Experimental Hematology 2008;36:15731584

Erythropoietins: A common mechanism of action

Steve Elliotta, Elizabeth Phama, and Iain C. Macdougallb
a

Amgen Inc, Thousand Oaks, Calif., USA; bRenal Unit, Kings College Hospital, London, UK (Received 20 June 2008; revised 20 June 2008; accepted 12 August 2008)

Clinical development of erythropoiesis-stimulating agents (ESAs) revolutionized the management of anemia. The major clinical benets 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 needlesticks 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 rst 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. 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Erythropoiesis, a complex physiologic process maintaining homeostasis of oxygen (O2) levels in the body, is primarily regulated by erythropoietin (EPO), a 30-kDa, 165damino acid hematopoietic growth factor produced by the kidneys [1,2]. 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 [3,4]. The human EPO gene was cloned in 1983 [5], 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

S.E. and E.P. are employees of Amgen. I.C.M. has received support grants from Amgen Inc, Ortho Biotech, Roche, and Affymax, and is a consultant for Amgen Inc, Ortho Biotech, Roche, and Affymax. Offprint requests to: Steve Elliott, Ph.D., Amgen Inc., One Amgen Center, M/S 29-1-A, Thousand Oaks, CA 91320-1799; E-mail: selliott@ amgen.com

sugar prole [6]. 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 [710]. 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 [1115] 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

0301-472X/08 $see front matter. Copyright 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2008.08.003

1574

S. Elliott et al./ Experimental Hematology 2008;36:15731584

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 longerdterm adaptation to boost O2-carrying capacity by increasing the concentration of RBCs, and thus, hemoglobin (Hb) concentration. RBCs are the most abundant (w99%) circulating cells in the bloodstream, representing 40% to 45% of total blood volume. In a healthy human with w5 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 w100 to 120 days, with a daily loss of w0.8% to 1.0% of circulating RBCs. To match this loss, the body assumes a normal, prodigious production capacity of w2.5 1011 cells/day. RBC production results from a tightly controlled proliferation and differentiation pathway (Fig. 1). Early hematopoietic progenitors differentiate into burst-forming
Multipotent hematopoietic stem cell
IL-1 IL-3 + IL-5 IL-11 G-CSF SCF FLT-3/FLK-2
TGFMIP1-

uniterythroid cells, in which EPORs appear for the rst time; however, EPO is not required at this stage [16]. Burst-forming uniterythroid cells differentiate into colony-forming uniterythroid cells, which are dependent on EPO for survival, and there is a corresponding rise in expression of EPORs [17,18]. 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 [18]. 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 insufcient 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 broblasts produce EPO in the kidney [1921], while hepatocytes produce EPO in the liver [22]. Initially, EPO is synthesized as a 193-amino-acid precursor. A 27-amino-acid signal

CFU-GEMM
IL-3 + IL-9 GM-CSF SCF TPO

BFUe

IL-3 IL-9 IGF-1 SCF EPO

Cytokine receptor

EPO receptor
EPO

Erythrocyte

Reticulocyte

Erythroblast

CFUe

Figure 1. Erythropoiesis. BFUe 5 burst-forming uniterythroid; CFUe 5 colony-forming uniterythroid; Epo 5 erythropoietin; FLK 5 fetal liver kinase; FLT 5 fetal liver tyrosine kinase; G-CSF 5 granulocyte-colony stimulating factor; GEMM 5 granulocyte, erythrocyte, monocyte, megakaryocyte; GMCSF 5 granulocyte macrophage CSF; IL 5 interleukin; MIP 5 macrophage inammatory protein; SCF 5 stem cell factor; TGF 5 transforming growth factor; TPO 5 thrombopoietin.

