vitamin D or iron. Collectively, 39 patients were recruited to the study - 32 with IBD and 7 patients constituting the control group (CG). Quantitative real-time polymerase chain reaction was used to assess the expression of vitamin D receptor (VDR), hepcidin (HAMP) and cathelicidin antimicrobial peptide (CAMP) in colon mucosa. The mean expression of VDR and CAMP was higher in patients with ulcerative colitis than other groups (VDR vs. CG, p<0.05). However, the mean HAMP expression reached higher values in CG than in groups with IBD. Further research to understand the relationship between the VDR, HAMP and CAMP may constitute the way for development of future diagnostic and therapeutic options in patients with IBD.

The Intersection of Hepcidin and Polycystic Ovary Syndrome: A Review of Current Understanding

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting up to 13% of reproductive-age women. It is characterized by a constellation of symptoms including menstrual irregularities, hyperandrogenism, and polycystic ovaries, often intertwined with metabolic disturbances such as insulin resistance and dyslipidemia. Hepcidin is a liver-produced peptide hormone that regulates iron homeostasis, with a significant role in the pathophysiology of PCOS. Hepcidin controls iron absorption and storage by binding to ferroportin, leading to its degradation and reducing iron export from enterocytes and macrophages. In PCOS patients, hepcidin dysregulation is linked to underlying hormonal and metabolic abnormalities, including insulin resistance and hyperandrogenism. Studies consistently demonstrate decreased hepcidin levels in PCOS patients, resulting in altered iron metabolism parameters, such as increased serum iron and ferritin concentrations, leading to iron overload. This iron overload can exacerbate complications like anemia. The relationship between hepcidin, anemia, and PCOS is influenced by several mechanisms. Hyperandrogenism in PCOS inhibits hepcidin synthesis, reducing iron sequestration and increasing serum iron levels, contributing to erythropoiesis and potentially mitigating anemia, while also posing a risk of iron overload. Hyperinsulinemia associated with insulin resistance further decreases hepcidin levels, enhancing dietary iron absorption and increasing serum iron levels. Despite the chronic low-grade inflammation typical in PCOS, which usually increases hepcidin levels, the inflammation may not be sufficient to override the suppressive effects of hyperandrogenism and hyperinsulinemia on hepcidin expression. Oligomenorrhea or amenorrhea in PCOS leads to reduced menstrual blood loss, contributing to iron retention and further influencing hepcidin regulation. Genetic and epigenetic factors also significantly impact hepcidin expression and iron metabolism in PCOS.

Foxo1 is an iron-responsive transcriptional factor regulating systemic iron homeostasis

AbstractThe liver plays a crucial role in maintaining systemic iron homeostasis by secreting hepcidin, which is essential for coordinating iron levels in the body. Imbalances in iron homeostasis are associated with various clinical disorders related to iron deficiency or iron overload. Despite the clinical significance, the mechanisms underlying how hepatocytes sense extracellular iron levels to regulate hepcidin synthesis and iron storage are not fully understood. In this study, we identified Foxo1, a well-known regulator of macronutrient metabolism, which translocates to the nucleus of hepatocytes in response to high-iron feeding, holo-transferrin, and bone morphogenetic protein 6 (BMP6) treatment. Furthermore, Foxo1 plays a crucial role in mediating hepcidin induction in response to both iron and BMP signals by directly interacting with evolutionally conserved Foxo binding sites within the hepcidin promoter region. These binding sites were found to colocalize with Smad-binding sites. To investigate the physiological relevance of Foxo1 in iron metabolism, we generated mice with hepatocyte-specific deletion of Foxo1. These mice exhibited reduced hepatic hepcidin expression and serum hepcidin levels, accompanied by elevated serum iron and liver nonheme iron concentrations. Moreover, high-iron diet further exacerbated these abnormalities in iron metabolism in mice lacking hepatic Foxo1. Conversely, hepatocyte-specific Foxo1 overexpression increased hepatic hepcidin expression and serum hepcidin levels, thereby ameliorating iron overload in a murine model of hereditary hemochromatosis (Hfe−/− mice). In summary, our study identifies Foxo1 as a critical regulator of hepcidin and systemic iron homeostasis. Targeting Foxo1 may offer therapeutic opportunities for managing conditions associated with aberrant iron metabolism.

Understanding iron homeostasis in MDS: the role of erythroferrone.

