Project description:High levels of hepcidin, the main regulator of systemic iron metabolism leads to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies (mAbs) ABT-207 and h5F9-AM8 which inhibit hemojuvelin also known as repulsive guidance molecule c (RGMc) and downregulate hepcidin. After a single application of these antibodies hepcidin expression in liver and its concentration in serum were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of ~80 fold higher than ABT-207. When hepcidin levels were downregulated iron deposition in the liver was visible histologically one week post application. The anitbody-mediated iron deposition was not associated with any toxicologically relevant effect at the doses and timepoints evaluate. Iron depositions seen after 14 weekly treatments with ABT-207 were partially reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease. Female Sprague-Dawley rats [Crl:CD®(SD)IGS BR], weighing ~200 g at study initiation were obtained from Charles River Laboratories, Inc. Rats were housed singly in ventilated, stainless steel, wire-bottom hanging cages, fed non-certified Rodent Chow and water ad libitum and acclimated for at least 5 days after arrival. Rats were randomly assigned to various treatment groups (5 rats/group) and were dosed once weekly by IV injection via tail vein with vehicle (30mM Histidine, 8% w/v Sucrose, pH6.0, + 0.02% Tween 80) or with 0.02, 0.2, or 20 mg/kg of RGMc antibody for a total of 4 doses. All rats were sacrificed under isoflurane anesthesia 24 hours after final dose. Liver and spleen waere flash frozen in liquid nitrogen and stored at 80°C until processing for gene expression profiling on the Affymetrix platform. total of 12 samples, 3 biological replicates (rats) from each of 4 treatments groups 1. vehicle control (30mM Histidine, 8% w/v Sucrose, pH6.0, + 0.02% Tween 80) 2. 0.02 mg/kg RGMC Ab 3. 0.2 mg/kg RGMC Ab 4. 20 mg/kg RGMC Ab; rats were dosed by IV injection via tail vein once weekly for 4 total doses, dose volume 2mL/kg/week

Project description:Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP) and interleukin-6 (IL-6) via effects on Smad4 or Stat3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone genistein from 28−52 hours post-fertilization in zebrafish embryos enhanced Hepcidin transcript levels as assessed by whole mount in situ hybridization and quantitative realtime RT-PCR. Genistein’s stimulatory effect was conserved in human hepatocytes: genistein treatment of HepG2 cells increased both Hepcidin transcript levels and Hepcidin promoter activity. We found that genistein’s effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the BMP response elements or the Stat3 binding site in the Hepcidin promoter. RNA-sequencing of transcripts from genistein-treated hepatocytes indicated that genistein upregulated 68% of the transcripts that were upregulated by BMP6, however genistein raised the levels of several transcripts involved in Stat3 signaling that were not upregulated by BMP6. Chromatin-immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION: Genistein is the first small molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. RNA-seq of HepG2 cells treated with DMSO 1%, BMP6 50 ng/ml, or genistein 10 micromolar. The numbers of biological replicates were 3, 2, and 3.

Project description:Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP) and interleukin-6 (IL-6) via effects on Smad4 or Stat3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone genistein from 28−52 hours post-fertilization in zebrafish embryos enhanced Hepcidin transcript levels as assessed by whole mount in situ hybridization and quantitative realtime RT-PCR. Genistein’s stimulatory effect was conserved in human hepatocytes: genistein treatment of HepG2 cells increased both Hepcidin transcript levels and Hepcidin promoter activity. We found that genistein’s effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the BMP response elements or the Stat3 binding site in the Hepcidin promoter. RNA-sequencing of transcripts from genistein-treated hepatocytes indicated that genistein upregulated 68% of the transcripts that were upregulated by BMP6, however genistein raised the levels of several transcripts involved in Stat3 signaling that were not upregulated by BMP6. Chromatin-immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION: Genistein is the first small molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

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3 Week IV Study of RGMC Ab in Female Sprague Dawley Rats

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