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Erythropoietins: A common mechanism of action

Open ArchivePublished:October 15, 2008DOI:https://doi.org/10.1016/j.exphem.2008.08.003
      Clinical development of erythropoiesis-stimulating agents (ESAs) revolutionized the management of anemia. The major clinical benefits of ESAs are effective treatment of anemia and avoidance of blood transfusion risks. Erythropoietin (EPO) interacts directly with the EPO receptor on the red blood cell (RBC) surface, triggering activation of several signal transduction pathways, resulting in the proliferation and terminal differentiation of erythroid precursor cells and providing protection from RBC precursor apoptosis. The magnitude of increase in RBC concentration in response to administration of recombinant human EPO products (rhEPO) is primarily controlled by the length of time EPO concentrations are maintained, not by the EPO concentration level. Subcutaneous (SC) EPO administration results in slower absorption than intravenous (IV) administration, leading to lower peak plasma levels and an apparent extended terminal half-life. However, SC administration requires additional needle-sticks and is associated with an increased risk of immunogenicity compared with IV administration. Multiple pathways may play a role in EPO clearance from the body. Epoetin alfa was the first rhEPO produced and approved for pharmaceutical use, followed by several related products and by newer ESAs with the same mechanism but more prolonged action. Darbepoetin alfa is a hyperglycosylated EPO analog with an extended terminal half-life and a greater relative potency compared with rhEPO at extended dosing intervals. PEGylation of EPO (addition of polyethylene glycol) has been used to further extend the terminal half-life. Also, new strategies are under investigation for stimulating erythropoiesis through activation of the EPO receptor.
      Erythropoiesis, a complex physiologic process maintaining homeostasis of oxygen (O2) levels in the body, is primarily regulated by erythropoietin (EPO), a 30-kDa, 165—amino acid hematopoietic growth factor produced by the kidneys [
      • Erslev A.
      Humoral regulation of red cell production.
      ,
      • Lai P.H.
      • Everett R.
      • Wang F.F.
      • Arakawa T.
      • Goldwasser E.
      Structural characterization of human erythropoietin.
      ]. Under normal conditions, endogenous EPO levels change with O2 tension. In the presence of EPO, bone marrow erythroid precursor cells proliferate and differentiate into red blood cells (RBCs). In its absence, these cells undergo apoptosis [
      • Kelley L.L.
      • Green W.F.
      • Hicks G.G.
      • Bondurant M.C.
      • Koury M.J.
      • Ruley H.E.
      Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.
      ,
      • Koury M.J.
      • Bondurant M.C.
      Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.
      ].
      The human EPO gene was cloned in 1983 [
      • Lin F.K.
      • Suggs S.
      • Lin C.H.
      • et al.
      Cloning and expression of the human erythropoietin gene.
      ], allowing for clinical development of recombinant human EPO (rhEPO), a biotechnological advance that revolutionized anemia treatment. Endogenous EPO and rhEPO share the same amino acid sequence, with slight differences in the sugar profile [
      • Skibeli V.
      • Nissen-Lie G.
      • Torjesen P.
      Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin.
      ]. In clinical practice, rhEPO is typically administered as a bolus injection, and the dose is titrated to give the desired effect. Administration of rhEPO initially corresponded to clinical practice patterns, with treatments being synchronized to dialysis frequencies or chemotherapy cycle schedules.
      Attempts to improve or “reengineer” rhEPO to meet the demands of patients and caregivers resulted in additional erythropoiesis-stimulating agents (ESAs) with increased serum half-lives (compared with rhEPO), as well as different receptor binding properties and in vivo biological potencies [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ,
      • Delorme E.
      • Lorenzini T.
      • Giffin J.
      • et al.
      Role of glycosylation on the secretion and biological activity of erythropoietin.
      ,
      • Egrie J.C.
      • Dwyer E.
      • Browne J.K.
      • Hitz A.
      • Lykos M.A.
      Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
      ,
      • Macdougall I.C.
      • Robson R.
      • Opatrna S.
      • et al.
      Pharmacokinetics and pharmacodynamics of intravenous and subcutaneous continuous erythropoietin receptor activator (C.E.R.A.) in patients with chronic kidney disease.
      ]. The characteristics and properties of these new ESAs allowed extension of the dosing intervals beyond the original thrice weekly (TIW) administration to weekly (QW), once every 2 weeks (Q2 W), once every 3 weeks (Q3 W), and even monthly (QM) administration [
      • Locatelli F.
      • Olivares J.
      • Walker R.
      • et al.
      Novel erythropoiesis stimulating protein for treatment of anemia in chronic renal insufficiency.
      ,
      • Nissenson A.R.
      • Swan S.K.
      • Lindberg J.S.
      • et al.
      Randomized, controlled trial of darbepoetin alfa for the treatment of anemia in hemodialysis patients.
      ,
      • Ling B.
      • Walczyk M.
      • Agarwal A.
      • Carroll W.
      • Liu W.
      • Brenner R.
      Darbepoetin alfa administered once monthly maintains hemoglobin concentrations in patients with chronic kidney disease.
      ,
      • Sulowicz W.
      • Locatelli F.
      • Ryckelynck J.P.
      • et al.
      Once-monthly subcutaneous C.E.R.A. maintains stable hemoglobin control in patients with chronic kidney disease on dialysis and converted directly from epoetin one to three times weekly.
      ,
      • Canon J.L.
      • Vansteenkiste J.
      • Bodoky G.
      • et al.
      Randomized, double-blind, active-controlled trial of every-3-week darbepoetin alfa for the treatment of chemotherapy-induced anemia.
      ]
      All ESAs share the same mechanism of action, binding to and activating the EPO receptor (EPOR), but differences in pharmacokinetic, pharmacodynamic, and receptor-binding properties affect their clinical use. In this review, we examine the biology of erythropoiesis and EPO and evaluate the limitations and opportunities afforded by new approaches to stimulating erythropoiesis through activation of the EPOR.

      Erythropoiesis

      A primary function of RBCs is to transport O2 from the lungs to O2-dependent tissues. Changes in O2 levels necessitate both acute and long-term physiologic adaptations. Acute adaptations include increases in respiration and heart rate, vasoconstriction, and changes in blood volume; however, these changes cannot be sustained. Erythropoiesis is a longer—term adaptation to boost O2-carrying capacity by increasing the concentration of RBCs, and thus, hemoglobin (Hb) concentration.
      RBCs are the most abundant (∼99%) circulating cells in the bloodstream, representing 40% to 45% of total blood volume. In a healthy human with ∼5 L blood, this represents approximately 2.5×1013 cells, a quantity substantial enough to provide the large O2 transport capacity needed to support aerobic respiration. In humans, the RBC lifespan is ∼100 to 120 days, with a daily loss of ∼0.8% to 1.0% of circulating RBCs. To match this loss, the body assumes a normal, prodigious production capacity of ∼2.5×1011 cells/day.
      RBC production results from a tightly controlled proliferation and differentiation pathway (Fig. 1). Early hematopoietic progenitors differentiate into burst-forming unit–erythroid cells, in which EPORs appear for the first time; however, EPO is not required at this stage [
      • Wu H.
      • Liu X.
      • Jaenisch R.
      • Lodish H.F.
      Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
      ]. Burst-forming unit–erythroid cells differentiate into colony-forming unit–erythroid cells, which are dependent on EPO for survival, and there is a corresponding rise in expression of EPORs [
      • Broudy V.C.
      • Lin N.
      • Brice M.
      • Nakamoto B.
      • Papayannopoulou T.
      Erythropoietin receptor characteristics on primary human erythroid cells.
      ,
      • Sawada K.
      • Krantz S.B.
      • Kans J.S.
      • et al.
      Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.
      ]. Continued stimulation with EPO triggers differentiation into erythroblasts, which enucleate to form reticulocytes and after a few days show loss of “reticulin,” resulting in RBCs. Reticulocytes and RBCs stop expressing EPOR and cease being responsive to EPO [
      • Sawada K.
      • Krantz S.B.
      • Kans J.S.
      • et al.
      Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.
      ].
      Figure thumbnail gr1
      Figure 1Erythropoiesis. BFUe=burst-forming unit–erythroid; CFUe=colony-forming unit–erythroid; Epo=erythropoietin; FLK=fetal liver kinase; FLT=fetal liver tyrosine kinase; G-CSF=granulocyte-colony stimulating factor; GEMM=granulocyte, erythrocyte, monocyte, megakaryocyte; GM-CSF=granulocyte macrophage CSF; IL=interleukin; MIP=macrophage inflammatory protein; SCF=stem cell factor; TGF=transforming growth factor; TPO=thrombopoietin.
      Disease states and environmental conditions often alter the tightly controlled balance between RBC production and destruction. When RBC loss exceeds gain, anemia results. Increased RBC loss can occur because of bleeding, enhanced destruction (chemically induced hematotoxicity), or reduced lifespan (sickle cell anemia). Potential causes of insufficient RBC production include defects in O2 sensing, excess of erythropoiesis inhibitors, and inadequate concentrations of ESAs.

