Comparative analysis of oral and intravenous iron therapy in rat models of inflammatory anemia and iron deficiency

Anemia is a major health issue and associated with increased morbidity. Iron deficiency anemia (IDA) is the most prevalent, followed by anemia of chronic disease (ACD). IDA and ACD often co-exist, challenging diagnosis and treatment. While iron supplementation is the first-line therapy for IDA, its optimal route of administration and the efficacy of different repletion strategies in ACD are elusive. Female Lewis rats were injected with group A streptococcal peptidoglycan-polysaccharide (PG-APS) to induce inflammatory arthritis with associated ACD and/or repeatedly phlebotomized and fed with a low iron diet to induce IDA, or a combination thereof (ACD/IDA). Iron was either supplemented by daily oral gavage of ferric maltol or by weekly intravenous (i.v.) injection of ferric carboxymaltose for up to 4 weeks. While both strategies reversed IDA, they remained ineffective to improve hemoglobin (Hb) levels in ACD, although oral iron showed slight amelioration of various erythropoiesis-associated parameters. In contrast, both iron treatments significantly increased Hb in ACD/IDA. In ACD and ACD/IDA animals, i.v. iron administration resulted in iron trapping in liver and splenic macrophages, induction of ferritin expression and increased circulating levels of the iron hormone hepcidin and the inflammatory cytokine interleukin-6, while oral iron supplementation reduced interleukin-6 levels. Thus, oral and i.v. iron resulted in divergent effects on systemic and tissue iron homeostasis and inflammation. Our results indicate that both iron supplements improve Hb in ACD/IDA, but are ineffective in ACD with pronounced inflammation, and that under the latter condition, i.v. iron is trapped in macrophages and may enhance inflammation

On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver

Iron is an essential component of the erythrocyte protein hemoglobin and is crucial to oxygen transport in vertebrates. In the steady state, erythrocyte production is in equilibrium with erythrocyte removal1. In various pathophysiological conditions, however, erythrocyte life span is severely compromised, which threatens the organism with anemia and iron toxicity2,3. Here we identify an on-demand mechanism that clears erythrocytes and recycles iron. We show that Ly-6Chigh monocytes ingest stressed and senescent erythrocytes, accumulate in the liver via coordinated chemotactic cues, and differentiate to ferroportin 1 (FPN1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1+ Tim-4neg macrophages are transient, reside alongside embryonically-derived Tim-4high Kupffer cells, and depend on Csf1 and Nrf2. The spleen likewise recruits iron-loaded Ly-6Chigh monocytes, but these do not differentiate into iron-recycling macrophages due to the suppressive action of Csf2. Inhibiting monocyte recruitment to the liver leads to kidney and liver damage. These observations identify the liver as the primary organ supporting rapid erythrocyte removal and iron recycling and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity

Preclinical Modeling of ACVR1-Dependent Hepcidin Production and Anemia By Momelotinib