Myelodysplastic neoplasms (MDS) are a heterogenous group of clonal stem cell disorders characterized by dysplasia and cytopenia in one or more cell lineages. Anemia is a very common symptom that is often treated with blood transfusions and/or erythropoiesis stimulating factors. Iron overload results from a combination of these factors together with the disease-associated ineffective erythropoiesis, that is seen especially in MDS cases with SF3B1 mutations. A growing body of research has shown that erythroferrone is an important regulator of hepcidin, the master regulator of systemic iron homeostasis. Consequently, it is of interest to understand how this molecule contributes to regulating the iron balance in MDS patients. This short review evaluates our current understanding of erythroferrone in general, but more specifically in MDS and seeks to place in context how the current knowledge could be utilized for prognostication and therapy.

Regulation of iron metabolism in HEC-1A endometrium cells by macrophage-derived factors and fractalkine.

Macrophages in the endometrium promote receptivity and implantation by secreting proinflammatory cytokines and other factors like fractalkine (FKN). Macrophages are closely linked to regulating iron homeostasis and can modulate iron availability in the tissue microenvironment. It has been revealed that the iron metabolism of the mother is crucial in fertility. Iron metabolism is strictly controlled by hepcidin, the principal iron regulatory protein. The inflammatory cytokines can modulate hepcidin synthesis and, therefore, the iron metabolism of the endometrium. It was proven recently that FKN, a unique chemokine, is implicated in maternal-fetal communication and may contribute to endometrial receptivity and implantation. In the present study, we investigated the effect of activated THP-1 macrophages and FKN on the iron metabolism of the HEC-1A endometrial cells. We established a noncontact coculture with or without recombinant human FKN supplementation to study the impact of the macrophage-derived factors and FKN on the regulation of hepcidin synthesis and iron release and storage of endometrial cells. Based on our findings, the conditioned medium of the activated macrophages could modify hepcidin synthesis via the nuclear factor kappa-light-chain-enhancer of activated B cells, the signal transducer and activator of transcription 3, and the transferrin receptor 2/bone morphogenetic protein 6/suppressor of mothers against decapentaplegic 1/5/8 signaling pathways, and FKN could alter this effect on the endometrial cells. It was also revealed that the conditioned macrophage medium and FKN modulated the iron release and storage of HEC-1A cells. FKN signaling may be involved in the management of iron trafficking of the endometrium by the regulation of hepcidin. It can contribute to the iron supply for fetal development at the early stage of the pregnancy.

Ochratoxin A Induces Renal Cell Ferroptosis by Disrupting Iron Homeostasis and Increasing ROS.

Mycotoxin ochratoxin A (OTA) is a critical food safety concern due to its nephron-toxic effects and is detected in a wide range of food and feedstuffs. OTA nephrotoxicity is related to oxidative stress and damage. However, the mediator(s) of the excessive oxidative stress is unclear. The current study used human kidney cell lines to investigate whether and how intracellular iron contributed to OTA-induced ROS accumulation and how OTA-induced iron-dependent ferroptotic cell death. Our results showed that OTA treatment affected the cell viability and induced the typical characteristics of cell ferroptosis. Furthermore, gene and protein expression results indicated that OTA disrupted iron homeostasis by upregulating the expression levels of iron importer TFR1 and FTH, while downregulating the expression level of iron exporter FPN and dramatically increasing its negative regulator Hepcidin. The changes were consistent with the induction of intracellular iron accumulation and elevated levels of oxidative stress and lipid peroxidation. Additionally, co-treatment with OTA and an iron chelator significantly improved cell viability, reduced cellular total iron and ROS, and reversed OTA-induced changes in iron metabolism gene expression levels. Interestingly, the addition of a ROS scavenger also reversed cell death and changes in mRNA and protein expression levels of iron metabolism genes but to a lesser degree than that of the iron-chelating agent. Our results revealed that OTA induced ferroptosis in renal cells by disrupting iron homeostasis and increasing ROS.