S. Elliott et al./ Experimental Hematology 2008;36:15731584

1575

peptide and C-terminal arginine are removed, and carbohydrate is added to three N-linked glycosylation sites and one O-linked glycosylation site [23]. The secreted protein contains 165 amino acids and is heavily glycosylated, with w40% of its mass composed of carbohydrate. The structure of rhEPO is a compact globular bundle that contains four a helices (Fig. 2). Generally, serum EPO concentrations of 10 to 25 mU/ mL [24] maintain Hb levels within the normal range of 12 to 17 g/dL [25]. The terminal half-life (t1/2) of EPO is w5 hours [26], which requires an average EPO production rate of w2 U/kg/day. The EPO production rate per cell appears constant [27], with uctuations 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 1000dfold because of a logarithmic increase in the number of cells producing EPO [24,27]. Other factors affecting EPO levels include iron availability, nutritional status, disease or comorbidities, environmental conditions, and genetic factors (congenital polycythemias). EPO-RBC timeresponse relationship A direct correlation exists between RBC production and serum EPO concentrations [28,29]. However, the rate of erythropoiesis change (w4dfold) [29]) is small compared to the larger change in EPO concentrations (w1000dfold) [30]. 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 w5 hours) [26]. In turn, EPO stimulates formation of enucleated reticulocytes (t1/2 5 15 days) [31,32], which rapidly mature into RBCs that have a long lifespan (100120 days) [33]. Thus, a short duration of EPO exposure results in a prolonged increase in RBC concentration. 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 [4]. The EPO-binding component on cells was rst detected by measuring physical attachment of radiolabeled EPO to erythroid precursor cells [18,34]. The EPOR gene was subsequently identied by expression cloning and found to be a single gene with no apparent homologs [35,36]. While other components may mediate afnity 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 [3740], which induces a conformational change in the receptor, triggering downstream signal transduction [4143]. Receptor afnity and in vitro potency The afnity (Kd) of EPO for its receptor on human cells is w100 to 200 pM [17,44,45], which is sufcient for low concentrations of EPO to maintain a Hb of w14 g/dL in healthy subjects. Normal circulating concentrations of EPO are w2 to 5 pM [24], signicantly below the EPO:EPOR Kd. At the half-maximal effective dose (ED50; w70 mU [28,46]) 6.8% of the receptors are occupied [46], 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 a in which the rate of hematocrit rise showed dosedependent increases to a plateau at a 200- to 500-U/kg dose [29]. 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 [4], with apoptosis beginning in as little as 2 to 8 hours following removal of EPO from the culture

Figure 2. The nuclear magnetic resonance minimized average structure of human erythropoietin.

1576

S. Elliott et al./ Experimental Hematology 2008;36:15731584

Serum level Reticulocyte response Hemoglobin response

Baseline level (or MEC) A duration of serum residence B duration of reticulocyte response Administration of ESA C duration of hemoglobin response

Time axis
Figure 3. Disproportion between half-life of recombinant human erythropoietin and lifespan of red blood cells. MEC 5 minimum effective dose. Adapted from Molineux [108] with permission.

[3,4,47]. Formation of erythroblasts from colony-forming uniterythroid cells can take up to a week. Thus, a single EPOEPOR binding event is insufcient 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 nal stages of erythropoiesis is EPO no longer required for RBC survival [46,48,49]. EPO derivatives or analogs with reduced receptor afnity may require higher concentrations to maintain an effective number of occupied EPORs. Although low binding afnity can be overcome with higher dosing and the rate of erythropoiesis corresponds to the duration of EPO exposure [28], 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 afnity, 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 [50]. 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 difcult, as low circulating EPO levels made protein purication difcult, a primary source of EPO mRNA was not obvious, and

mRNA was difcult to obtain. Once small quantities of puried 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 l phage library containing sheared human genomic DNA, and the human EPO gene was cloned [5]. rhEPOdThe rst ESA Successful cloning of the EPO gene in 1983 [5] allowed for the large-scale production of rhEPO and its subsequent clinical use [29]. Epoetin alfa (Epogen; Amgen Inc., Thousand Oaks, CA, USA; Procrit; Ortho Biotech Products, L.P., Bridgewater, NJ, USA) was the rst rhEPO commercialized in the United States, followed by a second epoetin a (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 [51]. An epoetin produced in baby hamster kidney cells, epoetin omega, differs from previous epoetins in the glycosylation prole. More recently, epoetin delta (Dynepo, Shire plc, Basingstoke, UK), produced from an engineered human brosarcoma cell line HT1080, has been described [52]. As patents for epoetins a and b expire, follow-on or biosimilar epoetins (Binocrit [Sandoz International GmbH, Holzkirchen, Germany]; epoetin a 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 [53], similar to that of endogenous EPO (average t1/2 5 5.2 hours) [26]. The volume of

S. Elliott et al./ Experimental Hematology 2008;36:15731584

1577

Figure 4. Signal 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 IkB, allowing the release of the transcription factor nuclear factor (NF)-kB 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]).

distribution was generally similar to the plasma volume (4060 mL/kg), indicating limited extravascular distribution. Subcutaneous (SC) administration resulted in slower absorption, leading to lower peak plasma levels (510% of those seen with IV administration) and an apparent extended t1/2 (w2025 hours) [53]. 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 [53]. 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 [53]. Clearance The mechanism of rhEPO clearance and the site of degradation still are not denitively characterized. The observation that rhEPO clearance is dose-dependent and saturable