Abstract Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Elevated levels of hepcidin in MF patients suggest that deregulated iron homeostasis is a driver of anemia in MF (Pardanani et al., Am J Hematol, 2013). A phase 2 study in MF patients demonstrated that momelotinib (MMB) treatment resulted in improvement of anemia (Pardanani et al., Blood, 2013, ASH Suppl). We have demonstrated that in addition to inhibiting JAK1/2, MMB also inhibits the bone morphogenetic protein receptor kinase, activin A receptor type 1 (ACVR1), a key regulator of hepcidin production in hepatocytes (Asshoff et al., Blood, 2015, ASH Suppl). This work supports a model by which the improvement of anemia observed in MF patients with MMB treatment results from the inhibition of ACVR1-mediated hepcidin expression in the liver, increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis. Here we demonstrate that MMB treatment in anemic rats resulted in a reduction of hepcidin and amelioration of anemia; whereas ruxolitinib dosing affected neither hepcidin nor anemia. Short-term MMB oral treatment in anemic rats resulted in a dose responsive inhibition of serum hepcidin induction with complete suppression at 25 mg/kg QD, 82% inhibition at 10 mg/kg QD, and 55% inhibition at 5 mg/kg QD (Table 1). Interestingly, only the two higher doses show significant time above EC50 levels, whereas the 5 mg/kg dose (which most closely approximates MMB human clinical exposures) only shows significant time above EC20. This suggests that systemic drug concentrations alone may not accurately predict activity in the model as 5 mg/kg demonstrated a 55% reduction in serum hepcidin. In a rat quantitative whole body autoradiography tissue distribution study, MMB levels have been demonstrated to be 3-4 fold higher in the liver relative to blood (Figure 1). Thus, the significant inhibiton of hepcidin levels in animals dosed with 5 mg/kg MMB may be due to higher concentrations in the liver. MMB concentrations would be expected to be higher in the liver relative to blood in humans as well. Our results provide preclinical evidence for a direct role of MMB in regulating iron homeostasis through the ACVR1/hepcidin axis. Evaluation of the activity of MMB on the ACVR1/hepcidin axis is being done in ongoing MMB clinical studies in patients with MF. Table 1 MMB exposure levels and % inhibition of serum hepcidin induction in ACD rats. Human (healthy volunteer) and ACD rat exposure levels for MMB. Table 1. MMB exposure levels and % inhibition of serum hepcidin induction in ACD rats. Human (healthy volunteer) and ACD rat exposure levels for MMB. Figure 1 Tissue distribution of radioactivity in rats following a single oral dose of [14C]-MMB. LOQ is 4, 159 ng-eq/g. 24h rat blood levels were below LOQ. Figure 1. Tissue distribution of radioactivity in rats following a single oral dose of [14C]-MMB. LOQ is 4, 159 ng-eq/g. 24h rat blood levels were below LOQ. Disclosures Warr: Gilead Sciences: Employment, Equity Ownership. Zheng:Gilead Sciences: Employment, Equity Ownership. Sharma:Gilead Sciences: Employment, Equity Ownership. Maciejewski:Gilead Sciences: Employment, Equity Ownership. Theurl:Gilead Sciences: Research Funding. Whitney:Gilead Sciences: Employment, Equity Ownership. </jats:sec

The Jak1/Jak2 Inhibitor Momelotinib Inhibits Alk2, Decreases Hepcidin Production and Ameliorates Anemia of Chronic Disease (ACD) in Rodents

Abstract Many patients with myelofibrosis (MF) develop anemia and are, or become, dependent on frequent red blood cell transfusions [1]. Results from the phase 2 studies for the treatment of myelofibrosis (MF) with the Jak1/2 inhibitor momelotinib (MMB) demonstrated that MMB treatment provided an anemia benefit, in addition to providing a durable spleen response and improvement in constitutional symptoms in MF patients [2]. MMB's anemia benefit was an unexpected outcome as erythropoietin (EPO)-mediated JAK2 signaling is essential for erythropoiesis [3, 4]. Systemic iron homeostasis is controlled by the peptide hormone hepcidin produced by the liver. Hepcidin reduces iron export from duodenal enterocytes and splenic and hepatic macrophages by binding to and down-regulating the iron exporter ferroportin [5-7]. In chronic disease, there is a significant increase in hepcidin levels which can result in severe anemia; this pathologic condition is termed anemia of chronic disease (ACD). Recently, MF patients have been shown to have elevated serum hepcidin levels and this increase is associated with inferior overall survival in these patients [8]. This study aimed to determine whether MMB's clinical anemia benefit is driven by direct activity of MMB on the hepcidin pathway. We assayed MMB inhibitory activity on the BMP-receptor kinase pathway (the central driver of hepcidin transcription in hepatocytes) and assessed the activity of MMB in a rodent model of anemia of chronic disease (ACD). We demonstrate that MMB inhibits BMP6-induced in vitro production of hepcidin in cultured hepatocytes (HepG2 cells) with an EC50 = 651 +/- 203 nM (n=3). This inhibitory activity is mediated by direct suppresion of the BMP-receptor kinase Alk2 as MMB inhibits Alk2 enzymatic activity with an IC50 = 8.4 +/- 1.5 nM (n=3). Ruxolitinib has no activity on either Alk2 or the BMP-receptor kinase pathway. To understand whether MMB could modulate hepcidin levels in vivo and ameliorate anemia in vivo we assessed the effect of MMB in a peptidoglycan-polysaccharide fragment (PG-APS)-induced rat ACD model. Treatment with clinically relevant exposure levels of MMB for 3 days resulted in a dose dependent reduction in both liver RNA and serum protein hepcidin levels and caused an increase in serum iron. Furthermore, long-term treatment with MMB for 21 days increased the numbers and percent of reticulocytes and mature red blood cells in the bone marrow and increased Hgb and hematocrit to normal levels in the blood. Our data suggest that MMB's clinical anemia benefit results from inhibition of ALK2-mediated expression of hepcidin in the liver, which results in increased release of iron from sequestered cellular stores and enhanced erythropoiesis. A phase 2 translational study in anemic subjects with MPNs is scheduled to confirm this mechanism. The Alk2-mediated activity on iron metabolism through hepcidin could prove beneficial in a number of additional indications, and facilitate the combination of MMB with myelosuppressive agents. 1. Tefferi, A., et al., One thousand patients with primary myelofibrosis: the mayo clinic experience. Mayo Clin Proc, 2012. 87 (1): p. 25-33. 2. Pardanani, A., et al., Update On The Long-Term Efficacy and Safety Of Momelotinib, a JAK1 and JAK2 Inhibitor, For The Treatment Of Myelofibrosis. Vol. 122. 2013. 108-108. 3. Verstovsek, S., et al., Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med, 2010. 363 (12): p. 1117-27. 4. Verstovsek, S., et al., A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012. 366 (9): p. 799-807. 5. Nemeth, E., et al., Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 2004. 306 (5704): p. 2090-3. 6. Theurl, I., et al., Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease. Blood, 2006. 107 (10): p. 4142-8. 7. Theurl, I., et al., Autocrine formation of hepcidin induces iron retention in human monocytes. Blood, 2008. 111 (4): p. 2392-9. 8. Pardanani, A., et al., Associations and prognostic interactions between circulating levels of hepcidin, ferritin and inflammatory cytokines in primary myelofibrosis. Am J Hematol, 2013. 88 (4): p. 312-6. Disclosures Warr: Gilead Science: Employment. Maciejewski:Gilead Science: Employment. Fowles:Gilead Science: Employment. Whitney:Gilead Sciences: Employment. Theurl:Gilead Science: Research Funding. </jats:sec

