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 >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.

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Expression of hepcidin in hereditary hemochromatosis: evidence for a regulation in response to the serum transferrin saturation and to non-transferrin-bound iron

Blood ◽

10.1182/blood-2002-11-3610 ◽

2003 ◽

Vol 102 (1) ◽

pp. 371-376 ◽

Cited By ~ 167

Author(s):

Sven G. Gehrke ◽

Hasan Kulaksiz ◽

Thomas Herrmann ◽

Hans-Dieter Riedel ◽

Karin Bents ◽

...

Keyword(s):

Iron Overload ◽

Serum Ferritin ◽

Iron Homeostasis ◽

Iron Status ◽

Hereditary Hemochromatosis ◽

Transferrin Saturation ◽

Serum Transferrin ◽

Transcript Levels ◽

Hepcidin Expression

Abstract Experimental data suggest the antimicrobial peptide hepcidin as a central regulator in iron homeostasis. In this study, we characterized the expression of human hepcidin in experimental and clinical iron overload conditions, including hereditary hemochromatosis. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we determined expression of hepcidin and the most relevant iron-related genes in liver biopsies from patients with hemochromatosis and iron-stain-negative control subjects. Regulation of hepcidin mRNA expression in response to transferrin-bound iron, non-transferrin-bound iron, and deferoxamine was analyzed in HepG2 cells. Hepcidin expression correlated significantly with serum ferritin levels in controls, whereas no significant up-regulation was observed in patients with hemochromatosis despite iron-overload conditions and high serum ferritin levels. However, patients with hemochromatosis showed an inverse correlation between hepcidin transcript levels and the serum transferrin saturation. Moreover, we found a significant correlation between hepatic transcript levels of hepcidin and transferrin receptor-2 irrespective of the iron status. In vitro data indicated that hepcidin expression is down-regulated in response to non-transferrin-bound iron. In conclusion, the presented data suggest a close relationship between the transferrin saturation and hepatic hepcidin expression in hereditary hemochromatosis. Although the causality is not yet clear, this interaction might result from a down-regulation of hepcidin expression in response to significant levels of non-transferrin-bound iron. (Blood. 2003;102:371-376)

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Cardiac Hepcidin Expression Associates with Injury Independent of Iron

American Journal of Nephrology ◽

10.1159/000449419 ◽

2016 ◽

Vol 44 (5) ◽

pp. 368-378 ◽

Cited By ~ 9

Author(s):

G. Fenna van Breda ◽

Lennart G. Bongartz ◽

Wenqing Zhuang ◽

Rachel P.L. van Swelm ◽

Jeanne Pertijs ◽

...

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Messenger Rna ◽

Iron Homeostasis ◽

Quantitative Polymerase Chain Reaction ◽

Cardiac Injury ◽

Tissue Growth ◽

Hepcidin Expression ◽

Sham Surgery ◽

Hepcidin Gene

Background: Hepcidin regulates systemic iron homeostasis by downregulating the iron exporter ferroportin. Circulating hepcidin is mainly derived from the liver but hepcidin is also produced in the heart. We studied the differential and local regulation of hepcidin gene expression in response to myocardial infarction (MI) and/or chronic kidney disease (CKD). We hypothesized that cardiac hepcidin gene expression is induced by and regulated to severity of cardiac injury, either through direct (MI) or remote (CKD) stimuli, as well as through increased local iron content. Methods: Nine weeks after subtotal nephrectomy (SNX) or sham surgery (CON), rats were subjected to coronary ligation (CL) or sham surgery to realize 4 groups: CON, SNX, CL and SNX + CL. In week 16, the gene expression of hepcidin, iron and damage markers in cardiac and liver tissues was assessed by quantitative polymerase chain reaction and ferritin protein expression was studied by immunohistochemistry. Results: Cardiac hepcidin messenger RNA (mRNA) expression was increased 2-fold in CL (p = 0.03) and 3-fold in SNX (p = 0.01). Cardiac ferritin staining was not different among groups. Cardiac hepcidin mRNA expression correlated with mRNA expression levels of brain natriuretic peptide (β = 0.734, p < 0.001) and connective tissue growth factor (β = 0.431, p = 0.02). In contrast, liver hepcidin expression was unaffected by SNX and CL alone, while it had decreased 50% in SNX + CL (p < 0.05). Hepatic ferritin immunostaining was not different among groups. Conclusions: Our data indicate differences in hepcidin regulation in liver and heart and suggest a role for injury rather than iron as the driving force for cardiac hepcidin expression in renocardiac failure.