1578

S. Elliott et al./ Experimental Hematology 2008;36:15731584

is consistent with at least two clearance pathways [5457], and emerging data suggest that multiple pathways play some role in clearance [58]. Clearance was rst thought to be mediated primarily through the liver and kidney [59] or via the EPOR on receptor-expressing cells. However, subsequent research indicated neither the liver [59,60] nor the kidney [6163] 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 [34,64]. Chemotherapy, which reduces the number of EPOR-bearing cells, reduces EPO clearance [54,6567]. 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 nondEPOR pathways in vitro, and absent or reduced binding activity of some rhEPO analogs with the EPOR resulted in only modest reductions in clearance [58]. In healthy men, only a small amount of intact radiolabeled epoetin b (!5% of the dose) was found to be excreted in the urine, suggesting that rhEPO is degraded elsewhere in the body [62]. Therefore, one important pathway may be degradation or metabolism in the interstitium, possibly via cells in the reticuloendothelial scavenging pathway or lymphatic system [68]. Consistent with this hypothesis, the lymphatic system is believed to play an important role in the reduced bioavailability after SC administration of proteins [69]. In addition, only small peptides or free 125I, and not intact material, are detected in tissues following IV administration of 125Iddarbepoetin a, suggesting that degradation may occur in tissue [70]. SC vs IV treatment efciency 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 [71]. 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 afnity, serum t1/2, clearance, bioavailability, and in vivo potency. Together, these characteristics shape the clinical efcacy 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 benet. 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 afnity for the EPOR may affect the dose-response relationship, while insufcient 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-efcient 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 efcacy 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 fasterdclearing molecules is one strategy, but this can be inefcient because of the high doses required. A more dose-conserving strategy is to increase serum t1/2 [7,9,72]. Administration of a longacting ESA may extend serum residence time, resulting in an increased relative biological response over time [28]. Early attempts to improve rhEPO included enhancing molecular stability or increasing afnity for EPOR [73]. However, increased afnity did not necessarily increase in vivo biological potency, because serum t1/2 was the primary driver of the degree of response [9,28,74]. The exception was modications that yielded molecules with an afnity 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) [7,75,76]. Successful approaches took advantage of the well-established safety and efcacy prole of rhEPO by

S. Elliott et al./ Experimental Hematology 2008;36:15731584

1579

modifying the nonfunctional regions without loss of structure or substantial reductions in afnity 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 afnity for EPOR may occur due to steric hindrance from the attachments (e.g., PEG [77,78]) or changes in electrostatic properties of the molecule (hyperglycosylation) [28,79], which directly or indirectly inhibit binding. Hyperglycosylation Both endogenous and recombinant EPO have microheterogeneity in carbohydrate structures with variation in sialic acid contentdup to 14 attached sialic acid residues [74,8082]. 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 [74]. The molecules with increased sialic acid content had reduced afnity for the EPOR and increased serum t1/2, suggesting that a longer t1/2 was a stronger determinant of potency than was receptor afnity. Consequently, the mixture of glycoforms was important in dening 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 (O14 residues), might prolong the serum t1/2 and enhance potency compared with rhEPO [7]. Darbepoetin a Darbepoetin a is a hyperglycosylated analog that contains two additional N-linked carbohydrate chains at positions 30 and 88, as a result of ve amino acid substitutions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr)

(Fig. 5) [7]. 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. Compared with epoetin a, darbepoetin a had a threefold longer serum t1/2, a lower receptor-binding afnity, and enhanced in vivo bioactivity in multiple species [7,28,72,74]. In a phase I trial in rhEPO-na ve patients receiving peritoneal dialysis [83], darbepoetin a 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 6 8 vs 132 6 88 ng h/mL), and a fourfold lower biphasic clearance (1.6 6 0.3 vs 4.0 6 0.3 mL/h/kg). The volume of distribution was similar for the two molecules (52.4 6 2.0 and 48.7 6 2.1 mL/kg, respectively). The mean terminal t1/2 for darbepoetin a SC was longer than that for IV administration (w49 hours); Cmax averaged about 10% of the IV value, Tmax averaged 54 6 5 hours, and mean bioavailability was 37%. Relative potency of darbepoetin a compared with rhEPO increased when the dosing interval was extended. At TIW dosing, three times more rhEPO was needed than darbepoetin a to maintain or elicit a similar erythropoietic response in normal mice [9,72]. 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 a. In humans receiving rhEPO or darbepoetin a QW, the dose of rhEPO needed to be 45% higher than that of darbepoetin a to maintain Hb levels in CKD patients [84]. The differences in relative potencies likely resulted from the threefold longer serum t1/2 of darbepoetin a, allowing for a prolonged period of time for erythroid cell exposure to the ESA [28].