Serum Erythroferrone Levels in a Large General Population: Towards a Better Understanding of Connections between Erythropoiesis and Iron Homeostasis

Introduction: Iron is essential for various biological functions [ Ganz T., Physiol Rev 2013], especially for erythropoiesis which is the most iron-consumer process daily. Hepcidin is the key hormone of iron homeostasis, preventing both iron deficiency and excess, by modulating iron absorption, recycling, and mobilization [ Camaschella C. et al, Haematologica 2020]. Erythroferrone (ERFE) constitutes the principal erythroid regulator of hepcidin: in the presence of enhanced erythropoiesis, expanding erythroblasts produce ERFE which, by impeding bone morphogenetic proteins (BMPs) signaling in hepatocytes, suppress hepcidin, in turn increasing iron availability for the bone marrow [ Ganz T., Free Rad Biol Med 2019]. Increased ERFE levels have been demonstrated in expanded/ineffective erythropoiesis (IE) (e.g., in thalassemia intermedia), where chronic ERFE-mediated hepcidin suppression may cause progressive iron accumulation even in subjects not regularly transfused. In view of this, ERFE has been proposed as a potential biomarker of IE and an appealing target for treating iron overload deriving from IE [ Srole D.N. and Ganz T., J Cell Physiol 2020]. However, studies that fully elucidate the role of ERFE in the general population under physiological conditions are still lacking. Our project evaluated age- and sex-related variations in ERFE levels, and their correlations with hematological and iron status parameters, in a large subpopulation from the Cooperative Health Research In South Tyrol (CHRIS) study. Subjects and methods: The CHRIS study is a population-based study carried out in South Tyrol (Northern Italy) aimed at investigating acquired and genetic basis of common chronic conditions associated with aging [ Pattaro C. et al, J Transl Med 2015]. Blood samples were tested for several biochemical and molecular parameters, including those related to iron status, such as ferritin, transferrin saturation (TSat), soluble transferrin receptor (sTfR), hepcidin, and C282Y and H63D mutations. Serum ERFE was measured in stored aliquots by the Human Erythroferrone IETM ELISA kit (Intrinsic Lifesciences - The BioIron Company TM, USA), according to manufacturer instructions. Results: We measured ERFE in 3688 subjects aged &amp;gt;18 years (55% female). A total of 777 subjects (21% of the whole population; 57% females) had ERFE concentrations below the lower limit of detection for our method (we assigned them an arbitrary value of 0.16 for statistical analyses). Figure 1 illustrates age- and sex-dependent variations of ferritin, sTfR, hepcidin, and ERFE in the whole population. After stratification by hemoglobin (Hb) levels, we observed that ERFE was significantly higher in anemic (i.e., Hb&amp;lt;12 g/dl in females and &amp;lt;13 g/dl in males; n=100) than in non-anemic subjects (respectively 3.74 and 0.65 ng/ml; p&amp;lt;0.0001). In anemic subjects, ERFE showed a strong negative correlation with Hb (r = -0.5; p &amp;lt; 0.001), TSat (r = -0.58; p &amp;lt; 0.001), ferritin (r = -0.48; p &amp;lt; 0.001) and hepcidin (r = -0.32; p = 0.001), and a positive correlation with sTfR (r = 0.58; p &amp;lt; 0.001). Moreover, ERFE was higher in iron-deficient (ID) subjects (i.e., ferritin &amp;lt;30 mg/l; n=773) than in non-ID subjects (0.92 vs. 0.63 ng/ml; p&amp;lt;0.0001). Although ID subjects had lower Hb levels than non-ID subjects (13.7 vs 15 g/dl; p&amp;lt;0.0001), Hb levels were broadly within the normal range.To better explore the relationship between ERFE and iron status, we divided the population into 4 subgroups, according to sTfR and ERFE levels. Considering the skewed distribution of the variables, we used sTfR above or below the 3° tertile (1316 ng/ml), in order to identify subjects having higher or lower iron needs, respectively. Analogously, we further divided the population in subjects with ERFE above or below the 3° tertile (1.21 ng/ml). As illustrated in Figure 2, after stratification, we observed that hepcidin reduced, in subjects with high ERFE levels, when iron requirement was high (0.94 vs 2.10 nM; p &amp;lt; 0.0001), but it didn't when iron requirement was low (3.35 vs. 3.05 nM; p = 0.72), even in presence of normal Hb levels (Hb 14.3 g/dl). Discussion: our data confirmed an association among ERFE, Hb and iron status parameters under conditions of anemia (when erythropoiesis is stimulated); moreover, if confirmed in further studies, it could suggest an effect of ERFE on hepcidin regulation could be linked to depletion of iron stores, even before anemia develops.