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Aln-TMP: A Subcutaneously Administered RNAi Therapeutic Targeting Tmprss6 For The Treatment Of β-Thalassemia

Blood ◽

10.1182/blood.v122.21.2260.2260 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2260-2260 ◽

Cited By ~ 2

Author(s):

James Butler ◽

Shannon Fishman ◽

Tim Racie ◽

Julia Hettinger ◽

Brian R Bettencourt ◽

...

Keyword(s):

Gene Expression ◽

Iron Overload ◽

Serum Iron ◽

Total Serum ◽

Gene Suppression ◽

Ineffective Erythropoiesis ◽

Therapeutic Targeting ◽

Hepcidin Expression ◽

Secondary Iron Overload ◽

Hepcidin Gene

Abstract The b-Thalassemias are a group of hereditary blood disorders resulting from insufficient beta globin production, ultimately giving rise to the signature clinical sequelae associated with β-Thalassemia which includes anemia, ineffective erythropoiesis, and secondary iron overload. Previously, we demonstrated that intravenous administration of an siRNA targeting hepatic Tmprss6 expression significantly ameliorated the disease phenotype in the Hbbth3/+ mouse model of Thalassemia Intermedia (Blood. 2013; 14;121(7):1200-8). The Tmprss6 gene encodes for the protein Matriptase-2 which negatively regulates Hepcidin gene expression by cleaving the Hepcidin regulatory protein Hemojuvelin; RNAi-mediated suppression of Tmprss6 removes this negative regulator, ultimately leading to an increase in Hepcidin expression. Increased Hepcidin expression leads to a significant decrease in serum iron concentration and Transferrin Saturation (TfSat), which in the β-Thalassemia disease setting, corrects ineffective erythropoiesis, ameliorates anemia, and mitigates secondary iron overload. The role of Tmprss6 in iron metabolism has been extensively characterized in animal and human studies and, together with the observation in the Hbbth3/+ mouse, represents an attractive therapeutic target for the treatment of β-Thalassemia. To this end, we developed ALN-TMP, a subcutaneous RNAi therapeutic targeting hepatic Tmprss6 for the treatment of β-Thalassemia. ALN-TMP employs the GalNAc conjugate siRNA delivery platform that safely and effectively delivers siRNA to the liver for hepatic gene silencing. Preclinical animal data demonstrate ALN-TMP exhibits robust and durable dose-dependent gene suppression as single dose administration of ALN-TMP leads to > 80% Tmprss6 gene suppression for up to 3 weeks post-dose. This leads to concomitant increases in Hepcidin gene expression (>2x baseline levels) and subsequent decreases in total serum iron and TfSat (>50% decrease from baseline). The degree to which ALN-TMP modulates Hepcidin and serum iron mobilization is nearly identical to that observed in the previous Hbbth3/+ mouse studies and suggests ALN-TMP is a potent RNAi therapeutic with the potential of producing disease modifying effects in β-Thalassemia. Disclosures: Butler: Alnylam: Employment. Fishman:Alnylam: Employment. Racie:Alnylam Pharmaceutical, Inc: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Bettencourt:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Fitzgerald:Alnylam: Employment.

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Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice

Blood ◽

10.1182/blood-2011-03-339952 ◽

2011 ◽

Vol 118 (15) ◽

pp. 4224-4230 ◽

Cited By ~ 123

Author(s):

Andrea U. Steinbicker ◽

Thomas B. Bartnikas ◽

Lisa K. Lohmeyer ◽

Patricio Leyton ◽

Claire Mayeur ◽

...

Keyword(s):

Gene Expression ◽

Iron Overload ◽

Hereditary Hemochromatosis ◽

Bmp Signaling ◽

Type I ◽

Anemia Of Chronic Disease ◽

Hepcidin Expression ◽

Hepatic Expression ◽

Bmp Receptors ◽

Hepcidin Gene

Abstract Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

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

Blood ◽

10.1182/blood-2020-141720 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 47-48

Author(s):

Roopa Taranath ◽

Gregory Bourne ◽

Jie Zhang ◽

Brian Frederick ◽

Tran T Tran ◽

...