Figure 5. Molecular structures of rhEPO (A) and darbepoetin alfa (B). Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology [7], 2003. rhEPO 5 recombinant human erythropoietin.

1580

S. Elliott et al./ Experimental Hematology 2008;36:15731584

The apparent paradoxdthat darbepoetin a has lower receptor-binding afnity but increased in vivo activitydwas explained by the counteracting effects of sialic acidcontaining carbohydrate on clearance [9,28]. At extended time intervals, the darbepoetin a relative concentration greatly exceeded that of rhEPO. Thus, prolonged exposure more than compensated for the reduced receptor-binding afnity, resulting in increased in vivo activity. Unlike rhEPO, studies with darbepoetin a in humans demonstrated that the dose efciency after SC and IV administration was approximately equivalent, with similar doses providing similar efcacy (i.e., similar Hb increases) [85]. The apparent t1/2 of darbepoetin a administered SC is extended approximately twofold relative to IV administration, which compensates for decreased bioavailability [83]. 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 [86]. 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 [86]. 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 [87]. One issue with drugs made by solution or solid-phase chemistry is poor specicity 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 [88]. Consequently, it is difcult to target specic 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 [8991]. Low afnity 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 specicity to the sulfhydryl group with retention of in vitro activity [89,92,93]. Another strategy is to produce PEGylated EPO synthetically. During synthesis, a PEGdconjugated 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 [77,94]. 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 [89,90,95]. Methoxy polyethylene glycol-epoetin b (PEG-epoetin b; Mircera; F. HoffmannLa Roche Ltd., Basel, Switzerland), recently approved by regulatory agencies in the United States and Europe, is a PEGylated form of epoetin b. Pharmacokinetic parameters of PEG-epoetin b were measured in patients receiving peritoneal dialysis [10]. Mean t1/2 was 134 hours when PEG-epoetin b 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 [95]. PEGdepoetin b did not display ip-op kinetics (absorption constant much slower than elimination constant) after SC administration because of the signicantly decreased systemic clearance and corresponding increase in t1/2. Other PEGylated ESAs include PEGylated darbepoetin a [58] and Hematide (Affymax Inc., Palo Alto, CA, USA) [96]. Mean t1/2 in rats of a PEG-darbepoetin was reported to be 24.3 hours compared with 17.5 hours for darbepoetin a [58]. 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 [97]. Impact of PEG ylation and hyperglycosylation on clearance Both hyperglycosylation and PEGylation of ESAs can reduce receptor-binding afnity, although the reduction is substantially greater for PEGylated vs hyperglycosylated rhEPO (50- to 100-fold vs 5-fold) [7,9,28,78,90,91]. 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 receptormediated endocytosis and degradation, which has been reported in vitro [64,91]. 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 [58,89,94]. Hyperglycosylated or PEGylated rhEPO and analogs have increased hydrodynamic size due to attached hydrated carbohydrate or PEG [86]. Thus, the reduced clearance of these molecules may be partially explained by steric factors. For example, the transport of ESAs from the blood

S. Elliott et al./ Experimental Hematology 2008;36:15731584

1581

to the clearing organs may be reduced as the size of the molecule increases, as was reported for unconjugated PEG [86]. 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 [98103]. 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 [104]. 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 rst 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 efcient. Thus, subsequent clinical studies have investigated different dosing schedules (QW, Q2 W, Q3 W, and QM) across different clinical settings [11 14,105,106]. 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) [107]. 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 a) 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-benet 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 renement of this understanding to probe the trade-off between the receptor afnity 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.

Table 1. Hemoglobin and erythropoiesis-stimulating agent (ESA) therapy in prevalent hemodialysis patients from the Dialysis Outcomes and Practice Patterns Studya Hemoglobin (median) ESA therapy (mean 6 SEM) % of Patients treated SC 75 6 1.8 77 6 1.73 86 6 1.44 91 6 1.25 92 6 1.3 84 6 0.71 Doses per week (% of patients)

Country France Germany Italy Spain UK Total

n 521 565 556 508 440 2590

Hb (g/dL) 11.1 10.9 10.8 11.6 11.0 11.1

n 581 586 595 551 454 2767

Dose (units/kg/week) 102 6 4.81 86 6 2.92 132 6 4.04 114 6 3.7 107 6 3.6 109 6 1.76

n 442 467 527 510 422 2368

3 47 71 50 49 60 55

2 38 17 30 36 31 30

1 14 11 18 15 8 14

ESA 5 erythropoiesis-stimulating agent; Hb, hemoglobin; SC 5 subcutaneous; SEM, standard error of the mean. a Modied from Locatelli et al. [107].