Single Ascending Doses of REGN7999, a Monoclonal Antibody Inhibitor of TMPRSS6, Increase Serum Hepcidin and Cause Deep, Sustained Reductions in Serum Iron in Healthy Human Volunteers

Background: Iron overload (IOL) develops in dyserythropoieses such as beta-thalassemia and myelodysplastic syndromes due both to red blood cell transfusions as well as inappropriate regulation of the hormone hepcidin, a key regulator of iron homeostasis. Hepcidin, produced in the liver, sequesters iron mainly in enterocytes and macrophages. In the setting of hepcidin down-regulation, toxic levels of labile iron can accumulate in organs such as the heart, liver, bone marrow, pancreas, and pituitary gland. The current standard-of-care for the treatment of IOL is iron chelation. Chelation solubilizes iron for renal excretion, but is associated with toxicities and limited patient satisfaction, severely limiting adoption and efficacy in certain disorders of IOL. There is a high unmet need for novel therapies which can safely reduce, and even prevent, toxic iron accumulation in organs in patients with IOL. Transmembrane serine protease 6 (TMPRSS6) is a membrane-bound serine protease whose expression is highly restricted to hepatocytes, where its only known role is inhibiting hepcidin transcription by cleaving the hemojuvelin co-receptor and thus reducing the bone morphogenetic protein (BMP) signaling pathway driving hepcidin production. Aims: REGN7999, an investigational monoclonal antibody targeting TMPRSS6, is being developed for the treatment of IOL. We have conducted a phase 1, first-in-human, placebo-controlled, single ascending dose study in healthy volunteers (NCT05481333) to explore the safety, tolerability, pharmacokinetics, and pharmacologic effects associated with REGN7999 administration. Methods: Healthy participants at a single center were randomized 6:2 to receive R7999 or placebo in 5 ascending IV and 3 ascending SC cohorts. Key inclusion criteria included hemoglobin, hematocrit, serum iron, and transferrin saturation at or above the lower limit of normal for age and sex. Female participants were required to be non-menstruating. The primary objective of the study was to identify the safety and tolerability of single doses of REGN7999. Key secondary and exploratory objectives included characterizing the drug concentration profile of REGN7999, and evaluating the effects of REGN7999 on serum biomarkers of iron homeostasis. Data reported uses the cutoff date of June 13, 2023. Results: In all cohorts, a total of 64 subjects were exposed. REGN7999 was well-tolerated, with no serious adverse events reported, and no participants developed anemia. Single doses of REGN7999 (ranging from 10 to 900 mg) resulted in acute 2-fold to 12-fold increases in serum hepcidin among all the REGN7999 dose cohorts, as well as acute 50-70% reductions from baseline in serum iron with the longest sustained effects (up to 7 weeks) in the highest IV and SC dose cohorts. Conclusion: Single doses of REGN7999 are well tolerated by healthy volunteers and lead to sustained reductions in serum iron with no associated safety concerns. These findings support continued development of REGN7999 for the treatment of IOL.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

Maternal prenatal, with or without postpartum, vitamin D3 supplementation does not improve maternal iron status at delivery or infant iron status at 6 months of age: secondary analysis of a randomised controlled trial

BackgroundVitamin D may modify iron status through regulation of hepcidin and inflammatory pathways. This study aimed to investigate effects of maternal vitamin D supplementation on iron status in pregnancy and...

Hepcidin's Antimalarial Arsenal: Safeguarding the Host

Malaria remains a significant global health threat, causing hundreds of thousands of deaths annually. Understanding the host's protective mechanisms against this parasitic infection is crucial for developing effective prevention and treatment strategies. Hepcidin, a central regulator of iron homeostasis, has emerged as a key player in the host's defense against malaria. This comprehensive review explores the multifaceted roles of hepcidin in safeguarding the host from malaria infection. We delve into the molecular mechanisms of hepcidin regulation and its impact on the intricate interplay between iron, the immune system, and Plasmodium species. Additionally, we discuss the potential therapeutic implications of targeting hepcidin to enhance antimalarial responses. By shedding light on hepcidin's antimalarial arsenal, this review aims to contribute to the development of innovative strategies for combating malaria. Keywords: Hepcidin, antimalaria, malaria and host

Estrogens and Prolactin Do Not Regulate Maternal and Embryo Iron Homeostasis during Pregnancy

