3 Week IV Study of RGMC Ab in Female Sprague Dawley Rats
ABSTRACT: 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:We used NGS sequencing of RNA (RNA-Seq) in livers of mice treated with a 3 week low iron (2-6 parts per million) diet, and treatment with either RGFP966 (an HDAC3 specific inhibitor) or vehicle. Our primary interest is in the role of HDAC3 in expression of the iron regulatory hormone hepcidin (Hamp1 in mice). Overall design: C57Bl/6 mice were treated with 3 weeks low iron diet, then treated with 2 doses of either RGFP966 20mg/kg i.p. or vehicle.
Project description:Tmprss6 is the master inhibitor of hepcidin and its inactivation causes iron refractory iron deficiency anemia both in human and in mice. Mice with iron deficiency anemia (IDA)-low hepcidin show a pro-inflammatory response that is blunted in iron deficienct-high hepcidin Tmprss6 null mice. We investigated the transcriptional response associated with chronic hepcidin overexpression by comparing whole genome transcription profiling of the liver of Tmprss6 KO mice and IDA animals, irrespective of iron deficiency. Total liver RNA obtained from Tmprss6 KO mice were compared to wild type (iron deficient) animals, under basal conditions and after LPS challenge
Project description:This study is designed to compare and contrast the temporal and spatial changes in bone formation rates and transcriptional profiles in cortical and cancellous bone cell populations enriched by laser capture microdissection (LCM) in ovariectomized rats administered Scl-Ab by subcutaneous injection for up to 26 consecutive weeks, followed by a recovery period of up to 18 weeks. Six-month-old Sprague-Dawley female rats were OVX, left untreated for 8 weeks. Rats were assigned to three treatment groups using a computerized blocking procedure designed to achieve body weight balance across treatment groups and then divided into three groups and administered by s.c. injection VEH or 3 or 50 mg/kg/wk of a Scl-Ab engineered to be less immunogenic in rats up to d183. Rats were administered either VEH or 3 or 50 mg/kg of Scl-Ab once weekly by s.c. injection. Up to 15 rats per group were euthanized at d8, 29, 85, and 183. To assess effects following treatment withdrawal, additional groups were maintained through a treatment-free period (TFP) and euthanized at d197 and 267 for VEH and 3 mg/kg groups and at d237 and 309 for VEH and 50 mg/kg groups. To label active bone-forming surfaces and facilitate osteoblast (OB) and lining cell (LC) enrichment during laser capture microdissection (LCM), 10 mg/kg calcein green was administered s.c. 13 and 3 days prior to scheduled euthanasia. At necropsy, the first and second lumbar vertebral (L1 and L2) bodies were isolated for histomorphometry and pQCT analyses, respectively. Cryosection preparation of L3 vertebral body, LCM procedure and validation of cell enrichment, RNA extraction and amplification, TaqMan, and initial microarray analysis was performed. LCM was performed on 4–6 ?m cryosections of vertebrae from the first 5 animals per treatment group per timepoint to enrich for OB, LC, and osteocyte (OCy) subpopulations for transcriptional analyses using Affmetrix Rat 230_2 microarrays
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:Mouse Iron Distribution Dynamics
Dynamic model of iron distribution in mice. This model attempts to fit the radioiron tracer data from Lopes et al. 2010 for mice fed iron deficient and rich diets by adjusting the rate of iron intake (vDiet) and the hepcidin synthesis rate (vhepcidin) independently for each experiment. All other parameters are those that provide the best fit for the adequate diet.
This model includes the radioiron tracer species.
Differences in parameter values between deficient, rich, and adequate diets:
Project description:Iron-related disorders are among the most prevalent diseases worldwide. Systemic iron homeostasis requires hepcidin (Hamp), a hepatic-derived hormone that controls iron mobilization through its molecular target, ferroportin (FPN), the only known mammalian iron exporter. Here, we took a transcriptomic approach to to compare the duodenal transcriptome during systemic iron demand to that of hepcidin-deficiency iron overload. Hampfl/fl (control) and AlbCreERT2;Hampfl/fl mice were placed on iron-replete and low-iron diets and were sacrificed two weeks following tamoxifen treatment. Duodenum RNA expression was compared across genotypes and across iron-replete and low iron diets.