1582

S. Elliott et al./ Experimental Hematology 2008;36:15731584 18. Sawada K, Krantz SB, Kans JS, et al. Purication of human erythroid colony-forming units and demonstration of specic binding of erythropoietin. J Clin Invest. 1987;80:357366. 19. Koury ST, Bondurant MC, Koury MJ. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood. 1988;71:524527. 20. Lacombe C, Da Silva JL, Bruneval P, et al. Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest. 1988;81:620623. 21. Maxwell PH, Osmond MK, Pugh CW, et al. Identication of the renal erythropoietin-producing cells using transgenic mice. Kidney Int. 1993;44:11491162. 22. Koury ST, Bondurant MC, Koury MJ, Semenza GL. Localization of cells producing erythropoietin in murine liver by in situ hybridization. Blood. 1991;77:24972503. 23. Browne JK, Cohen AM, Egrie JC, et al. Erythropoietin: gene cloning, protein structure, and biological properties. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):693702. 24. Erslev AJ. Erythropoietin titers in health and disease. Semin Hematol. 1991;28:27. 25. Groopman JE, Itri LM. Chemotherapy-induced anemia in adults: incidence and treatment. J Natl Cancer Inst. 1999;91:16161634. 26. Eckardt KU, Boutellier U, Kurtz A, Schopen M, Koller EA, Bauer C. Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia. J Appl Physiol. 1989;66:17851788. 27. Koury ST, Koury MJ, Bondurant MC, Caro J, Graber SE. Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration. Blood. 1989;74: 645651. 28. Elliott S, Egrie J, Browne J, et al. Control of rHuEPO biological activity: the role of carbohydrate. Exp Hematol. 2004;32:11461155. 29. Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med. 1987;316:7378. 30. Erslev AJ, Wilson J, Caro J. Erythropoietin titers in anemic, nonuremic patients. J Lab Clin Med. 1987;109:429433. 31. Finch CA, Harker LA, Cook JD. Kinetics of the formed elements of human blood. Blood. 1977;50:699707. 32. Hillman RS, Finch CA. Erythropoiesis: normal and abnormal. Semin Hematol. 1967;4:327336. 33. Smith JA. Exercise, training and red blood cell turnover. Sports Med. 1995;19:931. 34. Sawyer ST, Krantz SB, Goldwasser E. Binding and receptormediated endocytosis of erythropoietin in Friend virus-infected erythroid cells. J Biol Chem. 1987;262:55545562. 35. Jones SS, DAndrea AD, Haines LL, Wong GG. Human erythropoietin receptor: cloning, expression, and biologic characterization. Blood. 1990;76:3135. 36. DAndrea AD, Lodish HF, Wong GG. Expression cloning of the murine erythropoietin receptor. Cell. 1989;57:277285. 37. Syed RS, Reid SW, Li C, et al. Efciency of signalling through cytokine receptors depends critically on receptor orientation. Nature. 1998;395:511516. 38. Philo JS, Aoki KH, Arakawa T, Narhi LO, Wen J. Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-afnity and one low-afnity interaction. Biochemistry. 1996;35:16811691. 39. Elliott S, Lorenzini T, Yanagihara D, Chang D, Elliott G. Activation of the erythropoietin (EPO) receptor by bivalent anti-EPO receptor antibodies. J Biol Chem. 1996;271:2469124697. 40. Elliott S, Lorenzini T, Chang D, Barzilay J, Delorme E. Mapping of the active site of recombinant human erythropoietin. Blood. 1997;89: 493502.