Iron is essential for maternal and fetal health but the molecular mechanisms ensuring increased iron availability during pregnancy are not well understood. Hepcidin is the key iron-regulatory hormone and functions by blocking iron absorption, recycling and mobilization from stores, effectively decreasing plasma iron levels. In healthy human and rodent pregnancies, maternal hepcidin decreases starting in the second trimester and is nearly undetectable by late pregnancy. This decrease in hepcidin allows for greater iron availability for transfer to the fetus. If hepcidin is not appropriately suppressed during pregnancy, it results in fetal anemia, intrauterine growth restriction, and even fetal demise in severe cases 1. Unrelated to pregnancy, hormones estrogen and prolactin have been previously implicated in hepcidin regulation 2,3, although with conflicting results on the direction of hepcidin change 4-6. The levels of both hormones increase dramatically over the course of pregnancy, thus we hypothesized that they may modulate iron homeostasis during pregnancy. Studying the role of estrogens and prolactin during pregnancy is challenging as both hormones are required for pregnancy. Additionally, mice with global knockout (KO) of either estrogen receptor alpha or prolactin receptor are infertile. Thus, to determine the contribution of estrogens and prolactin on maternal hepcidin suppression during pregnancy, we generated liver-specific knockouts of either estrogen receptor alpha ( Er- α f/f;Alb-Cre +) or prolactin receptor ( Prlr f/f;Alb-Cre +), here abbreviated as ER-α KO and PRLR KO. Female ER-α KO, PRLR KO and WT mice were set up for timed pregnancy, and liver hepcidin mRNA, as well as maternal and embryo iron and hematological parameters were measured at E18.5 . Hepcidin suppression in ER-α KO and PRLR KO dams was not different from that in WT dams, suggesting that estrogens and prolactin are not major mediators of maternal hepcidin suppression. Additionally, maternal and embryo hematological parameters and liver iron concentrations were similar between WT, ER-α KO, PRLR KO pregnancies, further supporting our observation that estrogens and prolactin do not regulate iron homeostasis in pregnancy. The mechanisms by which maternal hepcidin is suppressed during pregnancy remain to be determined. 1Sangkhae V et al, Blood, 2020 2Yang Q et al, Endocrinology, 2012 3Wang J et al, Nanomedicine: nanotechnology, biology and medicine, 2015 4Zhen et al, Hepatology, 2013 5Ikeda Y et al, PloS one, 2012 6Lehtihet M et al, PloS one, 2016

ALK2 and JAK2 Inhibition for Improved Treatment of Anemia in Myelofibrosis Patients: Preclinical Profile of an ALK2 Inhibitor Zilurgisertib in Combination with Ruxolitinib

Introduction: Anemia is a common occurrence in patients with myelofibrosis (MF) and is associated with the need for red blood cell transfusion and poor clinical prognosis. JAK inhibitors such as ruxolitinib are used extensively to treat symptoms of MF and improve quality of life and overall survival. However, JAK inhibitors may also contribute to myelosuppression. Therapeutic interventions that allow for optimization of JAK inhibition with no concern of anemia would benefit patients with MF and potentially lower the rate of treatment discontinuation or suboptimal dosing. Recent studies have demonstrated that inhibition of ACVR1/ALK2, a bone morphogenetic protein receptor that is upstream of the transcriptional regulation of hepcidin, could reduce serum hepcidin levels in patients with MF and improve anemia (Verstovsek S et al. Lancet 2023; Oh S et al. Clin Lymphoma Myeloma Leuk 2022). Reducing levels of hepcidin, a key regulator of plasma iron levels, and restoring erythropoiesis would broadly benefit patients with MF. Based on these recent data, we developed zilurgisertib, a potent and selective ALK2 inhibitor that could be administered in combination with ruxolitinib at doses titrated to the needs of patients with MF. Methods and Results: In biochemical and cellular assays, zilurgisertib inhibited ALK2 kinase activity and SMAD1/5 phosphorylation with IC 50 values of 15 nM and 63 nM, respectively. In Huh-7 cells stimulated with BMP-6, zilurgisertib inhibited hepcidin production with an IC 50 of 20 nM, demonstrating the compound is a potent ALK2 inhibitor capable of regulating iron homeostasis via hepcidin. To assess possible off-target effects of zilurgisertib, kinome profiling at 10 μM ATP was performed at Reaction Biology (Malvern, PA) to determine the overall specificity across 356 kinases (see Figure). At 200 nM, zilurgisertib only inhibited ALK2, ALK1 (to 50%), and ALK6 (to 48%). In addition, zilurgisertib at 20 μM did not affect viability of HEK293 cells, a human cell line commonly used to assess general cell health and compound toxicity. Similarly, zilurgisertib did not affect viability of human fibroblasts or endothelial cells at concentrations up to 5 μM. To test whether the combination of ruxolitinib and zilurgisertib could suppress hepcidin and restore erythropoiesis in an in vivo mouse model of cancer-induced anemia, B16F10 cells were injected intraperitoneally, mimicking a metastatic tumor that leads to anemia 1 week after injection. In a dose-dependent manner, zilurgisertib improved hemoglobin by 2 to 3 g/dL and red blood cell counts, while reducing both liver pSMAD and circulating hepcidin levels by 50% or more compared with vehicle control. The combination of zilurgisertib with ruxolitinib had no effect on this activity, suggesting that inhibition of JAK2 does not inhibit erythropoiesis restored following ALK2 inhibition. Conclusion: Taken together, the potent and selective on-target activity of zilurgisertib suggests that ALK2 inhibition could reduce hepcidin and improve anemia, and that the combination of zilurgisertib with ruxolitinib is a rational and attractive approach to mitigate anemia in patients with MF. The combination of ruxolitinib and zilurgisertib is currently being evaluated in a phase 1 clinical trial in patients with anemia due to myeloproliferative disorders (NCT04455841).