Project description:Iron is an essential trace element whose absorption is usually tightly regulated in the duodenum. HFE-related hereditary hemochromatosis (HH) is characterized by abnormally low expression of the iron-regulatory hormone, hepcidin, which results in increased iron absorption. The liver is crucial for iron homeostasis as it is the main production site of hepcidin. The aim of this study was to explore and compare the genome-wide transcriptome response to Hfe deficiency and dietary iron overload in murine liver and duodenum. Overall design: C57BL/6 male mice were used following the next scheme: 3 Hfe knockout mice and 2 wild type mice as their controls; 3 mice with dietary iron overload and 2 mice fed a standard diet as their controls. 2 tissues were analyzed from all the described mice, liver and duodenum. As an exception, the duodenum samples of only 2 Hfe knockout mice, instead of 3, were used. This makes a total of 19 samples: 10 liver samples and 9 duodenum samples.
Project description:This study was initiated to characterize early DOX-induced changes in cardiac gene expression in order to identify potential additional areas for clinical intervention. Male spontaneously hypertensive rats (SHR) received 3 mg/kg DOX or vehicle (iv) 30 minutes following pretreatment with 50 mg/kg DZR or saline (ip) weekly for 1, 2 or 3 weeks.
Project description:Grange2001 - PK interaction of L-dopa and benserazide
A pharmacokinetics of L-dopa in rats after administration of L-dopa alone (BIOMD0000000321) or L-dopa combined with a peripheral AADC (amino-acid-decarboxylase) inhibitor (this model: BIOMD0000000320) has been studied using noncompartmental analysis.
This model is described in the article:
A pharmacokinetic model to predict the PK interaction of L-dopa and benserazide in rats.
Grange S, Holford NH, Guentert TW
Pharmaceutical Research [2001, 18(8):1174-1184]
To study the PK interaction of L-dopa/benserazide in rats. METHODS: Male rats received a single oral dose of 80 mg/kg L-dopa or 20 mg/kg benserazide or 80/20 mg/kg L-dopa/benserazide. Based on plasma concentrations the kinetics of L-dopa, 3-O-methyldopa (3-OMD), benserazide, and its metabolite Ro 04-5127 were characterized by noncompartmental analysis and a compartmental model where total L-dopa clearance was the sum of the clearances mediated by amino-acid-decarboxylase (AADC), catechol-O-methyltransferase and other enzymes. In the model Ro 04-5127 inhibited competitively the L-dopa clearance by AADC.
The coadministration of L-dopa/benserazide resulted in a major increase in systemic exposure to L-dopa and 3-OMD and a decrease in L-dopa clearance. The compartmental model allowed an adequate description of the observed L-dopa and 3-OMD concentrations in the absence and presence of benserazide. It had an advantage over noncompartmental analysis because it could describe the temporal change of inhibition and recovery of AADC.
Our study is the first investigation where the kinetics of benserazide and Ro 04-5127 have been described by a compartmental model. The L-dopa/benserazide model allowed a mechanism-based view of the L-dopa/benserazide interaction and supports the hypothesis that Ro 04-5127 is the primary active metabolite of benserazide.
The volumes and variables in this model are taken for a rat with 0.25 kg. The inital dose for L_Dopa (L_Dopa_per_kg_rat) and Benserazide (Benserazide_per_kg_rat) are to be given in umole per kg. 80 mg/kg L-Dopa correspond to 404 umol/kg, 20 mg/kg benserazide to 78 umol/kg. To change the model to a different mass of rat the compartment volumes, and the parameters rat_body_mass and Q have to changed accordingly.