      EPO

      Erythropoiesis primarily occurs in the kidney, but other organs (liver, brain) also produce EPO. Interstitial fibroblasts produce EPO in the kidney [
      • Koury S.T.
      • Bondurant M.C.
      • Koury M.J.
      Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization.
      ,
      • Lacombe C.
      • Da Silva J.L.
      • Bruneval P.
      • et al.
      Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney.
      ,
      • Maxwell P.H.
      • Osmond M.K.
      • Pugh C.W.
      • et al.
      Identification of the renal erythropoietin-producing cells using transgenic mice.
      ], while hepatocytes produce EPO in the liver [
      • Koury S.T.
      • Bondurant M.C.
      • Koury M.J.
      • Semenza G.L.
      Localization of cells producing erythropoietin in murine liver by in situ hybridization.
      ]. Initially, EPO is synthesized as a 193-amino-acid precursor. A 27-amino-acid signal peptide and C-terminal arginine are removed, and carbohydrate is added to three N-linked glycosylation sites and one O-linked glycosylation site [
      • Browne J.K.
      • Cohen A.M.
      • Egrie J.C.
      • et al.
      Erythropoietin: gene cloning, protein structure, and biological properties.
      ]. The secreted protein contains 165 amino acids and is heavily glycosylated, with ∼40% of its mass composed of carbohydrate. The structure of rhEPO is a compact globular bundle that contains four α helices (Fig. 2).
      Figure thumbnail gr2
      Figure 2The nuclear magnetic resonance minimized average structure of human erythropoietin.
      Generally, serum EPO concentrations of 10 to 25 mU/mL [
      • Erslev A.J.
      Erythropoietin titers in health and disease.
      ] maintain Hb levels within the normal range of 12 to 17 g/dL [
      • Groopman J.E.
      • Itri L.M.
      Chemotherapy-induced anemia in adults: incidence and treatment.
      ]. The terminal half-life (t1/2) of EPO is ∼5 hours [
      • Eckardt K.U.
      • Boutellier U.
      • Kurtz A.
      • Schopen M.
      • Koller E.A.
      • Bauer C.
      Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia.
      ], which requires an average EPO production rate of ∼2 U/kg/day. The EPO production rate per cell appears constant [
      • Koury S.T.
      • Koury M.J.
      • Bondurant M.C.
      • Caro J.
      • Graber S.E.
      Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration.
      ], with fluctuations in EPO synthesis resulting from changes in the number of cells producing the molecule. In cases of severe anemia, circulating EPO levels can increase up to 1000—fold because of a logarithmic increase in the number of cells producing EPO [
      • Erslev A.J.
      Erythropoietin titers in health and disease.
      ,
      • Koury S.T.
      • Koury M.J.
      • Bondurant M.C.
      • Caro J.
      • Graber S.E.
      Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration.
      ]. Other factors affecting EPO levels include iron availability, nutritional status, disease or comorbidities, environmental conditions, and genetic factors (congenital polycythemias).

      EPO-RBC time–response relationship

      A direct correlation exists between RBC production and serum EPO concentrations [
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ,
      • Eschbach J.W.
      • Egrie J.C.
      • Downing M.R.
      • Browne J.K.
      • Adamson J.W.
      Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.
      ]. However, the rate of erythropoiesis change (∼4—fold) [
      • Eschbach J.W.
      • Egrie J.C.
      • Downing M.R.
      • Browne J.K.
      • Adamson J.W.
      Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.
      ]) is small compared to the larger change in EPO concentrations (∼1000—fold) [
      • Erslev A.J.
      • Wilson J.
      • Caro J.
      Erythropoietin titers in anemic, nonuremic patients.
      ]. Thus, the magnitude of increase in RBC concentration is primarily controlled by the length of time EPO concentrations are maintained, and not by the EPO concentration level per se (Fig. 3). Increased EPO synthesis has a prolonged effect due to the disproportionate relationship between EPO t1/2 and RBC lifespan. Thirty minutes of hypoxia can result in production of EPO (t1/2 ∼5 hours) [
      • Eckardt K.U.
      • Boutellier U.
      • Kurtz A.
      • Schopen M.
      • Koller E.A.
      • Bauer C.
      Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia.
      ]. In turn, EPO stimulates formation of enucleated reticulocytes (t1/2=1–5 days) [
      • Finch C.A.
      • Harker L.A.
      • Cook J.D.
      Kinetics of the formed elements of human blood.
      ,
      • Hillman R.S.
      • Finch C.A.
      Erythropoiesis: normal and abnormal.
      ], which rapidly mature into RBCs that have a long lifespan (100–120 days) [
      • Smith J.A.
      Exercise, training and red blood cell turnover.
      ]. Thus, a short duration of EPO exposure results in a prolonged increase in RBC concentration.
      Figure thumbnail gr3
      Figure 3Disproportion between half-life of recombinant human erythropoietin and lifespan of red blood cells. MEC=minimum effective dose. Adapted from Molineux
      [
      • Molineux G.
      Biology of erythropoietin In: Molineux G.
      ]
      with permission.