Keyword(s):

Mouse Model ◽

Iron Overload ◽

Polycythemia Vera ◽

Serum Iron ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Iron Concentration ◽

Transferrin Saturation ◽

Private Company ◽

Liver Iron

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 >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.

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Effect of High Levels of Growth Differentiation Factor 15 (GDF15) On Hepcidin Expression in Monocytes of β-Thalassemia Intermedia Patients.

Blood ◽

10.1182/blood.v114.22.4061.4061 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4061-4061

Author(s):

Kleber Yotsumoto Fertrin ◽

Carolina Lanaro ◽

Carla Fernanda Franco-Penteado ◽

Dulcinéia M Albuquerque ◽

Mariana R. B. Mello ◽

...

Keyword(s):

Gene Expression ◽

Mononuclear Cells ◽

Positive Control ◽

Beta Thalassemia ◽

Iron Loading ◽

Thalassemia Intermedia ◽

Control Groups ◽

Hepcidin Expression ◽

Healthy Control ◽

Hepcidin Gene

Abstract Abstract 4061 Poster Board III-996 Ineffective erythropoiesis in thalassemia has been associated with inappropriate suppression of hepatic synthesis of the key iron regulatory peptide hepcidin, leading to spontaneous iron overload. Hepcidin mRNA was found to be suppressed in hepatocyte cultures by high levels of growth differentiation factor 15 (GDF15) detected in sera from patients with thalassemic syndromes. GDF15 may inhibit hepcidin production by antagonizing positive regulatory cytokines such as bone morphogenic protein 6 (BMP6), shown to stimulate hepatic hepcidin expression in mouse models. Although mainly produced in the liver, human hepcidin production occurs to a lesser extent in circulating monocytes. Studies with monocytes in patients with anemia of chronic disease showed increased hepcidin expression and iron retention, but to the best of our knowledge, monocyte-derived hepcidin has not yet been characterized in iron-loading anemias such as beta-thalassemia intermedia (TI). We evaluated GDF15 plasmatic levels and correlated these to hemoglobin (Hb) levels, reticulocyte counts and gene expressions in monocytes from transfusion-independent, non-chelated TI patients, homozygous for the IVS-I-6 T→C mutation (n=18), healthy, age-matched controls with no iron deficiency or overload (n=10) and transfusion-independent sickle cell anemia (SCA) patients in steady state (n=5), as a positive control group in which hyperexpression of hepcidin in mononuclear cells has been previously demonstrated. Total RNA was extracted from monocytes isolated from peripheral blood mononuclear cells and determination of BMP6 and hepcidin gene expression was performed by Real Time Polymerase Chain Reaction. Plasma GDF15 levels were determined by ELISA. Mean TI patient Hb and serum ferritin levels were 7.05±0.21g/dL and 1846±346.4μg/L, respectively. Mean absolute reticulocyte count in TI was 163.9±21.5×103/mm3. Mean GDF15 plasma levels differences were statistically significant among TI, SCA and healthy control groups (8390±827, 1780±460 and 196±21pg/mL, p<0.0001, respectively). Hepcidin gene expression did not differ significantly between TI and healthy control groups (0.007±0.006 vs. 0.05±0.03, p>0.05, respectively) but was elevated in our positive control SCA patient group (0.56±0.20; p=0.04). BMP6 gene expression was significantly decreased in TI patients compared to healthy controls (1.17±0.15 vs. 0.51±0.11, p=0.01, respectively). GDF15 concentrations correlated positively with reticulocyte counts (r=0.47; p=0.007) and negatively with hemoglobin levels (r=-0.74; p<0.0001) and BMP6 gene expression (r=-0.62; p=0.006). Our data show very high GDF15 plasma levels in a relatively homogenous population of patients with iron overload secondary to beta-thalassemia intermedia. Correlation of GDF15 with hematimetric parameters reinforces its relation to the degree of erythropoietic activity in beta thalassemia due to ineffective erythropoiesis. In addition, our study demonstrates that monocyte-derived hepcidin, like its hepatic counterpart, is inappropriately suppressed in iron-overloaded beta thalassemia intermedia patients in the presence of increased GDF15 production, correlated to decreased levels of BMP6 expression. This supports the possibility that GDF15/BMP interaction regulates hepcidin production in monocytes and hepatocytes in a similar manner, and further studies of monocyte-derived hepcidin regulation may prove to be a suitable, non-invasive tool for the investigation of human liver-derived hepcidin pathways in thalassemia and other iron-loading anemias. Disclosures: No relevant conflicts of interest to declare.