Phase 1/2 Study of the Activin Receptor-like Kinase-2 Inhibitor Zilurgisertib (INCB000928, LIMBER-104) As Monotherapy or with Ruxolitinib in Patients with Anemia Due to Myelofibrosis

Introduction:Elevated levels of the iron regulator hepcidin can cause functional iron deficiency anemia. Hepcidin dysregulation is central to anemia of chronic inflammation observed in several malignancies including myelofibrosis (MF). Activin receptor-like kinase-2 (ALK2 [ie, ACVR1]) contributes to MF-associated anemia via hepcidin upregulation. We evaluated the safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of zilurgisertib-a potent and selective oral ALK2 inhibitor-in patients (pts) with anemia due to MF. Methods:This ongoing open-label, multicenter, phase 1/2 dose-escalation/expansion INCB 00928-104 study (NCT04455841) is evaluating zilurgisertib alone (treatment group A [TGA]) or with ruxolitinib (RUX; treatment group B [TGB]). Eligible pts are ≥18 years old with primary/secondary MF of intermediate (Int)-1 (TGB only) or Int-2/high-risk (TGA and TGB) per the Dynamic International Prognostic Scoring System (DIPSS) and with anemia, including pts who are transfusion dependent (≥4 units of red blood cell transfusions during the 28 days or 8 weeks before Cycle [C] 1 Day [D] 1 for hemoglobin [Hb] &amp;lt;8.5 g/dL in the absence of bleeding or treatment-induced anemia) or are nontransfusion dependent with symptomatic anemia (Hb &amp;lt;10 g/dL during screening on 3 separate occasions ≥7 days apart and not meeting criteria for transfusion dependence). The zilurgisertib starting dose in TGA was 50 mg once daily (qd), with dose increases of ≤2-fold performed until a grade ≥2 toxicity with reasonable probability of being related to the treatment group was observed; subsequent dose increases were limited to ≤50% until the maximum tolerated dose (MTD) was reached or the recommended doses(s) for expansion were identified. In TGB, pts were on a stable regimen of RUX for ≥12 weeks before initiating zilurgisertib at 100 mg qd. The primary endpoints are safety and tolerability, including determination of dose-limiting toxicities (DLTs) and MTD. Secondary endpoints include efficacy (per anemia response parameters), PK, and PD (hepcidin measurement). Results:A total of36 pts were enrolled at the time of analysis (data cutoff, February 15, 2023), with 20 in TGA and 16 in TGB. Median (range) age was 73 (53-84) years for TGA and 75.5 (54-85) years for TGB; 65% of TGA pts had received prior RUX therapy. Overall, 89% had Int-2 risk DIPSS, and the remainder had high-risk DIPSS (1 in TGA, 3 in TGB). In total, 55% of pts in TGA and 25% in TGB were transfusion dependent at time of enrollment. Median (range) baseline Hb was 7.7 (7-10) g/dL in TGA and 8.0 (5-9) g/dL in TGB. Baseline median (range) hepcidin was high in both cohorts (TGA, 171 [18-535] ng/mL; TGB, 126 [7-421] ng/mL; normal range, 0-50 ng/mL). At the time of analysis, dose escalation was ongoing in both treatment groups. No DLTs occurred in either treatment group, and the MTD had not been reached. Treatment-emergent adverse events (TEAEs) were mainly low grade without apparent dose dependence. Grade ≥3 TEAEs occurred in 11 pts, with thrombocytopenia the most common (n=4). The PK profile for zilurgisertib at steady state was consistent with that observed with previous healthy volunteer studies, with observed time of maximum concentration at 2-4 hours after administration and predicted half-life of approximately 24 hours. Maximum reduction in hepcidin following zilurgisertib dosing in C1D1 was observed at 6-8 hours postdose; the highest doses tested in both TGA and TGB demonstrated more effective maintenance of hepcidin suppression over time compared with lower doses. Among non-transfusion-dependent pts, anemia improvement (Hb increase of ≥1.5 g/dL vs baseline) was observed in both treatment groups (Figure). Among transfusion-dependent pts, anemia response of transfusion independence had not been observed at data cutoff. All pts with anemia improvement have maintained Hb increase in the absence of transfusion and remain on study. Conclusions:Treatment with zilurgisertib monotherapy or in combination with RUX in this pt population was generally well tolerated, with predominantly grade 1/2 TEAEs. Reduced hepcidin levels were observed at all dose levels with both monotherapy and in combination with RUX, with greater control of hepcidin over time observed at higher zilurgisertib doses. Preliminary improvements in anemia were observed in non-transfusion-dependent pts during dose escalation, suggesting potential for therapeutic activity.