      EPO and EPOR

      The mechanism of action by which EPO stimulates erythropoiesis has been under extensive investigation. Early evidence indicated that EPO interacted with a protein on the cell surface, triggering activation of the JAK-signal transducers and activators of transcription, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase pathways (Fig. 4), resulting in the proliferation and terminal differentiation of erythroid precursor cells and providing protection from apoptosis [
      • Koury M.J.
      • Bondurant M.C.
      Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.
      ]. The EPO-binding component on cells was first detected by measuring physical attachment of radiolabeled EPO to erythroid precursor cells [
      • Sawada K.
      • Krantz S.B.
      • Kans J.S.
      • et al.
      Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.
      ,
      • Sawyer S.T.
      • Krantz S.B.
      • Goldwasser E.
      Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells.
      ]. The EPOR gene was subsequently identified by expression cloning and found to be a single gene with no apparent homologs [
      • Jones S.S.
      • D'Andrea A.D.
      • Haines L.L.
      • Wong G.G.
      Human erythropoietin receptor: cloning, expression, and biologic characterization.
      ,
      • D'Andrea A.D.
      • Lodish H.F.
      • Wong G.G.
      Expression cloning of the murine erythropoietin receptor.
      ].
      Figure thumbnail gr4
      Figure 4Signal transduction pathways of the erythropoietin receptor. Binding of erythropoietin (EPO) causes conformational changes to the EPO receptor, transphosphorylation of associated JAK2 molecules, phosphorylation of tyrosine residues in the cytoplasmic tail of the receptor, and phosphorylation or activation of signaling molecules. Phosphorylation of signal transducers and activators of transcription (STAT) 5 transcription factor (TF) causes homodimerization, translocation to the nucleus, and activation of genes for antiapoptotic molecules. Phosphorylated phosphatidylinositol 3-kinase (PI-3 kinase) phosphorylates protein kinase B (PKB)/Akt. PKB/Akt: 1) phosphorylates and inactivates proapoptotic molecules (Bad, caspase 9 or glycogen synthase kinase-3b [GSK-3b]); 2) phosphorylates FOXO TF, inhibiting translocation to the nucleus and activation of target genes (Fas ligand, Bim); and 3) phosphorylates IκB, allowing the release of the transcription factor nuclear factor (NF)-κB that then translocates into the nucleus and activates target genes encoding antiapoptotic molecules (XIAP, c-IAP2). Binding of EPO to its receptor also activates Hsp70, which binds to and inactivates proapoptotic molecules (apoptosis protease-activating factor-1 [Apaf-1], apoptosis-inducing factor [AIF]).
      While other components may mediate affinity or aid in signal transduction, the activation of signal transduction is initiated by an early, direct interaction of EPO with EPOR. Activation of EPOR occurs following cross-linking of two EPORs via one EPO ligand [
      • Syed R.S.
      • Reid S.W.
      • Li C.
      • et al.
      Efficiency of signalling through cytokine receptors depends critically on receptor orientation.
      ,
      • Philo J.S.
      • Aoki K.H.
      • Arakawa T.
      • Narhi L.O.
      • Wen J.
      Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction.
      ,
      • Elliott S.
      • Lorenzini T.
      • Yanagihara D.
      • Chang D.
      • Elliott G.
      Activation of the erythropoietin (EPO) receptor by bivalent anti-EPO receptor antibodies.
      ,
      • Elliott S.
      • Lorenzini T.
      • Chang D.
      • Barzilay J.
      • Delorme E.
      Mapping of the active site of recombinant human erythropoietin.
      ], which induces a conformational change in the receptor, triggering downstream signal transduction [
      • Constantinescu S.N.
      • Keren T.
      • Socolovsky M.
      • Nam H.
      • Henis Y.I.
      • Lodish H.F.
      Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain.
      ,
      • Kubatzky K.F.
      • Liu W.
      • Goldgraben K.
      • Simmerling C.
      • Smith S.O.
      • Constantinescu S.N.
      Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function.
      ,
      • Seubert N.
      • Royer Y.
      • Staerk J.
      • et al.
      Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer.
      ].

      Receptor affinity and in vitro potency

      The affinity (Kd) of EPO for its receptor on human cells is ∼100 to 200 pM [
      • Broudy V.C.
      • Lin N.
      • Brice M.
      • Nakamoto B.
      • Papayannopoulou T.
      Erythropoietin receptor characteristics on primary human erythroid cells.
      ,
      • Krantz S.B.
      • Sawyer S.T.
      • Sawada K.I.
      Purification of erythroid progenitor cells and characterization of erythropoietin receptors.
      ,
      • Sawada K.
      • Krantz S.B.
      • Sawyer S.T.
      • Civin C.I.
      Quantitation of specific binding of erythropoietin to human erythroid colony-forming cells.
      ], which is sufficient for low concentrations of EPO to maintain a Hb of ∼14 g/dL in healthy subjects. Normal circulating concentrations of EPO are ∼2 to 5 pM [
      • Erslev A.J.
      Erythropoietin titers in health and disease.
      ], significantly below the EPO:EPOR Kd. At the half-maximal effective dose (ED50; ∼70 mU [
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ,
      • Fraser J.K.
      • Lin F.K.
      • Berridge M.V.
      Expression of high affinity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL.
      ]) 6.8% of the receptors are occupied [
      • Fraser J.K.
      • Lin F.K.
      • Berridge M.V.
      Expression of high affinity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL.
      ], suggesting that only a fraction of the receptors need be occupied by EPO to achieve an adequate erythropoiesis maintenance rate.
      Increased EPOR occupancy does not increase the rate of cell division, but instead increases the rate of RBC formation by recruitment and differentiation of more erythroid precursor cells. However, the erythropoiesis rate is maximized when all available erythroid progenitors are actively dividing. This was evident from phase I clinical trials with epoetin α in which the rate of hematocrit rise showed dose-dependent increases to a plateau at a 200- to 500-U/kg dose [
      • Eschbach J.W.
      • Egrie J.C.
      • Downing M.R.
      • Browne J.K.
      • Adamson J.W.
      Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.
      ]. Higher doses did not further increase the rate of rise, but did increase the overall response by extending the exposure time and the duration of enhanced erythropoiesis.
      If EPOR occupancy is inadequate, apoptosis of precursor cells occurs [
      • Koury M.J.
      • Bondurant M.C.
      Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.
      ], with apoptosis beginning in as little as 2 to 8 hours following removal of EPO from the culture [
      • Kelley L.L.
      • Green W.F.
      • Hicks G.G.
      • Bondurant M.C.
      • Koury M.J.
      • Ruley H.E.
      Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.
      ,
      • Koury M.J.
      • Bondurant M.C.
      Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.
      ,
      • Somervaille T.C.
      • Linch D.C.
      • Khwaja A.
      Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax.
      ]. Formation of erythroblasts from colony-forming unit–erythroid cells can take up to a week. Thus, a single EPO–EPOR binding event is insufficient for stimulation of complete differentiation of early erythroid precursors. Instead, adequate EPO concentrations must be present during the entire process to ensure survival, proliferation, and differentiation to mature RBCs. Only during the final stages of erythropoiesis is EPO no longer required for RBC survival [
      • Fraser J.K.
      • Lin F.K.
      • Berridge M.V.
      Expression of high affinity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL.
      ,
      • Landschulz K.T.
      • Noyes A.N.
      • Rogers O.
      • Boyer S.H.
      Erythropoietin receptors on murine erythroid colony-forming units: natural history.
      ,
      • Mayeux P.
      • Billat C.
      • Jacquot R.
      The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage.
      ].
      EPO derivatives or analogs with reduced receptor affinity may require higher concentrations to maintain an effective number of occupied EPORs. Although low binding affinity can be overcome with higher dosing and the rate of erythropoiesis corresponds to the duration of EPO exposure [
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ], low receptor binding activity may be undesirable in some disease states, such as EPO resistance. In the case of longer-acting agents with very low receptor affinity, there may be low receptor occupancy for an extended period and consequently, a reduced rate of erythropoiesis, resulting in a slower rate of Hb rise.