The model has three species (A-dopa, A_B, A_M) whose initial concentrations are calculated from a listOfInitialAssignments
. While running for the first time the time-course (24hrs) for this model in COPASI (up to version 4.6, Build 33), the resulting graph displays only straight lines for all the species. Any subsequent runs should provide proper plots (i.e. without making any change to the model, just by clicking the "run" button again).
The above issue is caused by some initial assignments which are not calculated when COPASI imports the file. This issue should not be present in newer releases of COPASI.
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Project description:Background & Aims: Although hepcidin expression was shown to be induced by the BMP signaling pathway, it is not yet known how iron regulates hepcidin and which of the BMP molecules is the endogenous regulator of iron homeostasis in vivo. We therefore assessed liver transcription profiles of mice fed an iron-deficient or an iron-enriched diet and looked for genes that were regulated similarly to hepcidin in that context. Methods: Genome-wide liver expression profiles of mice of the B6 and D2 genetic backgrounds subjected to iron-deficient, -balanced, or -enriched diets were obtained using Agilent Whole Genome microarrays. Real-time quantitative-PCR and western-blots were used to confirm microarray results and compare gene expression variations induced by secondary iron deficiency or iron overload with those consecutive to Smad4 or Hamp1-deficiency. Results: Among 1419 transcripts significantly modulated by the dietary iron content, four were regulated similarly to the hepcidin genes Hamp1 and Hamp2. They are coding for Bmp6, the regulator of Bmp/Smad signal transduction Smad7, the negative regulator of basic helix-loop-helix (bHLH) proteins Id1, and a protein with a bHLH domain, Atoh8. The iron overload developed by Smad4 and Hamp1-deficient mice also increased Bmp6 transcription. Body iron stores influence Smad1/5/8 phosphorylation and, as shown by analysis of mice with liver-specific disruption of Smad4, the binding partner for the receptor-activated Smads is necessary for activation of Smad7, Id1, and Atoh8 transcription by iron. Conclusions: Liver expression of Bmp6, Smad7, Id1, and Atoh8 is regulated by body iron stores and may participate in hepcidin regulation through the Bmp/Smad pathway. Keywords: response to dietary iron content Overall design: Total RNA was extracted and purified using the RNeasy Lipid Tissue kit (Qiagen, Courtaboeuf, France). RNA quality was checked on RNA 6000 Nano chips using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). RNA samples used for chip experiments all had RNA Integrity Numbers (RIN) 16 greater than 9. Agilent’s Low RNA Input Linear Amplification Kit PLUS (One-color) was used to generate fluorescent cRNA. The amplified cyanine 3-labeled cRNA samples were then purified using Qiagen’s RNeasy mini spin colums and hybridized to Agilent Whole Mouse Genome Microarrays, 4x44K. Microarray slides were washed and scanned with an Agilent Scanner, according to the standard protocol of the manufacturer. Information from probe features was extracted from microarray scan images using the Agilent Feature Extraction software v.9.5.1. All the analyses were performed using Bioconductor, an open source software for the analysis of genomic data rooted in the statistical computing environment R. Normalization between arrays was performed using the quantile method. Genes for which the background-corrected signal intensities were not greater than 2.6 standard deviations above the average background in at least 3 B6 and 3 D2 mice were assumed not to be expressed in the liver and were excluded from further analysis. The R/MAANOVA package implemented in Bioconductor was used to perform a two-way analysis of variance in which the log2-transformed expression level was considered to be a function of diet, strain, and the effects of the interaction between these two factors. Fs tests, based on the James-Stein shrinkage estimates of the error variance, were computed on a gene-by-gene basis, and p values obtained by permutation analysis. The proportion of false positives among all the genes initially identified as being differentially expressed (FDR) was assessed using the procedure described by Storey. When influence of diet on expression levels was significant, t-tests were performed to investigate specific effects of iron deficiency and iron enrichment.