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

References
1. Erslev A. Humoral regulation of red cell production. Blood. 1953;8: 349357. 2. Lai PH, Everett R, Wang FF, Arakawa T, Goldwasser E. Structural characterization of human erythropoietin. J Biol Chem. 1986;261: 31163121. 3. Kelley LL, Green WF, Hicks GG, Bondurant MC, Koury MJ, Ruley HE. 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. Mol Cell Biol. 1994;14: 41834192. 4. Koury MJ, Bondurant MC. Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells. J Cell Physiol. 1988;137:6574. 5. Lin FK, Suggs S, Lin CH, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 1985;82:75807584. 6. Skibeli V, Nissen-Lie G, Torjesen P. Sugar proling proves that human serum erythropoietin differs from recombinant human erythropoietin. Blood. 2001;98:36263634. 7. Elliott S, Lorenzini T, Asher S, et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol. 2003;21:414421. 8. Delorme E, Lorenzini T, Gifn J, et al. Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry. 1992;31:98719876. 9. Egrie JC, Dwyer E, Browne JK, Hitz A, Lykos MA. Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp Hematol. 2003;31: 290299. 10. Macdougall IC, 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. Clin J Am Soc Nephrol. 2006;1:12111215. 11. Locatelli F, Olivares J, Walker R, et al. Novel erythropoiesis stimulating protein for treatment of anemia in chronic renal insufciency. Kidney Int. 2001;60:741747. 12. Nissenson AR, Swan SK, Lindberg JS, et al. Randomized, controlled trial of darbepoetin alfa for the treatment of anemia in hemodialysis patients. Am J Kidney Dis. 2002;40:110118. 13. 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. Clin Nephrol. 2005;63:327334. 14. Sulowicz W, Locatelli F, Ryckelynck JP, 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. Clin J Am Soc Nephrol. 2007;2:637646. 15. Canon JL, Vansteenkiste J, Bodoky G, et al. Randomized, doubleblind, active-controlled trial of every-3-week darbepoetin alfa for the treatment of chemotherapy-induced anemia. J Natl Cancer Inst. 2006;98:273284. 16. Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83:5967. 17. Broudy VC, Lin N, Brice M, Nakamoto B, Papayannopoulou T. Erythropoietin receptor characteristics on primary human erythroid cells. Blood. 1991;77:25832590.

S. Elliott et al./ Experimental Hematology 2008;36:15731584 41. Constantinescu SN, Keren T, Socolovsky M, Nam H, Henis YI, Lodish HF. Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain. Proc Natl Acad Sci U S A. 2001;98:43794384. 42. Kubatzky KF, Liu W, Goldgraben K, Simmerling C, Smith SO, Constantinescu SN. Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function. J Biol Chem. 2005;280:1484414854. 43. Seubert N, Royer Y, Staerk J, et al. Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer. Mol Cell. 2003;12:12391250. 44. Krantz SB, Sawyer ST, Sawada KI. Purication of erythroid progenitor cells and characterization of erythropoietin receptors. Br J Cancer Suppl. 1988;9:3135. 45. Sawada K, Krantz SB, Sawyer ST, Civin CI. Quantitation of specic binding of erythropoietin to human erythroid colony-forming cells. J Cell Physiol. 1988;137:337345. 46. Fraser JK, Lin FK, Berridge MV. Expression of high afnity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL. Exp Hematol. 1988;16: 836842. 47. Somervaille TC, Linch DC, Khwaja A. Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax. Blood. 2001;98:13741381. 48. Landschulz KT, Noyes AN, Rogers O, Boyer SH. Erythropoietin receptors on murine erythroid colony-forming units: natural history. Blood. 1989;73:14761486. 49. Mayeux P, Billat C, Jacquot R. The erythropoietin receptor of rat erythroid progenitor lens. Characterization and afnity cross-linkage. J Biol Chem. 1987;262:1398513990. 50. Perrotta PL, Snyder EL. Non-infectious complications of transfusion therapy. Blood Rev. 2001;15:6983. 51. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature. 2000;405:635. 52. Martin KJ. The rst human cell line-derived erythropoietin, epoetindelta (Dynepo), in the management of anemia in patients with chronic kidney disease. Clin Nephrol. 2007;68:2631. 53. Heatherington AC. Clinical pharmacokinetic properties of rHuEpo: a review. In: Molineux G, Foot MA, Elliott SG, eds. Erythropoietins and Erythropoiesis Molecular, Cellular, Preclinical, and Clinical Biology. Boston: Birkha user Basel; 2003. p. 87112. 54. Veng-Pedersen P, Chapel S, Al-Huniti NH, et al. Pharmacokinetic tracer kinetics analysis of changes in erythropoietin receptor population in phlebotomy-induced anemia and bone marrow ablation. Biopharm Drug Dispos. 2004;25:149156. 55. Veng-Pedersen P, Widness JA, Pereira LM, Schmidt RL, Lowe LS. A comparison of nonlinear pharmacokinetics of erythropoietin in sheep and humans. Biopharm Drug Dispos. 1999;20:217223. 56. Kinoshita H, Ohishi N, Kato M, Tokura S, Okazaki A. Pharmacokinetics and distribution of recombinant erythropoietin in rats. Arzneimittelforschung. 1992;42:174178. 57. Kato M, Kamiyama H, Okazaki A, Kumaki K, Kato Y, Sugiyama Y. Mechanism for the nonlinear pharmacokinetics of erythropoietin in rats. J Pharmacol Exp Ther. 1997;283:520527. 58. 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 [abstract]. Nephrol Dial Transplant. 2006;21: iv303iv304. Abstract MP013. 59. Dinkelaar RB, Engels EY, Hart AA, Schoemaker LP, Bosch E, Chamuleau RA. Metabolic studies on erythropoietin (EP): II. The role of liver and kidney in the metabolism of Ep. Exp Hematol. 1981;9:796803. 60. Jensen JD, Jensen LW, Madsen JK, Poulsen L. The metabolism of erythropoietin in liver cirrhosis patients compared with healthy volunteers. Eur J Haematol. 1995;54:111116.