      Anemia treatment and development of rhEPO

      Before development of rhEPO, blood transfusion was the most common treatment for patients with anemia. However, blood transfusions carry inherent risks, including risk of transmission of infectious agents and iron overload. Additionally, the blood supply is limited, and immune reactions developed after transfusion can make organ transplantation more problematic [
      • Perrotta P.L.
      • Snyder E.L.
      Non-infectious complications of transfusion therapy.
      ]. Iron supplementation was largely ineffective as a stand-alone treatment for anemia. The need for an effective anemia treatment option was obvious, and attempts to make and test rhEPO via cloning of the human EPO gene began.
      Successfully cloning the EPO gene was difficult, as low circulating EPO levels made protein purification difficult, a primary source of EPO mRNA was not obvious, and mRNA was difficult to obtain. Once small quantities of purified human EPO became available (10 mg from 1000 L urine from human patients with aplastic anemia), oligonucleotide probes for EPO were designed. Two different probes were used to screen a λ phage library containing sheared human genomic DNA, and the human EPO gene was cloned [
      • Lin F.K.
      • Suggs S.
      • Lin C.H.
      • et al.
      Cloning and expression of the human erythropoietin gene.
      ].

      rhEPO—The first ESA

      Successful cloning of the EPO gene in 1983 [
      • Lin F.K.
      • Suggs S.
      • Lin C.H.
      • et al.
      Cloning and expression of the human erythropoietin gene.
      ] allowed for the large-scale production of rhEPO and its subsequent clinical use [
      • Eschbach J.W.
      • Egrie J.C.
      • Downing M.R.
      • Browne J.K.
      • Adamson J.W.
      Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.
      ]. Epoetin alfa (Epogen; Amgen Inc., Thousand Oaks, CA, USA; Procrit; Ortho Biotech Products, L.P., Bridgewater, NJ, USA) was the first rhEPO commercialized in the United States, followed by a second epoetin α (Eprex; Ortho Biotech Products) and epoetin beta (NeoRecormon; F. Hoffmann-La Roche Ltd., Basel, Switzerland) in Europe. Epoetins alfa and beta, both produced by Chinese hamster ovary cells, have minor structural differences but the same physiological effects [
      • Lasne F.
      • de Ceaurriz J.
      Recombinant erythropoietin in urine.
      ]. An epoetin produced in baby hamster kidney cells, epoetin omega, differs from previous epoetins in the glycosylation profile. More recently, epoetin delta (Dynepo, Shire plc, Basingstoke, UK), produced from an engineered human fibrosarcoma cell line HT1080, has been described [
      • Martin K.J.
      The first human cell line-derived erythropoietin, epoetin-delta (Dynepo), in the management of anemia in patients with chronic kidney disease.
      ]. As patents for epoetins α and β expire, follow-on or biosimilar epoetins (Binocrit [Sandoz International GmbH, Holzkirchen, Germany]; epoetin α HEXAL [Hexal Biotech Forschungs GmbH, Oberhaching, Germany], and Abseamed [Medice Arzneimittel Puetter GmbH & Co. KG, Iserlohn, Germany]), have been approved in Europe.

      Pharmacokinetics

      In healthy volunteers, the t1/2 of intravenous (IV) rhEPO ranged from 5 to 11 hours [
      • Heatherington A.C.
      Clinical pharmacokinetic properties of rHuEpo: a review.
      ], similar to that of endogenous EPO (average t1/2=5.2 hours) [
      • Eckardt K.U.
      • Boutellier U.
      • Kurtz A.
      • Schopen M.
      • Koller E.A.
      • Bauer C.
      Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia.
      ]. The volume of distribution was generally similar to the plasma volume (40–60 mL/kg), indicating limited extravascular distribution. Subcutaneous (SC) administration resulted in slower absorption, leading to lower peak plasma levels (5–10% of those seen with IV administration) and an apparent extended t1/2 (∼20–25 hours) [
      • Heatherington A.C.
      Clinical pharmacokinetic properties of rHuEpo: a review.
      ]. Peak plasma levels are reached in most studies between 15 and 29 hours.
      Bioavailability estimates for SC rhEPO range from about 20% to 40%, suggesting a substantial loss of material during transport from the interstitial space to the lymphatic system and blood [
      • Heatherington A.C.
      Clinical pharmacokinetic properties of rHuEpo: a review.
      ]. The pharmacokinetic characteristics of rhEPO in healthy volunteers appear similar or comparable to those in several other populations, including chronic kidney disease (CKD), liver cirrhosis, and myelodysplastic syndrome patients [
      • Heatherington A.C.
      Clinical pharmacokinetic properties of rHuEpo: a review.
      ].

      Clearance

      The mechanism of rhEPO clearance and the site of degradation still are not definitively characterized. The observation that rhEPO clearance is dose-dependent and saturable is consistent with at least two clearance pathways [
      • Veng-Pedersen P.
      • Chapel S.
      • Al-Huniti N.H.
      • et al.
      Pharmacokinetic tracer kinetics analysis of changes in erythropoietin receptor population in phlebotomy-induced anemia and bone marrow ablation.
      ,
      • Veng-Pedersen P.
      • Widness J.A.
      • Pereira L.M.
      • Schmidt R.L.
      • Lowe L.S.
      A comparison of nonlinear pharmacokinetics of erythropoietin in sheep and humans.
      ,
      • Kinoshita H.
      • Ohishi N.
      • Kato M.
      • Tokura S.
      • Okazaki A.
      Pharmacokinetics and distribution of recombinant erythropoietin in rats.
      ,
      • Kato M.
      • Kamiyama H.
      • Okazaki A.
      • Kumaki K.
      • Kato Y.
      • Sugiyama Y.
      Mechanism for the nonlinear pharmacokinetics of erythropoietin in rats.
      ], and emerging data suggest that multiple pathways play some role in clearance [
      • Agoram B.
      • Molineux G.
      • Jang G.
      • et al.
      Effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: a minor role of erythropoietin receptor-mediated pathways.
      ].
      Clearance was first thought to be mediated primarily through the liver and kidney [
      • Dinkelaar R.B.
      • Engels E.Y.
      • Hart A.A.
      • Schoemaker L.P.
      • Bosch E.
      • Chamuleau R.A.
      Metabolic studies on erythropoietin (EP): II. The role of liver and kidney in the metabolism of Ep.
      ] or via the EPOR on receptor-expressing cells. However, subsequent research indicated neither the liver [
      • Dinkelaar R.B.
      • Engels E.Y.
      • Hart A.A.
      • Schoemaker L.P.
      • Bosch E.
      • Chamuleau R.A.
      Metabolic studies on erythropoietin (EP): II. The role of liver and kidney in the metabolism of Ep.
      ,
      • Jensen J.D.
      • Jensen L.W.
      • Madsen J.K.
      • Poulsen L.
      The metabolism of erythropoietin in liver cirrhosis patients compared with healthy volunteers.
      ] nor the kidney [
      • Macdougall I.C.
      • Roberts D.E.
      • Coles G.A.
      • Williams J.D.
      Clinical pharmacokinetics of epoetin (recombinant human erythropoietin).
      ,
      • Yoon W.H.
      • Park S.J.
      • Kim I.C.
      • Lee M.G.
      Pharmacokinetics of recombinant human erythropoietin in rabbits and 3/4 nephrectomized rats.
      ,
      • Widness J.A.
      • Veng-Pedersen P.
      • Schmidt R.L.
      • Lowe L.S.
      • Kisthard J.A.
      • Peters C.
      In vivo 125I-erythropoietin pharmacokinetics are unchanged after anesthesia, nephrectomy and hepatectomy in sheep.
      ] plays a major role in EPO clearance.
      Binding of EPO to the EPOR can lead to cellular internalization, during which the ligand may be degraded [
      • Sawyer S.T.
      • Krantz S.B.
      • Goldwasser E.
      Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells.
      ,
      • Gross A.W.
      • Lodish H.F.
      Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP).
      ]. Chemotherapy, which reduces the number of EPOR-bearing cells, reduces EPO clearance [
      • Veng-Pedersen P.
      • Chapel S.
      • Al-Huniti N.H.
      • et al.
      Pharmacokinetic tracer kinetics analysis of changes in erythropoietin receptor population in phlebotomy-induced anemia and bone marrow ablation.
      ,
      • Chapel S.
      • Veng-Pedersen P.
      • Hohl R.J.
      • Schmidt R.L.
      • McGuire E.M.
      • Widness J.A.
      Changes in erythropoietin pharmacokinetics following busulfan-induced bone marrow ablation in sheep: evidence for bone marrow as a major erythropoietin elimination pathway.
      ,
      • Chapel S.H.
      • Veng-Pedersen P.
      • Schmidt R.L.
      • Widness J.A.
      Receptor-based model accounts for phlebotomy-induced changes in erythropoietin pharmacokinetics.
      ,
      • Glaspy J.
      • Henry D.
      • Patel R.
      • et al.
      Effects of chemotherapy on endogenous erythropoietin levels and the pharmacokinetics and erythropoietic response of darbepoetin alfa: a randomised clinical trial of synchronous versus asynchronous dosing of darbepoetin alfa.
      ]. Thus, one clearance pathway may involve uptake and degradation of EPO via the EPOR-expressing cells, but it is unlikely to be the only one and may not necessarily be the major pathway. Bone marrow cells can deplete ESAs through non—EPOR pathways in vitro, and absent or reduced binding activity of some rhEPO analogs with the EPOR resulted in only modest reductions in clearance [
      • Agoram B.
      • Molineux G.
      • Jang G.
      • et al.
      Effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: a minor role of erythropoietin receptor-mediated pathways.
      ].
      In healthy men, only a small amount of intact radiolabeled epoetin β (<5% of the dose) was found to be excreted in the urine, suggesting that rhEPO is degraded elsewhere in the body [
      • Yoon W.H.
      • Park S.J.
      • Kim I.C.
      • Lee M.G.
      Pharmacokinetics of recombinant human erythropoietin in rabbits and 3/4 nephrectomized rats.
      ]. Therefore, one important pathway may be degradation or metabolism in the interstitium, possibly via cells in the reticuloendothelial scavenging pathway or lymphatic system [
      • Flaharty K.K.
      Clinical pharmacology of recombinant human erythropoietin (r-HuEPO).
      ]. Consistent with this hypothesis, the lymphatic system is believed to play an important role in the reduced bioavailability after SC administration of proteins [
      • Porter C.J.
      • Charman S.A.
      Lymphatic transport of proteins after subcutaneous administration.
      ]. In addition, only small peptides or free 125I, and not intact material, are detected in tissues following IV administration of 125I—darbepoetin α, suggesting that degradation may occur in tissue [
      • Agoram B.
      • Sutjandra L.
      • Molineux G.
      • Jang G.
      • Elliott S.
      Tissue distribution and excretion of 125I-darbepoetin alfa in Sprague Dawley rats following a single subcutaneous or intravenous administration abstract].
      ].

