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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),
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).
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.
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.
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 Agents
Stage of Development
Dimeric pegylated peptide
Synthetic peptide chemistry
Completed Phase 3
Prolyl hydroxylase inhibitor
Planning phase 1
EPO gene therapy (EPODURE)
Skin cells (microdermis) transfected with the EPO gene
Biopump technology, harvesting skin biopsies and using adenovirus as vector
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.
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.
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) .
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.
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.
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.
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).
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.
Correction 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 alfa
Correction study: peginesatide vs darbepoetin alfa in nondialysis patients (SC)
∼330 vs 165 (US and Europe)
Maintenance study: peginesatide vs epoetin alfa in dialysis patients (IV)
∼540 vs 270 (US)
Efficacy and safety of peginesatide noninferior to epoetin
Maintenance 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.
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) .
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.
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.
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).
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.
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.
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.
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.
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) .
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Erythropoietin Gene Therapy
Several years ago, a group of Israeli scientists developed a functional delivery system for the EPO gene using skin cells.
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.
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.
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.
Financial Disclosure: Professor Macdougall has received consultancy fees for advisory work, research grants, and honoraria for giving lectures from several manufacturers of ESAs, including those already licensed for clinical use and those in clinical development (Affymax, Amgen, Hospira, Ortho Biotech, Roche, Sandoz, and Takeda). He does not own stock or shares in any pharmaceutical company. There was no input from a medical writer or the pharmaceutical industry in the preparation of this article.
Working capacity is increased following recombinant human erythropoietin treatment.