1583

61. Macdougall IC, Roberts DE, Coles GA, Williams JD. Clinical pharmacokinetics of epoetin (recombinant human erythropoietin). Clin Pharmacokinet. 1991;20:99113. 62. Yoon WH, Park SJ, Kim IC, Lee MG. Pharmacokinetics of recombinant human erythropoietin in rabbits and 3/4 nephrectomized rats. Res Commun Mol Pathol Pharmacol. 1997;96:227240. 63. Widness JA, Veng-Pedersen P, Schmidt RL, Lowe LS, Kisthard JA, Peters C. In vivo 125I-erythropoietin pharmacokinetics are unchanged after anesthesia, nephrectomy and hepatectomy in sheep. J Pharmacol Exp Ther. 1996;279:12051210. 64. Gross AW, Lodish HF. Cellular trafcking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP). J Biol Chem. 2006;281:20242032. 65. Chapel S, Veng-Pedersen P, Hohl RJ, Schmidt RL, McGuire EM, Widness JA. Changes in erythropoietin pharmacokinetics following busulfan-induced bone marrow ablation in sheep: evidence for bone marrow as a major erythropoietin elimination pathway. J Pharmacol Exp Ther. 2001;298:820824. 66. Chapel SH, Veng-Pedersen P, Schmidt RL, Widness JA. Receptorbased model accounts for phlebotomy-induced changes in erythropoietin pharmacokinetics. Exp Hematol. 2001;29:425431. 67. 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. Eur J Cancer. 2005;41:11401149. 68. Flaharty KK. Clinical pharmacology of recombinant human erythropoietin (r-HuEPO). Pharmacotherapy. 1990;10:9S14S. 69. Porter CJ, Charman SA. Lymphatic transport of proteins after subcutaneous administration. J Pharm Sci. 2000;89:297310. 70. 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]. Nephrol Dial Transpl. 2006;21: iv304. Abstract MP014. 71. Schellekens H. Bioequivalence and the immunogenicity of biopharmaceuticals. Nat Rev Drug Discov. 2002;1:457462. 72. Sasu BJ, Hartley C, Schultz H, et al. Comparison of epoetin alfa and darbepoetin alfa biological activity under different administration schedules in normal mice. Acta Haematol. 2005;113:163174. 73. Elliott SG. New molecules and formulations of recombinant human erythropoietin. In: Molineux G, Foot MA, Elliott SG, eds. Erythropoietins and Erythropoiesis Molecular, Cellular, Preclinical, and Clinical Biology. Boston: Birkha user Basel; 2003. p. 241258. 74. Egrie J, Browne J. Darbepoetin alfa is more potent in vivo and can be administered less frequently than rHuEPO. Br J Cancer. 2002;87: 476477. 75. Macdougall IC, Eckardt KU. Novel strategies for stimulating erythropoiesis and potential new treatments for anaemia. Lancet. 2006; 368:947953. 76. Bunn HF. New agents that stimulate erythropoiesis. Blood. 2007;109: 868873. 77. Chen SA, Sawchuk RJ, Brundage RC, et al. Plasma and lymph pharmacokinetics of recombinant human interleukin-2 and polyethylene glycol-modied interleukin-2 in pigs. J Pharmacol Exp Ther. 2000; 293:248259. 78. Kochendoerfer G. Chemical and biological properties of polymermodied proteins. Expert Opin Biol Ther. 2003;3:12531261. 79. Darling RJ, Kuchibhotla U, Glaesner W, Micanovic R, Witcher DR, Beals JM. Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions. Biochemistry. 2002;41: 1452414531. 80. Rush RS, Derby PL, Strickland TW, Rohde MF. Peptide mapping and evaluation of glycopeptide microheterogeneity derived from endoproteinase digestion of erythropoietin by afnity high-performance capillary electrophoresis. Anal Chem. 1993;65:18341842.