      SC vs IV treatment efficiency

      While potency and dose of rhEPO are drug-related considerations in treatment choice, other considerations arise as a result of practice patterns and the patient population. SC administration requires additional needle-sticks and is associated with an increased risk of immunogenicity [
      • Schellekens H.
      Bioequivalence and the immunogenicity of biopharmaceuticals.
      ]. IV administration may be preferred in hemodialysis patients who already have IV access enabled.

      Development of additional ESAs

      Introduction of epoetins into clinical practice represented a milestone in anemia treatment, yet opportunities for further improvement in certain patient populations and clinical settings remained. The initial TIW dosing regimen for hemodialysis patients proved to be burdensome and inconvenient in other clinical settings such as CKD and oncology. Thus, research and development of additional treatment opportunities continued.

      Design considerations

      While all ESAs have the same mechanism regarding EPOR activation, they differ in molecular structure, receptor binding affinity, serum t1/2, clearance, bioavailability, and in vivo potency. Together, these characteristics shape the clinical efficacy and safety of these agents, as well as their versatility, especially in terms of dosing schedules.
      The clinical advantages of extended dosing regimens led to development of newer ESAs with a longer duration of action, but comparable clinical benefit. For example, extending the t1/2 may allow for extended-dosing schedules that offer patients and caregivers, especially those with CKD not on dialysis and cancer patients, the convenience of less frequent visits. Increased molecular stability may also decrease degradation products and the risk of immunogenicity, as well as allow for alternate storage opportunities or delivery systems (e.g., oral administration or slow delivery via pumps).
      Potential limitations or trade-offs need to be considered. Too long a t1/2 can result in loss of Hb control or Hb cycling, while too short a t1/2 may limit the range of extended dosing regimens. Too high an affinity for the EPOR may affect the dose-response relationship, while insufficient binding may result in a slower or incomplete response. Additionally, differences in IV vs SC potency may result in the need for an increased drug dose with the less-efficient route. These factors must be considered when developing new ESAs and will vary with patient needs, indication, region, and clinical practice. Ultimately, these newer molecules should offer patients efficacy and safety at least comparable with the original ESAs.

      Potential strategies

      Researchers have taken several approaches to reengineer and thereby extend treatment options with original ESAs. Prolonging the time of erythropoietic stimulation can increase response, which may allow for extending the dosing interval. Increasing the dose of faster—clearing molecules is one strategy, but this can be inefficient because of the high doses required. A more dose-conserving strategy is to increase serum t1/2 [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ,
      • Egrie J.C.
      • Dwyer E.
      • Browne J.K.
      • Hitz A.
      • Lykos M.A.
      Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
      ,
      • Sasu B.J.
      • Hartley C.
      • Schultz H.
      • et al.
      Comparison of epoetin alfa and darbepoetin alfa biological activity under different administration schedules in normal mice.
      ]. Administration of a long-acting ESA may extend serum residence time, resulting in an increased relative biological response over time [
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ].
      Early attempts to improve rhEPO included enhancing molecular stability or increasing affinity for EPOR [
      • Elliott S.G.
      New molecules and formulations of recombinant human erythropoietin.
      ]. However, increased affinity did not necessarily increase in vivo biological potency, because serum t1/2 was the primary driver of the degree of response [
      • Egrie J.C.
      • Dwyer E.
      • Browne J.K.
      • Hitz A.
      • Lykos M.A.
      Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
      ,
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ,
      • Egrie J.
      • Browne J.
      Darbepoetin alfa is more potent in vivo and can be administered less frequently than rHuEPO.
      ]. The exception was modifications that yielded molecules with an affinity that was too low, resulting in ineffective EPOR dimerization and activation.
      Many different approaches to extend the t1/2 have been considered, including addition of polyethylene glycol (PEG), hyperglycosylation, dimerization, and physical fusion of the peptide portion of the ESA to other molecules, such as antibodies or other proteins (e.g., albumin) [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ,
      • Macdougall I.C.
      • Eckardt K.U.
      Novel strategies for stimulating erythropoiesis and potential new treatments for anaemia.
      ,
      • Bunn H.F.
      New agents that stimulate erythropoiesis.
      ]. Successful approaches took advantage of the well-established safety and efficacy profile of rhEPO by modifying the “nonfunctional” regions without loss of structure or substantial reductions in affinity for EPOR. Any structural loss is of particular concern because of the increased potential for degradation or molecular instability of the drug with long-term storage, and hence, reduced potency and potentially increased risk of immunogenicity. Reduced affinity for EPOR may occur due to steric hindrance from the attachments (e.g., PEG [
      • Chen S.A.
      • Sawchuk R.J.
      • Brundage R.C.
      • et al.
      Plasma and lymph pharmacokinetics of recombinant human interleukin-2 and polyethylene glycol-modified interleukin-2 in pigs.
      ,
      • Kochendoerfer G.
      Chemical and biological properties of polymer-modified proteins.
      ]) or changes in electrostatic properties of the molecule (hyperglycosylation) [
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ,
      • Darling R.J.
      • Kuchibhotla U.
      • Glaesner W.
      • Micanovic R.
      • Witcher D.R.
      • Beals J.M.
      Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.
      ], which directly or indirectly inhibit binding.