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Role of BMP Signaling In the Anemia of Chronic Disease

Blood ◽

10.1182/blood.v116.21.2043.2043 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2043-2043

Author(s):

Andrea U. Steinbicker ◽

Ashley J. Vonner ◽

Chetana Sachidanandan ◽

Lisa Lohmeyer ◽

David T. Scadden ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Research Funding ◽

Critical Role ◽

Bmp Signaling ◽

Mrna Levels ◽

Anemia Of Chronic Disease ◽

Hepcidin Expression ◽

Hepcidin Gene ◽

Hepcidin Mrna

Abstract Abstract 2043 Introduction: Anemia of chronic disease (ACD) describes anemia associated with diverse chronic inflammatory, infectious, or neoplastic processes. These conditions are frequently associated with increased circulating levels of inflammatory cytokines such as interleukin 6 (IL-6). IL-6 regulates expression of the hormone hepcidin, which inhibits the release of iron from hepatocytes, macrophages, and enterocytes into the circulation. In addition to IL-6, hepcidin gene expression is known to be transcriptionally regulated by bone morphogenetic protein (BMP) signaling. Hypothesis: We hypothesized that BMP signaling is required for the induction of hepcidin gene expression by IL-6 and plays a critical role in the pathogenesis of ACD. Methods: We used a turpentine-dependent model of ACD in mice. Mice were challenged with weekly subcutaneous injections of turpentine, which induces anemia in an IL-6 dependent manner. This model was studied to determine hepcidin gene expression and rescue ACD using BMP inhibition. Moreover, we examined hepcidin gene expression in zebrafish injected with Pseudomonas aeruginosa, and in transgenic zebrafish overexpressing human IL-6. The regulation of hepcidin gene expression was also studied in the human hepatocarcinoma cell line (HepG2). Results: Injections of mice with IL-6 (0.8 μg/g ip) increased hepatic hepcidin mRNA levels expression at 24 hours and decreased serum iron concentrations. Both effects were prevented by a small molecule BMP type I receptor kinase inhibitor, LDN-193189, or protein BMP antagonists. Weekly turpentine injections induced microcytic anemia after 3 weeks with a decrease in hemoglobin levels from 12.8±0.3 to 9.7±1.7 g/dL (*p<0.01). Concurrent treatment with LDN-193189 prevented turpentine-induced anemia and microcytosis (*p<0.01 for both). In mice challenged with turpentine for 6 weeks, treatment with LDN-193189, beginning after anemia was established at week 3, led to an increase in hemoglobin levels at week 6 (10.9±0.1 vs 9.5±0.2 g/dL, LDN193189 vs vehicle, respectively; *p<0.05). In zebrafish, microinjection with Pseudomonas aeruginosa or overexpression of human IL-6 induced hepatic hepcidin expression, an effect which was blocked by LDN-193189. Incubation of HepG2 cells with IL-6 (100 ng/ml) increased hepcidin mRNA levels 2 to 5 fold. Pretreatment with LDN-193189, or recombinant protein BMP antagonists such as noggin, abrogated the induction of hepcidin expression by IL-6. Incubation of HepG2 cells with BMP6 (2.5 to 10 ng/ml) modestly increased hepcidin mRNA levels. However, the combination of IL-6 and BMP6 synergistically increased hepcidin gene expression (*p<0.05). Conclusion: BMP signaling appears to play a critical role in the pathogenesis of anemia in a mouse ACD model. Our findings support the concept that BMP signaling is required for the induction of hepcidin gene expression by IL-6 in vitro and in vivo. Moreover, manipulation of BMP signaling represents a potentially novel therapeutic approach to the treatment of anemia associated with inflammation. Disclosures: Steinbicker: Deutsche Forschungsgemeinschaft DFG: Research Funding. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Peterson:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Bloch:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Yu:Harvard Stem Cell Institute Seed Grant and the Howard Hughes Medical Institute Early Career Physician-Scientist Award: Honoraria, Research Funding; NHLBI 5K08HL079943: Research Funding.

