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New Anemia Therapies: Translating Novel Strategies From Bench to Bedside

2011; Elsevier BV; Volume: 59; Issue: 3 Linguagem: Inglês

10.1053/j.ajkd.2011.11.013

1523-6838

Iain C. Macdougall,

Hemoglobinopathies and Related Disorders

Recombinant human erythropoietin (epoetin) has been available for the treatment of renal anemia for more than 20 years, and within the last decade two molecularly engineered analogues darbepoetin alfa and pegylated epoetin beta were introduced as longer-acting erythropoiesis-stimulating agents. Recently, newer strategies for correcting anemia have been explored, some of which remain in the laboratory while others are translating across into clinical trials. Peginesatide has completed phase 3 clinical trials for the treatment of anemia associated with chronic kidney disease; this molecule is immunologically distinct from the erythropoietic proteins, with no cross-reactivity with anti-erythropoietin antibodies. HIF (hypoxia inducible factor) stabilization involves the pharmacologic inhibition of prolyl hydroxylation of HIF-α (the major transcription factor controlling erythropoietin gene expression), thereby preventing its degradation in the proteasome. Hepcidin is the master regulator of iron metabolism, and this peptide is upregulated in inflammatory conditions, including uremia; its antagonism has been shown to cause amelioration of inflammatory anemia in animal models. For the time being, erythropoiesis-stimulating agent therapy remains the mainstay of anemia management in chronic kidney disease, but it is possible that one or more of the strategies discussed in this review may have a future role in the treatment of this condition. Recombinant human erythropoietin (epoetin) has been available for the treatment of renal anemia for more than 20 years, and within the last decade two molecularly engineered analogues darbepoetin alfa and pegylated epoetin beta were introduced as longer-acting erythropoiesis-stimulating agents. Recently, newer strategies for correcting anemia have been explored, some of which remain in the laboratory while others are translating across into clinical trials. Peginesatide has completed phase 3 clinical trials for the treatment of anemia associated with chronic kidney disease; this molecule is immunologically distinct from the erythropoietic proteins, with no cross-reactivity with anti-erythropoietin antibodies. HIF (hypoxia inducible factor) stabilization involves the pharmacologic inhibition of prolyl hydroxylation of HIF-α (the major transcription factor controlling erythropoietin gene expression), thereby preventing its degradation in the proteasome. Hepcidin is the master regulator of iron metabolism, and this peptide is upregulated in inflammatory conditions, including uremia; its antagonism has been shown to cause amelioration of inflammatory anemia in animal models. For the time being, erythropoiesis-stimulating agent therapy remains the mainstay of anemia management in chronic kidney disease, but it is possible that one or more of the strategies discussed in this review may have a future role in the treatment of this condition. Recombinant human erythropoietin was introduced as a treatment for the anemia associated with chronic kidney disease (CKD) in 1989 (in the United States) and 1990 (in Europe). It has transformed the lives of millions of patients, particularly those on dialysis therapy who were transfusion dependent, iron overloaded, and severely debilitated from the symptoms associated with having an average hemoglobin level of ∼6-7 g/dL. Many younger patients were denied the potential benefits of kidney transplantation because of sensitization to HLA antigens resulting from repeated blood transfusions. Erythropoietin therapy rendered many of these patients free of transfusions, and a plethora of studies (most were uncontrolled) documented the dramatic benefits on quality of life (particularly physical capacity),1Mayer G. Thum J. Cada E.M. Stummvoll H.K. Graf H. Working capacity is increased following recombinant human erythropoietin treatment.Kidney Int. 1988; 34: 525-528Crossref PubMed Scopus (163) Google Scholar general well-being, and other physiologic effects of increasing hemoglobin levels from ∼6 g/dL up to approximately 11-12 g/dL.2Macdougall I.C. Lewis N.P. Saunders M.J. et al.Long-term cardiorespiratory effects of amelioration of renal anaemia by erythropoietin.Lancet. 