Ultrafine Particle-Induced Oxidation of Estrogen Receptor Alpha Prevents its Interaction with the Methyltransferase G9a and Dysregulation of the Iron Regulator Hepcidin

Ultrafine Particle-Induced Oxidation of Estrogen Receptor Alpha Prevents its Interaction with the Methyltransferase G9a and Dysregulation of the Iron Regulator Hepcidin

The efficiency and safety evaluation of hemoglobin hydrolysate as a non-heme iron fortifier

Hemoglobin hydrolysate is derived from the enzymatic degradation of hemoglobin. This work aimed to evaluate whether hemoglobin hydrolysate promotes the absorption of non-heme iron and the safety of absorbed iron in mice by analyzing the iron binding content, iron circulation, and liver homeostasis. We found that hemoglobin hydrolysate promoted the absorption of non-heme iron with high efficiency in duodenum by spontaneously binding non-heme iron during digestion, and increased hepatic iron content by up-regulating divalent metal transporter 1, zinc transporter 14, but hepatic iron content only increased at 3 weeks. Duodenal iron entered the blood through ferroportin without restriction at 3 weeks, and excessive iron entered the liver and then affected the hepatocyte membranes permeability and lipid synthesis through oxidative stress. With the prolongation of dietary intervention, the up-regulated hepcidin acted on the ferroportin to restrict excess iron from entering the blood, and then the hepatic homeostasis recovered. In addition, hemoglobin hydrolysate enhanced the hepatic antioxidant capacity. Taken together, hemoglobin hydrolysate has a strong ability to promote the absorption of non-heme iron in vivo, and the absorbed iron is relatively safe due to the regulation of hepcidin.

Features of regulation of hepcidin and ferroportin in cancer patients (literary review)

Background. The pathways of iron acquisition, outflow, storage and regulation are disrupted in cancer, which suggests that the reprogramming of iron metabolism is one of the central aspects of the survival of tumor cells.Aim. Is to review and generalize modern literature data on the regulation of hepcidin, ferroportin and prospects for the correction of iron metabolism in cancer patients.Materials and Methods. The paper presents the results of international and domestic studies of the peculiarities of iron metabolism and the prospects for its correction in cancer patients. The search for relevant sources was carried out in the web of Science, PubMed, Medline, eLibrary.ru systems for 1988–2023. Of the analyzed studies 61, the most relevant, were used to write a systematic review.Results. Over the past decade, a new understanding has emerged of the role of proteins, in particular hepcidin and ferroportin, which regulate cellular iron in cancer growth, angiogenesis and metastasis. New treatment methods with hepcidin-modifying strategies and stabilizers of hypoxia-induced factors are emerging, but their therapeutic efficacy for correcting iron metabolism in cancer patients needs to be evaluated and clinical trials.Conclusion. Analysis of the literature data has shown the high relevance of studies of the regulation of hepcidin and ferroportin in cancer patients and the need for further study of this problem.

Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in the dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct the iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.