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Target TMPRSS6 for the Treatment of Hereditary Hemochromatosis

Blood ◽

10.1182/blood.v118.21.1047.1047 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1047-1047

Author(s):

Sheri Booten ◽

Daniel Knox ◽

Luis Alvarado ◽

Shuling Guo ◽

Brett P. Monia

Keyword(s):

Iron Overload ◽

Genetic Disorder ◽

Hereditary Hemochromatosis ◽

Genetic Defect ◽

Transferrin Saturation ◽

Common Mutation ◽

C282y Mutation ◽

Iron Storage ◽

Hepcidin Expression ◽

Iron Deficient

Abstract Abstract 1047 Hereditary hemochromatosis (HH) is a genetic disorder in which hyperabsorption of dietary iron leads to accumulation of iron in multiple tissues including liver and heart. A common clinical manifestation in HH patients is cirrhosis and hepatocellular carcinoma as a result of iron-mediated injury in liver. The most prevalent genetic defect for HH is the failure to up-regulate hepcidin, a peptide hormone that inhibits the absorption of iron in duodenum and the release of iron from intracellular iron storage such as macrophages. Mutations in a number of genes have been identified as the cause for HH, including hepcidin itself. However, the most common mutation is C282Y mutation in HFE, which is a positive regulator for hepcidin expression. C282Y mutation represents about 85% of the HH population. HFE C282Y HH is an autosomal recessive disease with a ∼50% penetrance. Currently, the only treatment available for iron overload is phlebotomy which will continue throughout the patient's life. Hepcidin is mainly expressed and secreted by the liver and its expression is regulated predominantly at the transcription level. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor for hepcidin expression. It's been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of TMPRSS6 in Hfe−/− mice could ameliorate the iron overload phenotype (Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg KE et al., Blood, 2011). Using second generation antisense technology, we identified antisense oligonucleotides (ASOs) targeting mouse TMPRSS6 for the treatment of HH. These compounds were first identified through in vitro screens in mouse primary hepatocytes. After 4 weeks of treatment in C57BL/6 mice on normal chow, we observed an 80% to 90% reduction of liver TMPRSS6 mRNA with a subsequent 2–3 fold induction of liver hepcidin mRNA. Serum iron and transferrin saturation levels were reduced by ∼50%. These ASOs are currently being evaluated in a diet-induced iron overload model and an Hfe−/− iron overload model. Our preliminary results demonstrate that targeting TMPRSS6 is a viable approach for the treatment of hereditary hemochromatosis and possibly other iron-loading diseases associated with suppressed hepcidin levels. Disclosures: Booten: Isis Pharmaceuticals: Employment. Knox:Isis Pharmaceuticals: Summer Intern. Alvarado:Isis Pharmaceuticals: Employment. Guo:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals: Employment.

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RNAi-Mediated Inhibition of Tmprss6 Elevates Hamp1 Expression and Reduces Serum Iron Levels in Mice

Blood ◽

10.1182/blood.v118.21.1045.1045 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1045-1045 ◽

Cited By ~ 1

Author(s):

Ivanka Toudjarska ◽

Zuhua Cai ◽

Tim Racie ◽

Stuart Milstein ◽

Brian R Bettencourt ◽

...

Keyword(s):

Iron Overload ◽

Serum Iron ◽

Iron Concentration ◽

Transferrin Saturation ◽

Elevated Serum ◽

High Serum ◽

Therapeutic Modality ◽

Deficiency Anemia ◽

Loss Of Function ◽

Hepcidin Expression

Abstract Abstract 1045 The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality. Disclosures: Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.

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Target TMPRSS6 Using Antisense Technology for the Treatment of Hereditary Hemochromatosis and β-Thalassemia

Blood ◽

10.1182/blood.v120.21.481.481 ◽

2012 ◽

Vol 120 (21) ◽

pp. 481-481 ◽

Cited By ~ 1

Author(s):

Shuling Guo ◽

Carla Casu ◽

Sara Gardenghi ◽

Sheri Booten ◽

Andy Watt ◽

...

Keyword(s):

Treatment Group ◽

Iron Overload ◽

Hereditary Hemochromatosis ◽

Iron Concentration ◽

Transferrin Saturation ◽

Control Group ◽

Mrna Levels ◽

Liver Iron ◽

Hepcidin Expression ◽

Antisense Technology

Abstract Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to <150ug/dl in treatment group), as well as liver iron accumulation. Histopathological evaluation and Prussian's Perl Blue staining indicated that iron was sequestered by macrophages, which led to an increase in spleen iron concentration. The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

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