1990; 335: 489-493Abstract PubMed Scopus (229) Google Scholar, 3Evans R.W. Rader B. Manninen D.L. The quality of life of hemodialysis recipients treated with recombinant human erythropoietin Cooperative Multicenter EPO Clinical Trial Group.JAMA. 1990; 263: 825-830Crossref PubMed Scopus (402) Google Scholar, 4Marsh J.T. Brown W.S. Wolcott D. et al.rHuEPO treatment improves brain and cognitive function of anemic dialysis patients.Kidney Int. 1991; 39: 155-163Crossref PubMed Scopus (214) Google Scholar The dramatic benefits of this therapy completely overshadowed the minor inconveniences of the need for regular administration 3 times a week and the need to inject this protein parenterally. The first of these limitations was challenged by the introduction of a longer acting erythropoietin analogue, darbepoetin alfa, in 2001, and later by the advent of pegylated epoetin beta in countries outside the United States in 2007 (Table 1).5Curran M.P. McCormack P.L. Methoxy polyethylene glycol-epoetin beta: a review of its use in the management of anaemia associated with chronic kidney disease.Drugs. 2008; 68: 1139-1156Crossref PubMed Scopus (57) Google ScholarTable 1Currently Available Erythropoiesis-Stimulating AgentsAgentActive CompoundManufacturing ProcessYear LicensedEpoetin alfa/beta (Epogen, Eprex, Erypo, NeoRecormon)Recombinant human EPORecombinant DNA technology; EPO cDNA/gene–transfected CHO cells1989 (Epogen, in US); 1990 (Eprex/Erypo/NeoRecormon, in Europe)Epoetin delta (Dynepo)Recombinant human EPORecombinant DNA technology; EPO cDNA/gene–transfected human cells2006 (outside of US); product withdrawn by Shire in 2009"Biosimilar" epoetins (Binocrit, Hexal, Retacrit, Silapo, Eporatio)Recombinant human EPORecombinant DNA technology; EPO cDNA/gene–transfected CHO cells2009 onwardNonapproved or locally approved "copy" epoetinsRecombinant human EPORecombinant DNA technology; EPO cDNA/gene-transfected human cellsAvailable in many countries outside of US and Europe, eg, India, China, Thailand, Argentina, BrazilDarbepoetin alfa (Aranesp)Hyperglycosylated recombinant human EPO analogueRecombinant DNA technology; mutated EPO cDNA–transfected CHO cells2001 (both US and Europe)C.E.R.A. (Mircera)Pegylated recombinant human EPO analogue2009 (outside of US only)Abbreviations: EPO, erythropoietin; cDNA, complementary DNA; C.E.R.A., continuous erythropoietin receptor activator; CHO, Chinese hamster ovary; US, United States. Open table in a new tab Abbreviations: EPO, erythropoietin; cDNA, complementary DNA; C.E.R.A., continuous erythropoietin receptor activator; CHO, Chinese hamster ovary; US, United States. Several studies then investigated the potential benefits of normalizing hemoglobin concentration to levels seen in the healthy general population,6Besarab A. Bolton W.K. Browne J.K. et al.The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and epoetin.N Engl J Med. 1998; 339: 584-590Crossref PubMed Scopus (1883) Google Scholar, 7Parfrey P.S. Foley R.N. Wittreich B.H. et al.Double-blind comparison of full and partial anemia correction in incident hemodialysis patients without symptomatic heart disease.J Am Soc Nephrol. 2005; 16: 2180-2189Crossref PubMed Scopus (323) Google Scholar, 8Drüeke T.B. Locatelli F. Clyne N. et al.Normalization of hemoglobin level in patients with chronic kidney disease and anemia.N Engl J Med. 2006; 335: 2071-2084Crossref Scopus (1786) Google Scholar, 9Singh A.K. Szczech L. Tang K.L. et al.Correction of anemia with epoetin alfa in chronic kidney disease.N Engl J Med. 2006; 335: 2085-2098Crossref Scopus (2282) Google Scholar, 10Pfeffer M.A. Burdmann E.A. Chen C.Y. et al.A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease.N Engl J Med. 2009; 361: 2019-2032Crossref PubMed Scopus (1652) Google Scholar but it became apparent that partial correction of anemia (to hemoglobin levels in the range of 10-12 g/dL) was a safer strategy, reducing the risk of increased arterial and venous thromboembolism and other possible harmful effects. Post hoc analyses of the major randomized controlled trials have suggested that the increased risk of harm may not be simply because of the target or achieved hemoglobin level, but also may be caused by excessive doses of erythropoiesis-stimulating agents (ESAs) in resistant patients who were administered escalating doses per the study protocol.11Kilpatrick R.D. Critchlow C.W. Fishbane S. et al.Greater epoetin alfa responsiveness is associated with improved survival in hemodialysis patients.Clin J Am Soc Nephrol. 2008; 3: 1077-1083Crossref PubMed Scopus (177) Google Scholar, 12Szczech L.A. Barnhart H.X. Inrig J.K. et al.Secondary analysis of the CHOIR trial epoetin-alpha dose and achieved hemoglobin outcomes.Kidney Int. 2008; 74: 791-798Crossref PubMed Scopus (431) Google Scholar, 13Solomon S.D. Uno H. Lewis E.F. et al.Trial to Reduce Cardiovascular Events with Aranesp Therapy (TREAT) InvestigatorsErythropoietic response and outcomes in kidney disease and type 2 diabetes.N Engl J Med. 2010; 363: 1146-1155Crossref PubMed Scopus (376) Google Scholar Although these post hoc analyses are inherently limited by confounding, it is not clear whether this latter subpopulation is a sicker group of patients or the escalating doses of ESA contributed to the increased morbidity and mortality. It is clear that there are pleiotropic effects of erythropoietin, and it is possible that some of the harmful effects of this treatment may be mediated by negative effects on endothelium-platelet interactions.14Vaziri N.D. Zhou X.J. Potential mechanisms of adverse outcomes in trials of anemia correction with erythropoietin in chronic kidney disease.Nephrol Dial Transplant. 