Barbra Sasu

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Hepcidin revisited, disulfide connectivity, dynamics, and structure.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys1–Cys8, Cys3–Cys6, Cys2–Cys4, and Cys5–Cys7. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS

INTRODUCTION Hepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation. An accurate assessment of human serum hepcidin is critical to understand its role in anemia. METHODS An LC-MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum. The method was validated to FDA criteria for bioanalytical assays. RESULTS The calibration curve was validated over the range of 2.5 to 500 ng/mL. Hepcidin was stable in serum for at least 16 h at room temperature, 90 days at -60 to -80 degrees C, and after three F/T cycles. Interday accuracy (% RE) and precision (%CV) were -11.2% and 5.6%, respectively at the LLOQ, and less than +/-7.0% and 9.2%, respectively for higher concentrations. The mean accuracy of quality control samples (5.00, 15.0, 100 and 400 ng/mL) in 21 analytical batches was between -0.7 and +2.1%, with mean precision between 5.1% and 13.4%. Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects, while the mean concentration was less than 10 ng/mL. Sepsis and chronic kidney disease patients had mean serum concentrations of 252 ng/mL (n=16, median 121 ng/mL) and 99 ng/mL (n=50, median 68 ng/mL), respectively. CONCLUSIONS A fully validated LC-MS/MS method has been described for the determination of hepcidin in human serum. This method was applied to the determination of hepcidin in over 1200 human samples.

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

Serum hepcidin but not prohepcidin may be an effective marker for anemia of inflammation (AI).

Anemia in cancer patients can result from a complex interaction of numerous factors including iron deficiency, inflammation, toxicity related to therapy and effect of cancer on the marrow. Determining effective anemia treatment can therefore be complex, requiring a combination of diagnostic tests. Research on iron metabolism has highlighted the importance of hepcidin and its potential role in development of anemia of inflammation (AI). Hepcidin is a peptide that controls iron flow, is induced by inflammation and is speculated to cause the sequestration of iron in patients with inflammation. In the present study, serum hepcidin concentration determined by LC-MS/MS was shown to correlate with inflammatory markers in patients with anemia of cancer (AoC). In the absence of a widely-available serum hepcidin detection assay, detection of prohepcidin using a commercial assay has been used for several years as a surrogate for measuring serum hepcidin concentration. Analysis of prohepcidin concentration did not reveal any correlation with hepcidin or with inflammatory markers in patient samples and our data suggest that prohepcidin may not be stable in serum. Algorithms to sub-classify AoC patients showed that hepcidin was strongly associated with the population subset with inflammation and without iron deficiency. Serum hepcidin concentrations may therefore be a good predictor of AI, useful in diagnosis of anemia etiology and in treatment determination.

Soluble Transferrin Receptor-1 Levels in Mice Do Not Affect Iron Absorption

Soluble transferrin receptor-1 (sTfR1) concentrations are increased in the plasma under two conditions that are associated with increased iron absorption, i.e. iron deficiency and increased erythropoiesis. To determine the possible role of sTfR1 as a signaling mechanism for iron absorption, a hydrodynamic gene transfer technique was established to express transfected plasmid constructs of human sTfR1 (hsTfR1) and murine sTfR1 (msTfR1) from the livers of C57BL/6 mice. Iron absorption, serum iron levels and hepcidin expression were then measured. The hydrodynamic gene transfer technique proved to be an effective approach to achieving sustained expression of sTfR1 in mice. Although expression of high levels of sTfR1 significantly increased serum iron levels, repeated experiments showed that neither hsTfR1 nor msTfR1 had any effect on iron absorption or hepcidin mRNA expression levels. Thus, despite its attractiveness as a potential modifier of iron absorption, sTfR1 levels do not exert a regulatory effect on iron absorption.

Oxidative folding of hepcidin at acidic pH

Hepcidin is a four disulfide 25‐residue peptide hormone which has a central role in the regulation of iron homeostasis. To support studies on hepcidin we have sought to establish reliable and robust synthetic methods for the preparation of correctly folded materials. While correctly‐folded hepcidin has good aqueous solubility, we have found that its direct synthetic precursor, linear (reduced) hepcidin peptide, is resistant to solubilization, prone to precipitation at pH ≥ 6, and thus difficult to fold efficiently. Attempts to directly fold either the crude or purified linear hepcidin peptide by air or DMSO oxidation methods under basic conditions were ineffective. However, addition of a glutathione redox pair system improved folding of purified linear hepcidin at mild basic pH (pH 7.5). Under acidic conditions, it was possible to oxidatively fold both crude and purified hepcidin using a polymer‐supported oxidizing strategy. Peptide precipitation was also avoided under acidic conditions. Isolated folding yields of human hepcidin under acidic polymer‐assisted conditions were superior to yields under basic folding conditions. These studies enabled identification of a reliable synthetic route for correctly‐folded hepcidin.

Comparison of Epoetin Alfa and Darbepoetin Alfa Biological Activity under Different Administration Schedules in Normal Mice

The unit of erythropoietic activity has long been the standard by which erythropoietic agents are judged, but the development of long-acting agents such as darbepoetin alfa has highlighted the shortcomings of this approach. To this point, we compared the in vivo activity of Epoetin alfa and darbepoetin alfa per microgram of protein core. Using the established mass-to-unit conversion for Epoetin alfa (1 µg ≅ 200 U), we then calculated darbepoetin alfa activity in units. Activity varied with treatment regimen (1 µg darbepoetin alfa ≅ 800 U for 3 times weekly dosing to 8,000 U for a single injection). This analysis reveals the inadequacy of evaluating darbepoetin alfa activity in terms of standard erythropoietic units. We therefore propose that for molecules with heightened biological activity, a more legitimate basis for comparison is the protein mass.

Serum hepcidin levels predict response to intravenous iron and darbepoetin in chemotherapy-associated anemia

To the editor: Patients undergoing chemotherapy for cancer frequently experience anemia, which may require red blood cell (RBC) transfusions.[1][1] The erythropoiesis-stimulating agents (ESAs) epoetin and darbepoetin reduce transfusion needs and increase hemoglobin levels in 40% to 70% of patients