Rusfertide, a Hepcidin Mimetic, for Control of Erythrocytosis in Polycythemia Vera.

BACKGROUND: Polycythemia vera is a chronic myeloproliferative neoplasm characterized by erythrocytosis. Rusfertide, an injectable peptide mimetic of the master iron regulatory hormone hepcidin, restricts the availability of iron for erythropoiesis. The safety and efficacy of rusfertide in patients with phlebotomy-dependent polycythemia vera are unknown. METHODS: In part 1 of the international, phase 2 REVIVE trial, we enrolled patients in a 28-week dose-finding assessment of rusfertide. Part 2 was a double-blind, randomized withdrawal period in which we assigned patients, in a 1:1 ratio, to receive rusfertide or placebo for 12 weeks. The primary efficacy end point was a response, defined by hematocrit control, absence of phlebotomy, and completion of the trial regimen during part 2. Patient-reported outcomes were assessed by means of the modified Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) patient diary (scores range from 0 to 10, with higher scores indicating greater severity of symptoms). RESULTS: Seventy patients were enrolled in part 1 of the trial, and 59 were assigned to receive rusfertide (30 patients) or placebo (29 patients) in part 2. The estimated mean (±SD) number of phlebotomies per year was 8.7±2.9 during the 28 weeks before the first dose of rusfertide and 0.6±1.0 during part 1 (estimated difference, 8.1 phlebotomies per year). The mean maximum hematocrit was 44.5±2.2% during part 1 as compared with 50.0±5.8% during the 28 weeks before the first dose of rusfertide. During part 2, a response was observed in 60% of the patients who received rusfertide as compared with 17% of those who received placebo (P = 0.002). Between baseline and the end of part 1, rusfertide treatment was associated with a decrease in individual symptom scores on the MPN-SAF in patients with moderate or severe symptoms at baseline. During parts 1 and 2, grade 3 adverse events occurred in 13% of the patients, and none of the patients had a grade 4 or 5 event. Injection-site reactions of grade 1 or 2 in severity were common. CONCLUSIONS: In patients with polycythemia vera, rusfertide treatment was associated with a mean hematocrit of less than 45% during the 28-week dose-finding period, and the percentage of patients with a response during the 12-week randomized withdrawal period was greater with rusfertide than with placebo. (Funded by Protagonist Therapeutics; REVIVE ClinicalTrials.gov number, NCT04057040.).

Administration of dietary recombinant hepcidin on grass carp (Ctenopharyngodon idella) against Flavobacterium columnare infection under cage aquaculture conditions.

Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare-challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture.

The Effect of Hepcidin on Cardiac Ischemia-Reperfusion Injury.

Background/aim: Hepcidin is the main hormone in the regulation of iron metabolism which is also released from the heart. The aim of our study was to investigate the effects of hepcidin on the cardiac ischemia-reperfusion injury.Materials and methods: In this study, 12 Wistar albino rats were divided into two groups (n = 6 each): 1) The ischemia-reperfusion group (Group 1); 2) Hepcidin-treated group (Group 2). Rat hearts were perfused on Langendorff system with KH (Krebs-Henseleit) and subjected to 30 min stabilization, 30 min global ischemia, and 30 min reperfusion. Hepcidin (- M) was applied to group 2 at the onset of ischemia. Malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NOx) levels were measured in heart tissue for NOx levels, viscosity, and ion content of perfusate were collected before ischemia and the 1st, 5th, 10th, 20th, and 30th minutes of reperfusion were determined. Apoptosis in heart was evaluated.Results: NOx and MDA levels significantly decreased in heart tissue in Hepcidin-treated group. NOx and viscosity of perfusate were not significantly different between the groups. Perfusate iron, calcium, magnesium, potassium, and sodium levels in group 2 were more homogeneous. Histologic structures of heart tissue were regularly in group 2. Apoptosis were increased in control group compared to hepcidin treated group.Conclusion: These results suggest that hepcidin may have a protective effect on the heart for the ischemia-reperfusion injury.

Chlorella sp. transgenic with Scy-hepc enhancing the survival of Sparus macrocephalus and hybrid grouper challenged with Aeromonas hydrophila.

Two marine antimicrobial peptides (AMPs), PC-hepc from large yellow croaker (Pseudosciaena crocea) and scygonadin from mud crab (Scylla serrata), are potently active against specific bacteria and thus they could be used as substitutes for antibiotics in aquaculture. However, how to utilize the AMPs feasibly for marine cultured animals has been so far confused. In our study, a 510 bp of the Scy-hepc sequence was cloned into pMDC85 expression vector, which was then electroporated into Chlorella sp., and thus a transgenic Chlorella, in which the Scy-hepc gene was effectively expressed, was developed. The Scy-hepc fusion protein was successfully expressed in Chlorella sp. and it showed obvious bactericidal activity. In addition, the in vivo efficacy of the transgenic Chlorella was evaluated using Sparus macrocephalus and the hybrid Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂). Results showed that the survival rate of S. macrocephalus fed with transgenic Chlorella (80 ± 10% after 72 h) was significantly higher than that of fish fed with the same dosage of wild-type Chlorella (33.33 ± 11.55% after 72 h). Similarly, results showed that the survival rate of the hybrid grouper fed with transgenic Chlorella (55 ± 5% after 36 h) was much higher than that of fish fed with the same dosage of wild-type Chlorella (25 ± 5% after 36 h). Therefore, in vitro and in vivo results indicated that the constructed transgenic Chlorella with the marine AMPs Scy-hepc could exert effective protection for fish against the Aeromonas hydrophila infection, providing an encouraging prospect for the expected use of transgenic Chlorella in aquaculture in future.

Hemocromatosis: a changing world.

Due to major advances in the understanding of iron metabolism as well as in the bioche- iuical, imaging, and genetic domains: i) The nosologicalframework of hemochromatosis (HC) encompasses not only HFE-HC, by far the most frequent HC form, but also non-HFE HC diseases which comprise essentially juvenile HC and the ferroportin disease. ii) The diagnostic approach has become totally non invasive, based on clinical, imaging and biological data. iii) The treatment remains, for most forms, based on venesections, but the innovative emerging therapeutic approach is represented by hepcidin supplementation.

The Use of a Liposomal Formulation Incorporating an Antimicrobial Peptide from Tilapia as a New Adjuvant to Epirubicin in Human Squamous Cell Carcinoma and Pluripotent Testicular Embryonic Carcinoma Cells.

This study aims to explore the effects and mechanisms of hepcidin, a potential antimicrobial peptide from Tilapia, and epirubicin (Epi), an antineoplastic agent, on the generation of reactive oxygen species (ROS) and link the ROS levels to the reversal mechanisms of multidrug resistance (MDR) by epirubicin and hepcidin in human squamous cell carcinoma SCC15 and human embryonal carcinoma NT2D1 cells. The cells, pretreated with hepcidin, epirubicin, or a combination of these compounds in PEGylated liposomes, were used to validate the molecular mechanisms involved in inhibiting efflux transporters and inducing apoptosis as evaluated by cytotoxicity, intracellular accumulation, mRNA levels, cell cycle distribution, and caspase activity of this combination. We found that hepcidin significantly enhanced the cytotoxicity of epirubicin in liposomes. The co-incubation of epirubicin with hepcidin in liposomes intensified the ROS production, including hydrogen peroxide and superoxide free radicals. Hepcidin significantly increased epirubicin intracellular uptake into NT2D1 and SCC15 cells, as supported by the diminished mRNA expressions of MDR1, MDR-associated protein (MRP) 1, and MRP2. Hepcidin and/or epirubicin in liposomes triggered apoptosis, as verified by the reduced mitochondrial membrane potential, increased sub-G1 phase of cell cycle, incremental populations of apoptosis using annexin V/PI assay, and chromatin condensation. As far as we know, this is the first example showing that PEGylated liposomal TH1-5 and epirubicin gives rise to cell death in human squamous carcinoma and testicular embryonic carcinoma cells through the reduced epirubicin efflux via ROS-mediated suppression of P-gp and MRPs and concomitant initiation of mitochondrial apoptosis pathway. Hence, hepcidin in PEGylated liposomes may function as an adjuvant to anticancer drugs, thus demonstrating a novel strategy for reversing MDR.

[IRON OVERLOAD: BETTER UNDERSTANDING, BETTER CARE]. / Surcharges en fer: mieux comprises, mieux prises en charge.

Chronic iron overload, either of genetic (hemochromatoses) or acquired (transfusions) origin, leads to frequent disorders, affecting both the quality of life and life expectancy. Major recent advances in the knowledge of iron metabolism, together with advances in biology, imaging and drug design have already significantly improved the diagnostic and therapeutic approaches. These conceptual and technological ameliorations should, in the near future, continue to benefit the clinical management of iron overloaded patients.

Characterization and bioactivity of hepcidin-2 in zebrafish: dependence of antibacterial activity upon disulfide bridges.

Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9µM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.

A novel mutation in the SLC40A1 gene associated with reduced iron export in vitro.

Ferroportin disease is an inherited disorder of iron metabolism and is caused by mutations in the ferroportin gene (SLC40A1). We present a patient with hyperferritinemia, iron overload in the liver with reticuloendothelial distribution and also in the spleen, and under treatment with erythropheresis. A molecular study of the genes involved in iron metabolism (HFE, HJV, HAMP, TFR2, SLC40A1) was undertaken. In vitro functional studies of the novel mutation found in the SLC40A1 gene was performed. The patient was heterozygous for a novel mutation, c.386T>C (p.L129P) in the SLC40A1 gene; some of his relatives were also heterozygous for this mutation. In vitro functional studies of the L129P mutation on ferroportin showed it impairs its capacity to export iron from cells but does not alter its sensitivity to hepcidin. These findings and the iron overload phenotype of the patient suggest that the novel mutation c.386T>C (p.L129P) in the SLC40A1 gene has incomplete penetrance and causes the classical form of ferroportin disease.

Functional properties of human ferroportin, a cellular iron exporter reactive also with cobalt and zinc.

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-enhanced green fluorescent protein fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with (55)Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing (55)Fe efflux up to 300-fold over control. Ferroportin-mediated (55)Fe efflux was saturable, temperature-dependent (activation energy, Ea ≈ 17 kcal/mol), maximal at extracellular pH ≈ 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity constant < 10(-7) M. Ferroportin expression also stimulated efflux of (65)Zn and (57)Co but not of (64)Cu, (109)Cd, or (54)Mn. Hepcidin treatment of oocytes inhibited efflux of (55)Fe, (65)Zn, and (57)Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.