      Hyperglycosylation

      Both endogenous and recombinant EPO have microheterogeneity in carbohydrate structures with variation in sialic acid content—up to 14 attached sialic acid residues [
      • Egrie J.
      • Browne J.
      Darbepoetin alfa is more potent in vivo and can be administered less frequently than rHuEPO.
      ,
      • Rush R.S.
      • Derby P.L.
      • Strickland T.W.
      • Rohde M.F.
      Peptide mapping and evaluation of glycopeptide microheterogeneity derived from endoproteinase digestion of erythropoietin by affinity high-performance capillary electrophoresis.
      ,
      • Sasaki H.
      • Bothner B.
      • Dell A.
      • Fukuda M.
      Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA.
      ,
      • Takeuchi M.
      • Kobata A.
      Structures and functional roles of the sugar chains of human erythropoietins.
      ]. The importance of sialic acid content for clearance was noted in experiments with rhEPO isoforms, revealing a direct and positive relationship between sialic acid content and in vivo potency [
      • Egrie J.
      • Browne J.
      Darbepoetin alfa is more potent in vivo and can be administered less frequently than rHuEPO.
      ]. The molecules with increased sialic acid content had reduced affinity for the EPOR and increased serum t1/2, suggesting that a longer t1/2 was a stronger determinant of potency than was receptor affinity. Consequently, the mixture of glycoforms was important in defining the biologic properties of the particular rhEPO products. This observation also led to the hypothesis that “reengineering” of the EPO molecule, by adding more carbohydrate chains and increasing the sialic acid content (>14 residues), might prolong the serum t1/2 and enhance potency compared with rhEPO [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ].

      Darbepoetin α

      Darbepoetin α is a hyperglycosylated analog that contains two additional N-linked carbohydrate chains at positions 30 and 88, as a result of five amino acid substitutions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr) (Fig. 5) [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ]. The new carbohydrates did not directly interfere with receptor binding or disrupt the structure or stability of the molecule. The carbohydrate portion increased from 40% to 51%, and the approximate molecular weight increased from 30 kDa to 37 kDa. The theoretical maximum number of sialic acid residues increased from 14 to 22.
      Figure thumbnail gr5
      Figure 5Molecular structures of rhEPO (A) and darbepoetin alfa (B). Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology
      [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ]
      , 2003. rhEPO=recombinant human erythropoietin.
      Compared with epoetin α, darbepoetin α had a threefold longer serum t1/2, a lower receptor-binding affinity, and enhanced in vivo bioactivity in multiple species [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ,
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ,
      • Sasu B.J.
      • Hartley C.
      • Schultz H.
      • et al.
      Comparison of epoetin alfa and darbepoetin alfa biological activity under different administration schedules in normal mice.
      ,
      • Egrie J.
      • Browne J.
      Darbepoetin alfa is more potent in vivo and can be administered less frequently than rHuEPO.
      ]. In a phase I trial in rhEPO-naïve patients receiving peritoneal dialysis [
      • Macdougall I.C.
      • Gray S.J.
      • Elston O.
      • et al.
      Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients.
      ], darbepoetin α administered IV had an approximately threefold longer mean terminal t1/2 compared with rhEPO (25.3 vs 8.5 hours, respectively), a more than twofold greater area under the curve (291±8 vs 132±88 ng h/mL), and a fourfold lower biphasic clearance (1.6±0.3 vs 4.0±0.3 mL/h/kg). The volume of distribution was similar for the two molecules (52.4±2.0 and 48.7±2.1 mL/kg, respectively). The mean terminal t1/2 for darbepoetin α SC was longer than that for IV administration (∼49 hours); Cmax averaged about 10% of the IV value, Tmax averaged 54±5 hours, and mean bioavailability was 37%.
      Relative potency of darbepoetin α compared with rhEPO increased when the dosing interval was extended. At TIW dosing, three times more rhEPO was needed than darbepoetin α to maintain or elicit a similar erythropoietic response in normal mice [
      • Egrie J.C.
      • Dwyer E.
      • Browne J.K.
      • Hitz A.
      • Lykos M.A.
      Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
      ,
      • Sasu B.J.
      • Hartley C.
      • Schultz H.
      • et al.
      Comparison of epoetin alfa and darbepoetin alfa biological activity under different administration schedules in normal mice.
      ]. With QW administration, the difference increased 13-fold. With a single injection, the rhEPO dose needed to be 30 to 40 times higher to match the effect of the lower dose of darbepoetin α. In humans receiving rhEPO or darbepoetin α QW, the dose of rhEPO needed to be 45% higher than that of darbepoetin α to maintain Hb levels in CKD patients [
      • Tolman C.
      • Richardson D.
      • Bartlett C.
      • Will E.
      Structured conversion from thrice weekly to weekly erythropoietic regimens using a computerized decision-support system: a randomized clinical study.
      ]. The differences in relative potencies likely resulted from the threefold longer serum t1/2 of darbepoetin α, allowing for a prolonged period of time for erythroid cell exposure to the ESA [
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ].
      The apparent paradox—that darbepoetin α has lower receptor-binding affinity but increased in vivo activity—was explained by the counteracting effects of sialic acid–containing carbohydrate on clearance [
      • Egrie J.C.
      • Dwyer E.
      • Browne J.K.
      • Hitz A.
      • Lykos M.A.
      Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
      ,
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ]. At extended time intervals, the darbepoetin α relative concentration greatly exceeded that of rhEPO. Thus, prolonged exposure more than compensated for the reduced receptor-binding affinity, resulting in increased in vivo activity.
      Unlike rhEPO, studies with darbepoetin α in humans demonstrated that the dose efficiency after SC and IV administration was approximately equivalent, with similar doses providing similar efficacy (i.e., similar Hb increases) [
      • Locatelli F.
      • Canaud B.
      • Giacardy F.
      • Martin-Malo A.
      • Baker N.
      • Wilson J.
      Treatment of anaemia in dialysis patients with unit dosing of darbepoetin alfa at a reduced dose frequency relative to recombinant human erythropoietin (rHuEpo).
      ]. The apparent t1/2 of darbepoetin α administered SC is extended approximately twofold relative to IV administration, which compensates for decreased bioavailability [
      • Macdougall I.C.
      • Gray S.J.
      • Elston O.
      • et al.
      Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients.
      ].

      PEGylation

      PEGylation, the addition of PEG to reactive regions of proteins or carbohydrates by either solution or solid-phase chemistry, has been used successfully to extend the serum t1/2 of many different recombinant proteins [
      • Yamaoka T.
      • Tabata Y.
      • Ikada Y.
      Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice.
      ]. PEGylated molecules have an increased hydrodynamic size because a “water shell” surrounds the molecule. The increased hydrodynamic size can result in reduced clearance because of a reduced rate of translocation from blood to extravascular tissues [
      • Yamaoka T.
      • Tabata Y.
      • Ikada Y.
      Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice.
      ]. PEG is thought to be relatively inert and nonimmunogenic and, thus, to be a suitable starting material for protein conjugate therapeutics. However, PEGylation of a molecule does not guarantee protection against an immune response [
      • Basser R.L.
      • O'Flaherty E.
      • Green M.
      • et al.
      Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor.
      ].
      One issue with drugs made by solution or solid-phase chemistry is poor specificity of conjugation in the chemical reaction, with generation of undesirable byproducts. Current chemistries typically target the reactive amino groups on lysine or the amino terminal amine [
      • Francis G.E.
      • Fisher D.
      • Delgado C.
      • Malik F.
      • Gardiner A.
      • Neale D.
      PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimisation of coupling techniques.
      ]. Consequently, it is difficult to target specific reactive amine groups. PEGylated rhEPO molecules typically contain mixtures of molecules with PEG attached to different reactive amines, each of which may have differential effects on activity and protein folding. For example, if lysines are proximal to active sites involved in receptor binding, PEGylation can reduce binding activity and, consequently, in vitro activity, either by altering essential amino acids important in EPOR binding or by steric hindrance [
      • Long D.L.
      • Doherty D.H.
      • Eisenberg S.P.
      • et al.
      Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.
      ,
      • Macdougall I.C.
      CERA (Continuous Erythropoietin Receptor Activator): a new erythropoiesis-stimulating agent for the treatment of anemia.
      ,
      • Jarsch M.
      • Brandt M.
      • Lanzendorfer M.
      • Haselbeck A.
      Comparative erythropoietin receptor binding kinetics of C.E.R.A. and epoetin-beta determined by surface plasmon resonance and competition binding assay.
      ].
      Low affinity for EPOR associated with amine chemistry may be overcome via other PEGylation strategies. Cysteine substitutions at targeted “nonfunctional” regions can allow addition of the conjugate with high specificity to the sulfhydryl group with retention of in vitro activity [
      • Long D.L.
      • Doherty D.H.
      • Eisenberg S.P.
      • et al.
      Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.
      ,
      • Goodson R.J.
      • Katre N.V.
      Site-directed pegylation of recombinant interleukin-2 at its glycosylation site.
      ,
      • He X.H.
      • Shaw P.C.
      • Tam S.C.
      Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation.
      ]. Another strategy is to produce PEGylated EPO synthetically. During synthesis, a PEG—conjugated amino acid is introduced in place of the unconjugated amino acid, allowing targeting of particular amino acid positions for PEG attachment, such as the glycosylation sites, and reducing the potential for loss of in vitro activity [
      • Chen S.A.
      • Sawchuk R.J.
      • Brundage R.C.
      • et al.
      Plasma and lymph pharmacokinetics of recombinant human interleukin-2 and polyethylene glycol-modified interleukin-2 in pigs.
      ,
      • Chen S.Y.
      • Cressman S.
      • Mao F.
      • et al.
      Synthetic erythropoietic proteins: tuning biological performance by site-specific polymer attachment.
      ]. Both strategies may allow for extended serum t1/2. However, it is unclear if these particular molecules will have similar stability, in vivo activity, and lack of immunogenicity to their glycosylated counterparts.