A Fully Human Anti-BMP6 Antibody Reduces the Need for Erythropoietin Stimulating Agent in Two Rodent Anemia of Chronic Disease Models

Abstract Anemia of chronic disease (ACD) is the most common cause of anemia in hospitalized patients. The underlying pathophysiological mechanisms are manifold, including reduced Erythropoietin (EPO) availability and sensitivity, direct negative effects of inflammatory cytokines on erythropoiesis and functional iron deficiency due to iron restriction mainly in the reticuloendothelial system (RES). The latter can be ascribed to increased hepcidin levels, a small liver derived peptide that has been found to be the key iron regulator. Up to date, most patients suffering from ACD are treated with a combination of both ESA (Erythropoietin Stimulating Agent) and intravenous (i.v.) iron to maintain hemoglobin (Hb) levels. Despite this combination therapy a significant number of patients require increasing doses of i.v. iron and ESA during their medical history - often resulting in continuous, potential toxic iron overload and ESA doses that exceed the acceptable range. As hepcidin is central to the iron-restrictive phenotype in ACD, several therapeutic approaches of hepcidin modulation have been investigated to overcome iron overload and EPO resistance. Some of these therapies are currently investigated in early clinical trials. We here report the effects of a fully human anti-BMP6 antibody on anemia, iron metabolism, erythropoiesis and ESA dosing in two different, well established rodent models of ACD. As BMPs, mainly BMP2 and BMP6, have been reported to be involved in hepcidin control, with knock out mice showing very low hepcidin levels even in an inflammatory setting, a fully human anti-BMP6 antibody has been developed to suppress hepcidin levels. We tested the antibody treatment in two distinct ACD animal models: First, a rat arthritis model (PG-APS in Lewis rats) and second, a Chronic Kidney Disease (CKD) mouse anemia model (Adenine model in C57BL/6 mice). Both models present with long-lasting anemia as seen in humans suffering from ACD. Mice and rats were treated with different doses of the anti-BMP6 antibody with and without ESA. Whole blood count, serum iron parameters (including hepcidin), bone marrow erythropoiesis determined by FACS analysis, cytokine expression and iron metabolism gene expression in spleen, liver and kidney were analyzed. Anti-BMP6 as well as ESA monotherapy resulted in a net increase in Hb level but only anti-BMP6 treatment significantly increased MCV and MCH, which can be ascribed to effective iron mobilization. In contrast, ESA therapy raised Hb levels by increasing red blood cell numbers. Of note, mere i.v. iron supplementation (sodium ferric gluconate), even at high doses, was not able to restore Hb levels to the same extent as the anti-BMP6 monotherapy. Strikingly, combination of both, ESA and anti-BMP6 treatment, had a synergistic effect on Hb levels, especially in the rat PG-APS model. Combination therapy of low ESA doses that only had a modest effect as a monotherapy, led to a dramatic increase in Hb levels, even exceeding those seen in healthy rats. Based on these results, additional experiments were performed to investigate the potential of this combination treatment to reduce ESA doses. Indeed, when anti-BMP6 was combined with a significantly reduced total ESA dose Hb levels were corrected to normal values in disease animals. Anti-BMP6 treatment also led to a significant decrease of iron deposition in the spleen with no iron deposition in parenchymal organs, indicating that the freed-up iron was effectively used for erythropoiesis and not just distributed elsewhere. In summary, anti-BMP6 therapy worked synergistically with ESA treatment in two different models of ACD leading to significantly increased Hb levels, a reduced ESA need as well as reduced iron overload in the RES. Furthermore, these experiments clearly show that treatment of ACD, being a complex multifactorial disease, benefits from using a combination of diversified approaches to overcome anemia and significantly reduce the dose of each therapeutic. Disclosures Wake: Kymab Ltd.: Employment. Bayliss:Kymab Ltd.: Employment. Papworth:Kymab Ltd.: Employment. Carvalho:Kymab Ltd.: Employment. Deantonio:Kymab Ltd.: Employment. Weiss:Kymab Ltd.: Consultancy. Germaschewski:Kymab Ltd.: Employment. Theurl:Kymab Ltd.: Consultancy, Research Funding. </jats:sec