2009; 24: 1082-1088Crossref PubMed Scopus (71) Google Scholar During the last few years, newer strategies for correcting anemia have been investigated (Table 2). Some of these (eg, peptide-based erythropoietic agents, HIF [hypoxia inducible factor] stabilization, and erythropoietin gene therapy) still require interaction of erythropoietin with its receptor, whereas other strategies, such as hepcidin modulation, examine a completely new strategy. Some, such as GATA-2 (GATA binding protein 2) inhibition and hepcidin modulation, have been tested in laboratory animals but have not yet been translated into clinical trials. Others, such as the erythropoietic peptide–based therapy peginesatide (completed phase 3 trials), HIF stabilization (phase 2), and erythropoietin gene therapy (phase 2), already have been tested in patients with anemia associated with CKD.Table 2Future Erythropoiesis-Stimulating AgentsAgentActive CompoundManufacturing ProcessStage of DevelopmentPeginesatide (Hematide)Dimeric pegylated peptideSynthetic peptide chemistryCompleted Phase 3HIF stabilizersProlyl hydroxylase inhibitorChemical synthesisPhase 1-2Hepcidin modulationVariousVariousPlanning phase 1GATA-2 inhibitorsSmall moleculeChemical synthesis??EPO gene therapy (EPODURE)Skin cells (microdermis) transfected with the EPO geneBiopump technology, harvesting skin biopsies and using adenovirus as vectorPhase 2Abbreviations: EPO, erythropoietin; GATA-2, GATA-binding protein 2; HIF, hypoxia inducible factor. Open table in a new tab Abbreviations: EPO, erythropoietin; GATA-2, GATA-binding protein 2; HIF, hypoxia inducible factor. The concept that a peptide could activate the erythropoietin receptor and stimulate erythropoiesis was described first by Wrighton et al15Wrighton N.C. Farrell F.X. Chang R. et al.Small peptides as potent mimetics of the protein hormone erythropoietin.Science. 1996; 273: 458-464Crossref PubMed Scopus (660) Google Scholar in 1996 in Science. This seminal work was a collaborative effort between scientists working in Johnson & Johnson and Affymax, who screened a large peptide library looking for possible ligands of the erythropoietin binding protein (EBP). Of several thousand candidate molecules, a few were selected for further study, and one of these, EMP-1 (erythropoietin-mimetic peptide 1), was characterized further in both cell culture experiments and animal models.15Wrighton N.C. Farrell F.X. Chang R. et al.Small peptides as potent mimetics of the protein hormone erythropoietin.Science. 1996; 273: 458-464Crossref PubMed Scopus (660) Google Scholar EMP-1 had low affinity for the erythropoietin receptor and low biological activity. Nevertheless, it was able to stimulate cellular proliferation of erythroid cells in culture in a dose-dependent manner and also increase reticulocyte counts in 2 distinct animal models of erythropoiesis, including the ex-hypoxic polycythemic mouse bioassay. These findings were all the more remarkable, given that the 10–amino acid sequence of EMP-1 was not contained within either native or recombinant erythropoietin. Despite this complete lack of homology in primary structure, EMP-1 seemed to share the same biological and functional characteristics of the native protein.15Wrighton N.C. Farrell F.X. Chang R. et al.Small peptides as potent mimetics of the protein hormone erythropoietin.Science. 1996; 273: 458-464Crossref PubMed Scopus (660) Google Scholar A group of scientists extended this concept further by examining methods to increase the biological potency of a peptide-based erythropoietin receptor agonist. Peginesatide is a dimeric peptide joined with a spacer linker to a pegylation chain to enhance its metabolic stability in vivo (Fig 1) .16Macdougall I.C. Hematide, a novel peptide-based erythropoiesis-stimulating agent for the treatment of anemia.Curr Opin Investig Drugs. 2008; 9: 1034-1047PubMed Google Scholar Cellular and animal data investigating peginesatide (Hematide) as an erythropoietic agent were published by Fan et al,17Fan Q. Leuther K.K. Holmes C.P. et al.Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia.Exp Hematol. 2006; 34: 1303-1311Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar and as with EMP-1, peginesatide was able to stimulate erythroid colony growth, as well as reticulocyte count and hematocrit, in both rats and cynomolgus monkeys.17Fan Q. Leuther K.K. Holmes C.P. et al.Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia.Exp Hematol. 2006; 34: 1303-1311Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Furthermore, because there is no structural homology between peginesatide and erythropoietin, antibodies against erythropoietin do not cross-react with peginesatide, and vice versa. Woodburn et al18Woodburn K.W. Fan Q. Winslow S. et al.Hematide is immunologically distinct from erythropoietin and corrects anemia induced by antierythropoietin antibodies in a rat pure red cell aplasia model.Exp Hematol. 2007; 35: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar tested this in a rat model of severe anemia induced by anti-erythropoietin antibodies and were able to show an increase in reticulocyte count and hematocrit with peginesatide that was not seen with vehicle alone. At the same time these experiments were being conducted, several patients, particularly in Europe, had developed antibody-mediated pure red cell aplasia caused by one or more of the commercially available erythropoietic proteins. This condition was caused by the development of anti-erythropoietin antibodies that neutralized not only the exogenous erythropoietic agent, but also all of the patients' own endogenous erythropoietin, effectively obliterating any meaningful erythropoiesis in bone marrow. Thus, these patients developed a severe transfusion-dependent anemia characterized by very low reticulocyte counts and the absence or near-absence of erythropoiesis in bone marrow. Given the results from the rat model of antibody-mediated pure red cell aplasia, a collaborative clinical study was set up across the United Kingdom, France, and Germany to investigate whether it was possible to "rescue" patients with this condition, many of whom were transfusion dependent, by administering peginesatide. Preliminary findings for the first 14 patients indicated that 13 of them achieved a hemoglobin concentration >11 g/dL without the need for further red blood cell transfusions.19Macdougall I.C. Rossert J. Casadevall N. et al.A peptide-based erythropoietin-receptor agonist for pure red-cell aplasia.N Engl J Med. 2009; 361: 1848-1855Crossref PubMed Scopus (130) Google Scholar One patient developed anti-peginesatide antibodies in addition to her anti-erythropoietin antibodies, and peginesatide therapy had to be stopped. Peginesatide has been developed as a once-monthly therapy that can be administered intravenously or subcutaneously. Phase 2 studies were conducted using a dose-escalation design, in the range of 0.025-0.075 mg/kg.20Macdougall I.C. Wiecek A. Tucker B. et al.Dose-finding study of peginesatide for anemia correction in chronic kidney disease patients.Clin J Am Soc Nephrol. 2011; 6: 2579-2586Crossref PubMed Scopus (22) Google Scholar The starting dose in the pure red cell aplasia study was 0.05 mg/kg.19Macdougall I.C. Rossert J. Casadevall N. et al.A peptide-based erythropoietin-receptor agonist for pure red-cell aplasia.N Engl J Med. 2009; 361: 1848-1855Crossref PubMed Scopus (130) Google Scholar The phase 3 clinical trial program for this agent has now been completed, and this involved 4 randomized controlled trials with either epoetin or darbepoetin alfa as a comparator therapy (Table 3). Two of these studies were in nondialysis patients (PEARL-1 and PEARL-2) and 2 of the trials were in dialysis patients (EMERALD-1 and EMERALD-2). All these studies involved a primary efficacy analysis in terms of anemia correction or hemoglobin level maintenance, and in all 4 trials, this objective was achieved.21Fishbane S. Besarab A. Schiller B. et al.Primary safety and efficacy results from four Phase 3 randomized, active-controlled, open-label studies of Hematide™/ Peginesatide among CKD dialysis and non-dialysis patients.in: Proceedings of the American Society of Nephrology Renal Week. November 16-21, 2010Google Scholar A composite cardiovascular safety end point for all 4 studies also was analyzed, and again, this met the prespecified noninferiority criteria. However, in a subanalysis of the PEARL studies, an increased risk of developing the cardiovascular composite was seen in patients receiving peginesatide versus the comparator ESA (with an overall hazard ratio of 1.32).21Fishbane S. Besarab A. Schiller B. et al.Primary safety and efficacy results from four Phase 3 randomized, active-controlled, open-label studies of Hematide™/ Peginesatide among CKD dialysis and non-dialysis patients.in: Proceedings of the American Society of Nephrology Renal Week. November 16-21, 2010Google Scholar The explanation for this somewhat unexpected outcome remains obscure. At the time of writing, an application has been made to the US Food and Drug Administration (FDA) for a product license for peginesatide in dialysis patients.Table 3Peginesatide Phase 3 Clinical Trials OverviewStudyDescriptionSample Size (region)OutcomesPEARL 1Correction study: peginesatide vs darbepoetin alfa in nondialysis patients (SC)∼330 vs 165 (US)Efficacy of peginesatide noninferior to darbepoetin; increased HR for composite safety end point at 1.32 for peginesatide vs darbepoetin alfaPEARL 2Correction study: peginesatide vs darbepoetin alfa in nondialysis patients (SC)∼330 vs 165 (US and Europe)EMERALD 1Maintenance study: peginesatide vs epoetin alfa in dialysis patients (IV)∼540 vs 270 (US)Efficacy and safety of peginesatide noninferior to epoetinEMERALD 2Maintenance study: peginesatide vs epoetin alfa or beta in dialysis patients (IV/SC)∼540 vs 270 (US and Europe)Abbreviations: EMERALD, Hematide Injection for Anemia in Chronic Hemodialysis Patients; HR, hazard ratio; IV, intravenous; PEARL, Safety and Efficacy of Hematide for the Correction of Anemia in Patients With Chronic Renal Failure; SC, subcutaneous; US, United States. Open table in a new tab Abbreviations: EMERALD, Hematide Injection for Anemia in Chronic Hemodialysis Patients; HR, hazard ratio; IV, intravenous; PEARL, Safety and Efficacy of Hematide for the Correction of Anemia in Patients With Chronic Renal Failure; SC, subcutaneous; US, United States. The manufacturing process for this peptide-based ESA involves much simpler synthetic peptide chemistry techniques compared with the complex biotechnological methods (recombinant DNA technology and cell culture) that are required for the manufacture of existing ESAs. Targeting the 3′ enhancer (HIF stabilization) and the 5′ promoter (GATA-2 inhibition) of the erythropoietin gene (EPO) has been the subject of detailed investigation during the last decade or so (Fig 2) .22Jelkmann W. Molecular biology of erythropoietin.Intern Med. 2004; 43: 649-659Crossref PubMed Scopus (288) Google Scholar Erythropoietin expression in both the kidney and liver is suppressed by oxygen-dependent degradation of the α subunit of HIF (HIF-1α), mediated by prolyl hydroxylation.23Schofield C.J. Ratcliffe P.J. Oxygen sensing by HIF hydroxylases.Nat Rev Mol Cell Biol. 2004; 5: 43-54Crossref Scopus (1620) Google Scholar, 24Nangaku M. Eckardt K.U. Pathogenesis of renal anemia.Semin Nephrol. 2006; 26: 261-268Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar When hydroxylated, HIF then couples with the von Hippel Lindau tumor suppressor protein, and the resultant complex is targeted for proteasomal degradation (Fig 3) . Thus, inhibiting prolyl hydroxylase results in stabilization of HIF and consequently transcription of the EPO gene. These agents that prevent degradation of HIF are termed HIF stabilizers and are 2-oxoglutarate analogues.25Nangaku M. Kojima I. Tanaka T. Ohse T. Kato H. Fujita T. Novel drugs and the response to hypoxia: HIF stabilizers and prolyl hydroxylase.Recent Patent Rev Cardiovasc Drug Disc. 2006; 1: 129-139Crossref PubMed Scopus (32) Google ScholarFigure 3Regulation of hypoxia inducible factor (HIF) activity. Abbreviations: EPO, erythropoietin; VHL, von Hippel Lindau protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Several HIF stabilizer compounds have been studied. One of the early candidate molecules was FG-2216, synthesized by Fibrogen. Administration of FG-2216 stabilizes HIF, thereby promoting EPO gene upregulation and increased erythropoietin synthesis.26Urquilla P. Fong A. Oksanen S. et al.Upregulation of endogenous EPO in healthy subjects by inhibition of HIF-PH [abstract].J Am Soc Nephrol. 2004; 15: 546AGoogle Scholar Thus, these molecules are able to increase endogenous erythropoietin levels without the need to administer exogenous ESA therapy. It is now clear that the anemia associated with CKD is not due simply to deficient erythropoietin production, but rather to defective EPO gene regulation, because it has been shown that hemodialysis patients can increase serum erythropoietin levels significantly. Although it is assumed that at least some of the erythropoietin is produced by the diseased kidneys, the fact that even anephric individuals can generate erythropoietin in response to HIF stabilization suggests that there are other (extrarenal) sites of erythropoietin production (eg, the liver).27Bernhardt W.M. Wiesener M.S. Scigalla P. et al.Inhibition of prolyl hydroxylases increases erythropoietin production in ESRD.J Am Soc Nephrol. 2010; 21: 2151-2156Crossref PubMed Scopus (271) Google Scholar The strategy of stimulating endogenous erythropoietin production is interesting not only for its lack of need for exogenous ESA therapy. For example, it is possible that increasing erythropoietin levels above a certain threshold on a pulsatile daily or thrice-weekly basis may prove to be safer than administering very high concentrations of erythropoietic protein in a pharmacologic manner. This hypothesis requires careful investigation in controlled clinical trials. There are 2 additional potential advantages of HIF stabilization therapy. First, these agents are orally active and thus there is potential for a noninjectable anemia therapy in the future. Second, these molecules are able to modulate a number of other genes involved in erythropoiesis (eg, the erythropoietin receptor, transferrin, transferrin receptor, ferroportin, and divalent metal transporter 1) in addition to the EPO gene.28Haase V.H. Hypoxic regulation of erythropoiesis and iron metabolism.Am J Physiol Renal Physiol. 2010; 299: F1-F13Crossref PubMed Scopus (230) Google Scholar Furthermore, there is evidence that HIF stabilization may downregulate the production of hepcidin (see next section). These latter features may be of particular value in patients who are most resistant to conventional ESA therapy as a result of acute or chronic inflammation. Traditionally, these are the patients who have been administered the highest doses of ESA. There are 2 potential downsides to prolyl hydroxylase inhibition. The first of these originates from trials of FG-2216. In a phase 2 clinical trial, a patient developed fatal hepatic necrosis, and this was related temporally to administration of the HIF stabilizer.29Astellas Pharma Inc.Adverse event of FG-2216 for the treatment of anemia Media Release May 07, 2007.http://www.astellas.com/global/about/news/2007/pdf/070507_eg.pdfGoogle Scholar As a result of this single death, as well as other patients who developed abnormal liver enzyme test results, the FDA suspended this clinical trial and no further experimental activity has been performed on humans with this molecule. The second-generation HIF stabilizer molecule from Fibrogen is FG-4592. This is now in phase 2 clinical trials, preliminary results of which were presented at the American Society of Nephrology Congress in Denver, CO, November 2010. In a CKD population, FG-4592 significantly increased hematocrits and also was found to decrease serum hepcidin levels in these patients.30Besarab A. Hulter H.N. Klaus S. et al.FG-4592, a novel oral HIF prolyl hydroxylase inhibitor, elevates hemoglobin in anemic stage 3/4 CKD patients.American Society of Nephrology Congress. 2010Google Scholar The second potential downside of prolyl hydroxylation is upregulation of several hundred other hypoxia-sensitive genes, including those involved in glucose regulation, angiogenesis, etc.31Toffoli S. Roegiers A. Feron O. et al.Intermittent hypoxia is an angiogenic inducer for endothelial cells: role of HIF-1.Angiogenesis. 2009; 12: 47-67Crossref PubMed Scopus (70) Google Scholar One of the most concerning has been the potential ability of these compounds to upregulate VEGF (vascular endothelial growth factor), which may have potential adverse effects on enhancing tumor growth and proliferative diabetic retinopathy. Thus, the ubiquitous nature of this new class of erythropoietic molecules requires careful evaluation. In addition to Fibrogen, several other companies are developing HIF stabilizers.32GlaxoSmithKline pipeline 1278863 prolyl hydroxylase inhibitor for anaemia associated with chronic renal disease.http://www.gsk.com/investors/product_pipeline/docs/gsk-pipeline-2011.pdfGoogle Scholar, 33Bayer pipeline news BAY 85-3934. HIF PH inhibitor in phase I trials for anemia in chronic kidney disease.http://www.press.bayer.com/baynews/baynews.nsf/id/2010-1515-eGoogle Scholar, 34Shalwitz R. Hartman C. Finn C. Shalwitz I. Logan D.K. AKB-6548, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor reduces hepcidin and ferritin while it increases reticulocyte production and total binding capacity in healthy adults.in: Proceedings of the American Society of Nephrology Kidney Week. November 8-13, 2011Google Scholar Hepcidin is a small defensin-like peptide produced largely by the liver, but also by other cells, such as the macrophage and adipocyte.35Verga Falzacappa M.V. Muckenthaler M.U. Hepcidin: iron-hormone and anti-microbial peptide.Gene. 2005; 364: 37-44Crossref PubMed Scopus (81) Google Scholar In addition to its antimicrobial properties, it is the master regulator of iron metabolism, controlling the amount of dietary iron absorbed from the duodenum and also the release of iron from cells in the reticuloendothelial system (Kupffer cells, splenic macrophages, etc; Fig 4) .36Babitt J.L. Lin H.Y. Molecular mechanisms of hepcidin regulation: implications for the anemia of CKD.Am J Kidney Dis. 2010; 55: 726-741Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar Hepcidin is upregulated by a variety of stimuli, such as inflammation and iron overload, and downregulated by anemia, hypoxia, and iron deficiency. It now is recognized that uremia, as a chronic inflammatory state, also upregulates hepcidin, and in particular, dialysis patients have much higher serum hepcidin levels than healthy individuals.37Macdougall I.C. Malyszko J. Hider R.C. Bansal S.S. Current status of the measurement of blood hepcidin levels in chronic kidney disease.Clin J Am Soc Nephrol. 2010; 5: 1681-1689Crossref PubMed Scopus (77) Google Scholar It currently is believed that this has a part in the pathogenesis of anemia in CKD by limiting iron availability to the bone marrow. At a molecular level, hepcidin binds to the main iron exporter protein ferroportin, which controls iron efflux from duodenal enterocytes, hepatocytes, and macrophages.38Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3632) Google Scholar The regulation of hepcidin is complex, but one of the major stimuli to its production is interleukin 6 (IL-6), produced as part of the inflammatory response. Other molecules, such as hemojuvelin and BMP-6 (bone morphogenetic protein 6), also have a role.39Kemna E.H. Tjalsma H. Willems H.L. Swinkels D.W. Hepcidin: from discovery to differential diagnosis.Haematologica. 2008; 93: 90-97Crossref PubMed Scopus (250) Google Scholar As with other inflammatory anemias, it has been hypothesized that antagonizing hepcidin may ameliorate the anemic state, and there is laboratory evidence to support this assumption. A group of scientists recently generated a monoclonal antibody against hepcidin and have shown that this improves anemia in an inflammatory mouse model.40Sasu B.J. Cooke K.S. Arvedson T.L. et al.Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.Blood. 2010; 115: 3616-3624Crossref PubMed Scopus (197) Google Scholar An RNA-based antagonist of hepcidin also has been created. It consists of a 44-nucleotide l-RNA oligonucleotide produced using so-called Spiegelmers technology (RNA molecules in which the ribose component is levorotatory, or the mirror image of the natural right-handed sugar moiety). The Spiegelmer is linked to a 40-kDa pegylation chain (NOX-H94), which has been shown to ameliorate the anemia of inflammation in cynomolgus monkeys.41Noxxon Pharma AG pipeline NOX-H94, 44-nucleotide l-RNA oligonucleotide linked to 40 kDa PEG.http://www.noxxon.com/index.php?option=com_content&view=article&id=88&Itemid=100Google Scholar Rather than antagonizing the hepcidin molecule per se, another strategy could be to inhibit the production of hepcidin. This could be achieved by using antisense oligonucleotides or silencing messenger RNA transcribed from the hepcidin gene (HAMP). None of the strategies to suppress hepcidin production or antagonize this peptide have been subjected to clinical trials. A theoretical concern could be that inhibition of hepcidin might exacerbate the risk of infections, given its endogenous antimicrobial properties. However, there are counterarguments to this suggestion, and it may be possible to suppress hepcidin to "safe" levels without obliterating hepcidin activity completely. The ability to upregulate the EPO gene by inhibiting GATA-2 also has been investigated.42Imagawa S. Yamamoto M. Miura Y. Negative regulation of the erythropoietin gene expression by the GATA transcription factors.Blood. 1997; 89: 1430-1439Crossref PubMed Google Scholar The GATA family of transcription factors inhibits EPO gene expression by acting on its promoter.43Ingley E. Tilbrook P.A. Klinken S.P. New insights into the regulation of erythroid cells IUBMB.Life. 2004; 56: 177-184PubMed Google Scholar Thus, it was hypothesized that inhibition of GATA would stimulate EPO gene expression and production and thereby enhance erythropoiesis. This has been reported with 2 GATA transcription factor inhibitors: K-7174 and K-11706.44Imagawa S. Nakano Y. Obara N. et al.A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA.FASEB J. 2003; 17: 1742-1744PubMed Google Scholar, 45Nakano Y. Imagawa S. Matsumoto K. et al.Oral administration of K-11706 inhibits GATA binding activity, enhances hypoxia-inducible factor 1 binding activity, and restores indicators in an in vivo mouse model of anemia of chronic disease.Blood. 2004; 104: 4300-4307Crossref PubMed Scopus (63) Google Scholar In the first series of experiments, Imagawa et al44Imagawa S. Nakano Y. Obara N. et al.A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA.FASEB J. 2003; 17: 1742-1744PubMed Google Scholar studied the effects of K-7174 in both a human hepatoma cell line (Hep3B cells in 1% oxygen) and an animal model of anemia and showed that this GATA-specific inhibitor potentiated erythropoietin protein production and EPO promoter activity that previously had been suppressed with IL-1β, TNF-α (tumor necrosis factor α), or NG-monomethyl l-arginine (L-NMMA). Electrophoretic mobility shift assays showed that the addition of K-7174 decreased GATA binding activity.44Imagawa S. Nakano Y. Obara N. et al.A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA.FASEB J. 2003; 17: 1742-1744PubMed Google Scholar Similarly, in the animal model, K-7174 was able to reverse the decrease in hemoglobin levels and reticulocyte counts induced by intraperitoneal injection of IL-1β or TNF-α in mice.44Imagawa S. Nakano Y. Obara N. et al.A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA.FASEB J. 2003; 17: 1742-1744PubMed Google Scholar The same group of Japanese scientists then investigated whether another molecule with GATA-inhibiting properties (K-11706) could improve erythropoietin production in the same cellular and animal models.45Nakano Y. Imagawa S. Matsumoto K. et al.Oral administration of K-11706 inhibits GATA binding activity, enhances hypoxia-inducible factor 1 binding activity, and restores indicators in an in vivo mouse model of anemia of chronic disease.Blood. 2004; 104: 4300-4307Crossref PubMed Scopus (63) Google Scholar As with K-7174, oral administration of K-11706 was able to reverse the decreases in hemoglobin and erythropoietin concentrations, reticulocyte counts, and numbers of erythroid colony-forming units induced by IL-1β or TNF-α.45Nakano Y. Imagawa S. Matsumoto K. et al.Oral administration of K-11706 inhibits GATA binding activity, enhances hypoxia-inducible factor 1 binding activity, and restores indicators in an in vivo mouse model of anemia of chronic disease.Blood. 2004; 104: 4300-4307Crossref PubMed Scopus (63) Google Scholar In comparing the 2 molecules, K-11706 was found to evoke greater hypoxic induction compared with K-7174, possibly through stimulation of HIF-1 binding activity in addition to GATA inhibition. Results from both these studies suggest a potential role for an orally administered GATA inhibitor in the treatment of anemia. However, as with the HIF stabilizers, there is concern that GATA inhibition will promote activation of other genes in addition to erythropoietin. Several years ago, a group of Israeli scientists developed a functional delivery system for the EPO gene using skin cells.46Brill-Almon E. Stern B. Afik D. et al.Ex vivo transduction of human dermal tissue structures for autologous implantation production and delivery of therapeutic proteins.Mol Ther. 2005; 12: 274-282Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar The early experiments were conducted in SCID mice, and the basic methodology involved extracting a microbiopsy specimen of dermal cells, harvesting them, and transducing them with the EPO gene (using an adenovirus vector in which the cytomegalovirus immediate early promoter drives EPO), and then reimplanting the preparation back into the SCID mice. The mice responded by producing increased levels of erythropoietin, and this was associated with an increase in hematocrit. No such effect was seen with the vector alone.46Brill-Almon E. Stern B. Afik D. et al.Ex vivo transduction of human dermal tissue structures for autologous implantation production and delivery of therapeutic proteins.Mol Ther. 2005; 12: 274-282Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar These animal data have now been translated into humans using Biopump technology (Medgenics, www.medgenics.com). A small group of patients with CKD in Israel have taken part in a proof-of-concept phase 1-2 clinical trial of this delivery system for the EPO gene. All patients showed increased erythropoietin production, with most showing sustained elevation of hemoglobin levels (the primary end point) in the target range of 10-12 g/dL for 6-12 months without receiving additional erythropoietin injections.47Medgenics press release April 27, 2010 Medgenics granted approval for extension of anaemia trial.http://www.medgenics.com/downloads/Announcement-MOH_270410.pdfGoogle Scholar One patient maintained hemoglobin levels in the normal range for more than 18 months without erythropoietin injections. Further evaluation of this novel therapeutic strategy is ongoing.47Medgenics press release April 27, 2010 Medgenics granted approval for extension of anaemia trial.http://www.medgenics.com/downloads/Announcement-MOH_270410.pdfGoogle Scholar This review summarizes our current knowledge about a variety of new strategies for stimulating erythropoiesis. These are not only of interest scientifically, but also could yield therapeutic agents in the future. As with all treatments for anemia, there will be both efficacy and safety considerations. The lessons we learned from recombinant human erythropoietin therapy were that although we proved early that this agent could increase hemoglobin levels, it took us nearly 20 years to realize the limitations of this therapy and the potential for harm if used too aggressively. None of the newer agents has outcomes data showing superiority to existing ESAs, and none has been tested in sufficient numbers of hyporesponsive patients to know whether the outcomes in these patients are different from those with conventional ESAs. They therefore will need to be subjected to the same degree of scientific investigation as the existing ESAs, and it may be many years before the true efficacy-safety balance of these novel scientific strategies is realized.