      Development of PEGylated erythropoiesis-stimulating agents

      PEGylated EPO molecules with potential clinical utility have been considered [
      • Long D.L.
      • Doherty D.H.
      • Eisenberg S.P.
      • et al.
      Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.
      ,
      • Macdougall I.C.
      CERA (Continuous Erythropoietin Receptor Activator): a new erythropoiesis-stimulating agent for the treatment of anemia.
      ,
      • Jolling K.
      • Ruixo J.J.
      • Hemeryck A.
      • Piotrovskij V.
      • Greway T.
      Population pharmacokinetic analysis of pegylated human erythropoietin in rats.
      ]. Methoxy polyethylene glycol-epoetin β (PEG-epoetin β; Mircera; F. Hoffmann-La Roche Ltd., Basel, Switzerland), recently approved by regulatory agencies in the United States and Europe, is a PEGylated form of epoetin β. Pharmacokinetic parameters of PEG-epoetin β were measured in patients receiving peritoneal dialysis [
      • Macdougall I.C.
      • Robson R.
      • Opatrna S.
      • et al.
      Pharmacokinetics and pharmacodynamics of intravenous and subcutaneous continuous erythropoietin receptor activator (C.E.R.A.) in patients with chronic kidney disease.
      ]. Mean t1/2 was 134 hours when PEG-epoetin β was administered IV and 139 hours when administered SC. Clearance was 0.49 and 0.90 mL/h/kg, respectively, and SC bioavailability was 52%. Another version of PEG-rhEPO was examined in rats, with similar conclusions [
      • Jolling K.
      • Ruixo J.J.
      • Hemeryck A.
      • Piotrovskij V.
      • Greway T.
      Population pharmacokinetic analysis of pegylated human erythropoietin in rats.
      ]. PEG—epoetin β did not display “flip-flop” kinetics (absorption constant much slower than elimination constant) after SC administration because of the significantly decreased systemic clearance and corresponding increase in t1/2.
      Other PEGylated ESAs include PEGylated darbepoetin α [
      • Agoram B.
      • Molineux G.
      • Jang G.
      • et al.
      Effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: a minor role of erythropoietin receptor-mediated pathways.
      ] and Hematide (Affymax Inc., Palo Alto, CA, USA) [
      • Stead R.B.
      • Lambert J.
      • Wessels D.
      • et al.
      Evaluation of the safety and pharmacodynamics of Hematide, a novel erythropoietic agent, in a phase 1, double-blind, placebo-controlled, dose-escalation study in healthy volunteers.
      ]. Mean t1/2 in rats of a PEG-darbepoetin was reported to be 24.3 hours compared with 17.5 hours for darbepoetin α [
      • Agoram B.
      • Molineux G.
      • Jang G.
      • et al.
      Effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: a minor role of erythropoietin receptor-mediated pathways.
      ]. Hematide is a synthetic PEGylated dimeric peptide that binds to and activates EPOR. Because of the rapid clearance of the peptides, Hematide was PEGylated to extend the serum t1/2, which was found to be 21 to 30 hours in rats [
      • Fan Q.
      • Leuther K.K.
      • Holmes C.P.
      • et al.
      Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia.
      ].

      Impact of PEG ylation and hyperglycosylation on clearance

      Both hyperglycosylation and PEGylation of ESAs can reduce receptor-binding affinity, although the reduction is substantially greater for PEGylated vs hyperglycosylated rhEPO (50- to 100-fold vs 5-fold) [
      • Elliott S.
      • Lorenzini T.
      • Asher S.
      • et al.
      Enhancement of therapeutic protein in vivo activities through glycoengineering.
      ,
      • Egrie J.C.
      • Dwyer E.
      • Browne J.K.
      • Hitz A.
      • Lykos M.A.
      Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
      ,
      • Elliott S.
      • Egrie J.
      • Browne J.
      • et al.
      Control of rHuEPO biological activity: the role of carbohydrate.
      ,
      • Kochendoerfer G.
      Chemical and biological properties of polymer-modified proteins.
      ,
      • Macdougall I.C.
      CERA (Continuous Erythropoietin Receptor Activator): a new erythropoiesis-stimulating agent for the treatment of anemia.
      ,
      • Jarsch M.
      • Brandt M.
      • Lanzendorfer M.
      • Haselbeck A.
      Comparative erythropoietin receptor binding kinetics of C.E.R.A. and epoetin-beta determined by surface plasmon resonance and competition binding assay.
      ]. The reduced EPOR binding activity results in corresponding decreases in in vitro potency with these molecules. It has been suggested that the reduced clearance of PEGylated and hyperglycosylated ESAs is due to their effect on receptor-mediated endocytosis and degradation, which has been reported in vitro [
      • Gross A.W.
      • Lodish H.F.
      Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP).
      ,
      • Jarsch M.
      • Brandt M.
      • Lanzendorfer M.
      • Haselbeck A.
      Comparative erythropoietin receptor binding kinetics of C.E.R.A. and epoetin-beta determined by surface plasmon resonance and competition binding assay.
      ]. However, clearance studies with rhEPO analogs with altered receptor-binding characteristics suggested PEGylated and hyperglycosylated ESAs have reduced clearance due to their impact on non-EPOR–mediated clearance pathways [
      • Agoram B.
      • Molineux G.
      • Jang G.
      • et al.
      Effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: a minor role of erythropoietin receptor-mediated pathways.
      ,
      • Long D.L.
      • Doherty D.H.
      • Eisenberg S.P.
      • et al.
      Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.
      ,
      • Chen S.Y.
      • Cressman S.
      • Mao F.
      • et al.
      Synthetic erythropoietic proteins: tuning biological performance by site-specific polymer attachment.
      ].
      Hyperglycosylated or PEGylated rhEPO and analogs have increased hydrodynamic size due to attached hydrated carbohydrate or PEG [
      • Yamaoka T.
      • Tabata Y.
      • Ikada Y.
      Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice.
      ]. Thus, the reduced clearance of these molecules may be partially explained by steric factors. For example, the transport of ESAs from the blood to the clearing organs may be reduced as the size of the molecule increases, as was reported for unconjugated PEG [
      • Yamaoka T.
      • Tabata Y.
      • Ikada Y.
      Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice.
      ].

      ESA use in clinical practice

      ESAs have been used successfully to treat anemia in many different patient populations, helping to prevent or minimize use of blood transfusions. ESAs have been used primarily to treat anemia associated with kidney failure and chemotherapy-induced anemia. Additional indications have been considered, including anemia associated with cancer (including myelodysplasia), anemia of chronic disease, anemia associated with AIDS treatment, and treatment in perisurgery indications. In some disease settings (e.g., end-stage renal disease and chronic kidney disease without dialysis), there is a consistent relationship between ESA treatment, increased hemoglobin, and improved quality of life [
      • Macdougall I.C.
      • Temple R.M.
      • Kwan J.T.
      Is early treatment of anaemia with epoetin-alpha beneficial to pre-dialysis chronic kidney disease patients? Results of a multicentre, open-label, prospective, randomized, comparative group trial.
      ,
      • Revicki D.A.
      • Brown R.E.
      • Feeny D.H.
      • et al.
      Health-related quality of life associated with recombinant human erythropoietin therapy for predialysis chronic renal disease patients.
      ,
      • Perlman R.L.
      • Finkelstein F.O.
      • Liu L.
      • et al.
      Quality of life in chronic kidney disease (CKD): a cross-sectional analysis in the Renal Research Institute-CKD study.
      ,
      • Roger S.D.
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      • Clarkson A.
      • et al.
      Effects of early and late intervention with epoetin alpha on left ventricular mass among patients with chronic kidney disease (stage 3 or 4): results of a randomized clinical trial.
      ,
      • Singh A.K.
      • Szczech L.
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      • et al.
      Correction of anemia with epoetin alfa in chronic kidney disease.
      ,
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      • Locatelli F.
      • Clyne N.
      • et al.
      Normalization of hemoglobin level in patients with chronic kidney disease and anemia.
      ]. This is particularly true for measures related to energy, physical function, and exercise. While results are mixed in the oncology setting, some studies suggest aspects of quality of life, such as fatigue, may be improved by the resultant increases in Hb levels [
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      ].
      Practice patterns vary with the indication and geographical region. Dosing frequency is often associated with visits to health care professionals and the duration of action of the ESA. When rhEPO first became available, it was used to treat anemia in patients on hemodialysis. Such patients may attend either a dialysis unit or a hospital twice or thrice weekly for hemodialysis, and frequent administration of ESAs that coincided with hemodialysis sessions was deemed practical. For CKD patients who are not on dialysis and for cancer patients receiving chemotherapy, extended dosing regimens may be more convenient and, in some cases, more cost efficient. Thus, subsequent clinical studies have investigated different dosing schedules (QW, Q2 W, Q3 W, and QM) across different clinical settings [
      • Locatelli F.
      • Olivares J.
      • Walker R.
      • et al.
      Novel erythropoiesis stimulating protein for treatment of anemia in chronic renal insufficiency.
      ,
      • Nissenson A.R.
      • Swan S.K.
      • Lindberg J.S.
      • et al.
      Randomized, controlled trial of darbepoetin alfa for the treatment of anemia in hemodialysis patients.
      ,
      • Ling B.
      • Walczyk M.
      • Agarwal A.
      • Carroll W.
      • Liu W.
      • Brenner R.
      Darbepoetin alfa administered once monthly maintains hemoglobin concentrations in patients with chronic kidney disease.
      ,
      • Sulowicz W.
      • Locatelli F.
      • Ryckelynck J.P.
      • et al.
      Once-monthly subcutaneous C.E.R.A. maintains stable hemoglobin control in patients with chronic kidney disease on dialysis and converted directly from epoetin one to three times weekly.
      ,
      • Provenzano R.
      • Bhaduri S.
      • Singh A.K.
      Extended epoetin alfa dosing as maintenance treatment for the anemia of chronic kidney disease: the PROMPT study.
      ,
      • Benz R.
      • Schmidt R.
      • Kelly K.
      • Wolfson M.
      Epoetin alfa once every 2 weeks is effective for initiation of treatment of anemia of chronic kidney disease.
      ]. Current practice patterns, even when limited to the nephrology setting, vary considerably. Data from the Dialysis Outcomes Practice Patterns trial show a broad spectrum of ESA dosing patterns and hemoglobin outcomes in different countries (Table 1) [
      • Locatelli F.
      • Pisoni R.L.
      • Combe C.
      • et al.
      Anaemia in haemodialysis patients of five European countries: association with morbidity and mortality in the Dialysis Outcomes and Practice Patterns Study (DOPPS).
      ].
      Table 1Hemoglobin and erythropoiesis-stimulating agent (ESA) therapy in prevalent hemodialysis patients from the Dialysis Outcomes and Practice Patterns Study
      Modified from Locatelli et al. [107].
      Hemoglobin (median)ESA therapy (mean±SEM)Doses per week (% of patients)
      CountrynHb (g/dL)n% of Patients treated SCDose (units/kg/week)n
      France52111.158175±1.8102±4.81442473814
      Germany56510.958677±1.7386±2.92467711711
      Italy55610.859586±1.44132±4.04527503018
      Spain50811.655191±1.25114±3.7510493615
      UK44011.045492±1.3107±3.642260318
      Total259011.1276784±0.71109±1.762368553014
      ESA=erythropoiesis-stimulating agent; Hb, hemoglobin; SC=subcutaneous; SEM, standard error of the mean.
      a Modified from Locatelli et al.
      • Locatelli F.
      • Pisoni R.L.
      • Combe C.
      • et al.
      Anaemia in haemodialysis patients of five European countries: association with morbidity and mortality in the Dialysis Outcomes and Practice Patterns Study (DOPPS).
      .
      In the oncology setting, patients were initially dosed twice or thrice weekly primarily because of the short serum t1/2 of rhEPO and the common use of these regimens in the nephrology setting. However, dose frequencies were subsequently extended to QW to minimize needle-sticks and unnecessary visits to health care professionals. Less frequent administration of longer-lived ESAs (e.g., darbepoetin α) that coincide with QW, Q2 W, and Q3 W chemotherapy cycles have also been explored.
      ESA therapy has transformed anemia management in both the renal and oncology settings. The major advantages of ESAs have been the treatment of anemia and avoidance or minimization of blood transfusions, as well as possible improvements in quality of life associated with higher Hb levels. However, there may be an Hb limit beyond which the risk-to-benefit ratio is no longer favorable for the patient. The frequency of dosing of the various ESAs is driven by a number of factors, including practice patterns and the duration of action of the ESA. While extending the dosing interval of ESA therapy is often possible, the major considerations from the clinical and pharmacoeconomic viewpoints are whether Hb levels can be effectively maintained within a target range, e.g., 11 to 12 g/dL, and whether there is any dose penalty (i.e., need for dose increases greater than those calculated by simple multiplication of time intervals) for extending the dosing-frequency schedule. We have expanded our understanding of the ways in which molecules stimulate erythropoiesis, including the interaction of EPO with its receptor and the effect on in vitro and in vivo activity. Further refinement of this understanding to probe the trade-off between the receptor affinity of ESAs and the circulating t1/2 of the molecule indicated that there is a balance between these biologic properties that translates into the clinical arena. It is to be hoped that greater appreciation of this phenomenon will foster high-quality randomized controlled trials to determine the optimal use for each ESA.

      Acknowledgments

      The authors wish to thank Michael Raffin and Beatrice Benoit of Nexus Communications, Inc. for their editorial assistance on this manuscript.

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