Comparative analysis of oral and intravenous iron therapy in rat models of inflammatory anemia and iron deficiency

Anemia is a major health issue and associated with increased morbidity. Iron deficiency anemia (IDA) is the most prevalent, followed by anemia of chronic disease (ACD). IDA and ACD often co-exist, challenging diagnosis and treatment. While iron supplementation is the first-line therapy for IDA, its optimal route of administration and the efficacy of different repletion strategies in ACD are elusive. Female Lewis rats were injected with group A streptococcal peptidoglycan-polysaccharide (PG-APS) to induce inflammatory arthritis with associated ACD and/or repeatedly phlebotomized and fed with a low iron diet to induce IDA, or a combination thereof (ACD/IDA). Iron was either supplemented by daily oral gavage of ferric maltol or by weekly intravenous (i.v.) injection of ferric carboxymaltose for up to 4 weeks. While both strategies reversed IDA, they remained ineffective to improve hemoglobin (Hb) levels in ACD, although oral iron showed slight amelioration of various erythropoiesis-associated parameters. In contrast, both iron treatments significantly increased Hb in ACD/IDA. In ACD and ACD/IDA animals, i.v. iron administration resulted in iron trapping in liver and splenic macrophages, induction of ferritin expression and increased circulating levels of the iron hormone hepcidin and the inflammatory cytokine interleukin-6, while oral iron supplementation reduced interleukin-6 levels. Thus, oral and i.v. iron resulted in divergent effects on systemic and tissue iron homeostasis and inflammation. Our results indicate that both iron supplements improve Hb in ACD/IDA, but are ineffective in ACD with pronounced inflammation, and that under the latter condition, i.v. iron is trapped in macrophages and may enhance inflammation.</jats:p

Mitochondrial Respiration in Response to Iron Deficiency Anemia: Comparison of Peripheral Blood Mononuclear Cells and Liver

Iron is an essential component for metabolic processes, including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity, and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA), which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and the liver. Furthermore, we evaluated whether the mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to the controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and the liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue.</jats:p

Mitochondrial Respiration in Response to Iron Deficiency Anemia: Comparison of Peripheral Blood Mononuclear Cells and Liver

Iron is an essential component for metabolic processes, including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity, and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA), which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and the liver. Furthermore, we evaluated whether the mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to the controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and the liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue