Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist.

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

The authors regret that an affiliation was left out of the Acknowledgments. Investigations were supported in part by National Institutes of Health (NIH) grants R01HL044491 and R01DK089439 (D.M. Wojchowski).The authors would like to apologize for any inconvenience caused. The authors regret that an affiliation was left out of the Acknowledgments. Investigations were supported in part by National Institutes of Health (NIH) grants R01HL044491 and R01DK089439 (D.M. Wojchowski). The authors would like to apologize for any inconvenience caused. Peginesatide and erythropoietin stimulate similar erythropoietin receptor–mediated signal transduction and gene induction eventsExperimental HematologyVol. 40Issue 7PreviewPeginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. Full-Text PDF Open Archive

Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. After stimulation with peginesatide or rHuEPO, cell lysates were prepared from UT-7/EPO cells. Liquid chromatography-tandem mass spectrometry and MesoScale arrays were used to quantify phosphorylation events. Transcriptional changes were analyzed using microarrays and quantitative reverse transcription polymerase chain reaction. Peginesatide and rHuEPO were found to regulate the tyrosine phosphorylation of an essentially equivalent set of protein substrates, and modulate the expression of a similar set of target genes. Consistent with their roles in stimulating erythropoiesis, peginesatide and rHuEPO regulate similar cellular pathways.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.

Abstract Abstract 2089 Peginesatide is a peptide-based erythropoietin receptor (EPOR) agonist with recent FDA approval for treating the anemia of chronic kidney disease among adult dialysis patients. Although peginesatide exhibits a 47.9h half-life when given IV in dialysis patients, it is administered once-monthly. Taken together, this predicts that additional functional properties contribute to peginesatide's durable erythropoiesis stimulating activity. Here we report on three such properties. During ex vivo murine bone marrow erythroid development, compared directly to rHuEPO, peginesatide first enhanced KitposCD71pos progenitor cell expansion (including KitposCD71lowCD36posCD13pos erythromyelo- progenitors). Second, peginesatide exhibited a 1300 minute EPOR residence time vs. 77 minutes for rHuEPO. Third, the culture of EPO-dependent human UT7epo cells in peginesatide led to substantial EPOR up-modulation, as well as an apparent lessening of the processing of mature EPOR's. Furthermore, in studies that compared peginesatide vs. rHuEPO effects on the erythroid development of human bone marrow-derived CD34pos progenitors, the following differences were observed. At days 6–10 of culture, peginesatide gave rise to increased frequencies (up to 200% as compared to rHuEPO) of KitposCD71pos co-positive erythroid progenitors. At early stages (d2-d4), cell-surface EPOR levels also were elevated among progenitors expanded in peginesatide. When levels of CD13posCD36pos co-positive cells were analyzed, frequencies of these erythromyelo-progenitors also were heightened up to three-fold. Analyses of cultures at later time-points (eg, d10 of culture) indicated that peginesatide and rHuEPO supported the formation of GPAhigh erythroblasts at similarly high frequencies. In addition, cytospin analyses revealed morphological distinctions for multicellular proerythroblast complexes formed in peginesatide. Peginesatide's persistent erythropoietic activity allowing for once-monthly dosing therefore is likely to involve novel effects on erythromyelo-progenitor recruitment, and increased EPOR cell surface expression including apparent increases in relative levels of full-length EPOR forms. Disclosures: Green: Affymax, Inc.: Employment. Leu:Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Prior employment Other. Schatz:Affymax, Inc.: Employment. Wojchowski:Affymax, Inc.: Membership on an entity's Board of Directors or advisory committees.

Peginesatide is a PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA) that was designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Clinical use of peginesatide is anticipated to result in chronic dosing in chronic kidney disease (CKD) patients, and the nonclinical data to support development should include an evaluation of carcinogenic potential evaluation. Peginesatide was not mutagenic or clastogenic in a standard genotoxicity battery of tests. Doses for a rasH2 transgenic mouse carcinogenicity assay were defined in a 28-day study in the wild-type littermates of the rasH2 transgenic mouse strain, using intravenous doses of 1-25 mg/kg on days 1 and 22. The findings were consistent with exaggerated pharmacology, including polycythemia, with associated increases in hemoglobin level and extramedullary hematopoiesis and bone marrow hypercellularity.

Abstract Abstract 682 Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion. Disclosures: Leu: Affymax, Inc.: Employment, own stock in Affymax, Inc. Young:Affymax, Inc.: Employment, own stock in Affymax, Inc. Schatz:Affymax, Inc.: Employment, own stock in Affymax, Inc. Green:Affymax, Inc.: Employment, own stock in Affymax, Inc.

Since the 1990's, cheating athletes have abused substances to increase their oxygen transport capabilities; among these substances, recombinant EPO is the most well known. Currently, other investigational pharmaceutical products are able to produce an effect similar to EPO but without having chemical structures related to EPO; these are the synthetic erythropoiesis stimulating agents (ESAs). Peginesatide (also known as Hematide™) is being developed by Affymax and Takeda and, if approved by regulatory authorities, could soon be released on the international market. To detect potential athletic abuse of this product and deter athletes who consider cheating, we initiated a collaboration to implement a detection test for anti-doping purposes. Peginesatide is a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. It is undetectable using current anti-doping tests due to its lack of sequence homology to EPO. To detect and deter potential abuse of peginesatide, we initiated an industry/antidoping laboratory collaboration to develop and validate screening and confirmation assays so that they would be available before peginesatide reaches the market. We describe a screening ELISA and a confirmation assay consisting of immune-purification followed by separation with SDS-PAGE and revelation with Western double blotting. Both assays can detect 0.5 ng/mL concentrations of peginesatide in blood samples, enabling detection for several days after administration of a physiologically relevant dose. This initial report describes experimental characterization of these assays, including testing with a blinded set of samples from a clinical study conducted in healthy volunteers.

Abstract Abstract 2046 The erythropoietin receptor mediates pro-erythroblast production; can promote erythrocytosis; and can cytoprotect select non-hematopoietic tissues. For certain key aspects of EPOR biology, knowledge nonetheless remains limited. This includes regulated EPOR trafficking, which we presently investigate using a novel panel of specific rabbit monoclonal anti-EPOR antibodies. In exponentially growing EPO-dependent UT7epo cells, a single high-Mr species was detected at low levels, and EPOR turnover appeared to predominate. The limiting of EPO led to the accumulation of two high Mr EPOR forms, EPOR-68K and EPOR-70K. EPO exposure rapidly converted an indicated cell surface EPOR-70K form to an EPOR-72K tyrosine-phosphorylated species (and to lower Mr fragments). In contrast, EPOR-68K was not rapidly modulated by EPO, and proved to comprise an intracellular, endoglycosidase-sensitive EPOR pool. At the cell surface, EPOR repopulation also was regulated and interestingly was accelerated in response to higher-dose EPO exposure. Constitutive EPOR turnover was observed, but as comparably limited ligand-independent trafficking. EPOR expression in primary erythroid progenitors also was analyzed. Flow cytometry and western blotting each unexpectedly revealed peak EPOR expression at a GPAhigh late erythroblast stage. These studies provide new insight into dynamic, EPO dose-dependent EPOR trafficking, together with an observed sustained expression of the EPOR within maturing primary human erythroblasts. Disclosures: Leu: Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Employment. Schatz:Affymax, Inc.: Employment. Green:Affymax, Inc.: Employment.

BACKGROUND:Aperi- and postnatal reproduction toxicity study was conducted in rats treated with Hematide, a synthetic PEGylated peptidic erythropoiesis stimulating agent (ESA).METHODS:Hematide, at IV doses of 0, 0.5, 3, and 15 mg/kg, was administered from implantation through lactation on gestation days (GDs) 5 and 18 and lactation day (LD) 13.RESULTS:Hematide induced pronounced polycythemia in all Hematide-treated dams. On LDs 2 and 21, hemoglobin (Hgb) increases above control levels were 3.1, 5.2, and 5.0 g/dL and 4.1, 5.1, and 5.5 g/dL at the 0.5, 3, and 15 mg/kg/dose, respectively. There were no effects on parturition, lactation, or maternal behavior in the F0 generation female rats. A slight decrease in pup viability on postpartum days 2-4 and lower body weights and/or body weight gain for the F1 generation were associated with pronounced polycythemia and decreases in maternal body weight gain and/or food consumption at > or =3 mg/kg/dose. Hematide fetal exposure was negligible. No Hematide effect, other than on growth and survival, was noted on developmental, functional, mating, and fertility end points in the F1 generation rats, and no effect on litter or fetal parameters was observed in the F2 generation. The maternal no-observed-adverse-effect level (NOAEL) for Hematide was 0.5 mg/kg, and the NOAEL for parturition and maternal behavior was 15 mg/kg. The NOAEL for F1 pup viability and growth was 0.5 mg/kg/dose.CONCLUSIONS:In conclusion, the Hematide-associated adverse findings were attributed to exaggerated erythropoiesis (pronounced and prolonged polycythemia) resulting from administration of an ESA to pregnant animals.

Abstract: Hematide™ is a synthetic peptide-based, PEGylated erythropoiesis-stimulating agent, which is being developed for the chronic treatment of anaemia associated with chronic renal failure. To support the safety of long-term dosing of chronic renal failure patients, a comprehensive toxicology programme was implemented including rat subchronic and chronic studies. Rats were administered 0, 0.1, 1 and 10 mg/kg of Hematide every 3 weeks for 3 months via subcutaneous injection or for 6 months via intravenous injection. The dosing period was followed by a 6-week follow-up period. The primary pharmacology of Hematide resulted in erythroid polycythemia as measured by elevated haemoglobin levels that were time- and dose-dependent. The pharmacology profiles were similar regardless of administration route. For example, for male rats at Day 90, subcutaneous dosing resulted in haemoglobin increases of 2.7, 4.5 and 6.9 g/dl for 0.1, 1 and 10 mg Hematide/kg respectively, compared to 2.8, 5.7 and 7.4 g/dl increases for intravenous dosing. Histopathological changes were related to the prolonged severe polycythemia induced in normocythemic animals administered an erythropoiesis-stimulating agent. The findings included extramedullary haematopoiesis in the spleen and liver, bone marrow hypercellularity and organ congestion. Microscopic findings were reversible, demonstrating a return towards control findings within 6 weeks following cessation of dosing. Systemic exposures, based on both area under the curve (AUC) and maximum concentration (C max ), were substantially greater for intravenous than subcutaneous administration. No Hematide-specific antibodies were detected. In conclusion, Hematide is a potent erythropoiesis-stimulating agent, and the studies provide support for the safety of clinical development, including chronic dosing, for the treatment of anaemia associated with chronic renal failure.

The subchronic toxicity of Hematide™, a synthetic PEGylated peptidic erythropoiesis-stimulating agent (ESA), was evaluated in CD-1 mice at intravenous doses of 0, 1, 5, 25, and 125 mg/kg administered once every 3 weeks for 3 months. Hematide displayed sustained plasma levels with reduced clearance and prolonged half-lives up to 59.4 hours that translated into sustained, pronounced polycythemia, bone marrow hyperplasia, and splenic and liver extramedullary hematopoiesis. Toxicological findings were considered to be secondary to exaggerated pharmacology, rather than a direct drug effect, and included mortality at ≥25 mg/kg/dose. The no-observed-adverse-effect-level was determined to be 5 mg/kg.