1584

S. Elliott et al./ Experimental Hematology 2008;36:15731584 96. Stead RB, 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. Blood. 2006;108:18301834. 97. Fan Q, Leuther KK, Holmes CP, et al. Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia. Exp Hematol. 2006;34:13031311. 98. Macdougall IC, Temple RM, Kwan JT. Is early treatment of anaemia with epoetin-alpha benecial to pre-dialysis chronic kidney disease patients? Results of a multicentre, open-label, prospective, randomized, comparative group trial. Nephrol Dial Transplant. 2007;22: 784793. 99. Revicki DA, Brown RE, Feeny DH, et al. Health-related quality of life associated with recombinant human erythropoietin therapy for predialysis chronic renal disease patients. Am J Kidney Dis. 1995; 25:548554. 100. Perlman RL, Finkelstein FO, Liu L, et al. Quality of life in chronic kidney disease (CKD): a cross-sectional analysis in the Renal Research Institute-CKD study. Am J Kidney Dis. 2005;45: 658666. 101. Roger SD, McMahon LP, 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. J Am Soc Nephrol. 2004;15:148156. 102. Singh AK, Szczech L, Tang KL, et al. Correction of anemia with epoetin alfa in chronic kidney disease. N Engl J Med. 2006;355: 20852098. 103. Drueke TB, Locatelli F, Clyne N, et al. Normalization of hemoglobin level in patients with chronic kidney disease and anemia. N Engl J Med. 2006;355:20712084. 104. Ross SD, Allen IE, Henry DH, Seaman C, Sercus B, Goodnough LT. Clinical benets and risks associated with epoetin and darbepoetin in patients with chemotherapy-induced anemia: a systematic review of the literature. Clin Ther. 2006;28:801831. 105. Provenzano R, Bhaduri S, Singh AK. Extended epoetin alfa dosing as maintenance treatment for the anemia of chronic kidney disease: the PROMPT study. Clin Nephrol. 2005;64:113123. 106. 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. Clin J Am Soc Nephrol. 2007;2:215221. 107. Locatelli F, Pisoni RL, Combe C, et al. Anaemia in haemodialysis patients of ve European countries: association with morbidity and mortality in the Dialysis Outcomes and Practice Patterns Study (DOPPS). Nephrol Dial Transplant. 2004;19:121132. 108. Molineux G. Biology of erythropoietin In: Molineux G. In: Foot MA, Elliott SG, eds. Erythropoietins and Erythropoiesis Molecular, Cellular, Preclinical, and Clinical Biology. Boston, MA: Birkha user Basel; 2003. p. 113132.

81. Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J Biol Chem. 1987;262:1205912076. 82. Takeuchi M, Kobata A. Structures and functional roles of the sugar chains of human erythropoietins. Glycobiology. 1991;1:337346. 83. Macdougall IC, Gray SJ, Elston O, et al. Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients. J Am Soc Nephrol. 1999;10:23922395. 84. 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. J Am Soc Nephrol. 2005;16:14631470. 85. 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). Nephrol Dial Transplant. 2003;18:362369. 86. Yamaoka T, Tabata Y, Ikada Y. Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice. J Pharm Sci. 1994;83:601606. 87. Basser RL, OFlaherty E, Green M, et al. Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor. Blood. 2002;99:25992602. 88. Francis GE, 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. Int J Hematol. 1998;68:118. 89. Long DL, Doherty DH, Eisenberg SP, et al. Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity. Exp Hematol. 2006;34:697704. 90. Macdougall IC. CERA (Continuous Erythropoietin Receptor Activator): a new erythropoiesis-stimulating agent for the treatment of anemia. Curr Hematol Rep. 2005;4:436440. 91. Jarsch M, Brandt M, Lanzendorfer M, Haselbeck A. Comparative erythropoietin receptor binding kinetics of C.E.R.A. and epoetinbeta determined by surface plasmon resonance and competition binding assay. Pharmacology. 2007;81:6369. 92. Goodson RJ, Katre NV. Site-directed pegylation of recombinant interleukin-2 at its glycosylation site. Biotechnology (N Y). 1990;8:343346. 93. He XH, Shaw PC, Tam SC. Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation. Life Sci. 1999;65:355368. 94. Chen SY, Cressman S, Mao F, et al. Synthetic erythropoietic proteins: tuning biological performance by site-specic polymer attachment. Chem Biol. 2005;12:371383. 95. Jolling K, Ruixo JJ, Hemeryck A, Piotrovskij V, Greway T. Population pharmacokinetic analysis of pegylated human erythropoietin in rats. J Pharm Sci. 2004;93:30273038.