Experimental Hematology 2008;36:1573–1584

Erythropoietins: A common mechanism of action

Steve Elliotta, Elizabeth Phama, and Iain C. Macdougallb

aAmgen Inc, Thousand Oaks, Calif., USA; bRenal Unit, Kings College Hospital, London, UK

(Received 20 June 2008; revised 20 June 2008; accepted 12 August 2008)

S.E. and E.P. are e

grants from Amgen In

sultant for Amgen Inc

Offprint requests t

Center, M/S 29-1-A,

amgen.com

0301-472X/08 $–see

doi: 10.1016/j.exph

Clinical development of erythropoiesis-stimulating agents (ESAs) revolutionized the manage-ment of anemia. The major clinical benefits of ESAs are effective treatment of anemia andavoidance of blood transfusion risks. Erythropoietin (EPO) interacts directly with the EPOreceptor on the red blood cell (RBC) surface, triggering activation of several signal transduc-tion pathways, resulting in the proliferation and terminal differentiation of erythroid precur-sor cells and providing protection from RBC precursor apoptosis. The magnitude of increasein 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 slowerabsorption than intravenous (IV) administration, leading to lower peak plasma levels and anapparent extended terminal half-life. However, SC administration requires additional needle-sticks and is associated with an increased risk of immunogenicity compared with IV adminis-tration. Multiple pathways may play a role in EPO clearance from the body. Epoetin alfa wasthe first rhEPO produced and approved for pharmaceutical use, followed by several relatedproducts and by newer ESAs with the same mechanism but more prolonged action. Darbepoe-tin alfa is a hyperglycosylated EPO analog with an extended terminal half-life and a greaterrelative 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 activationof the EPO receptor. � 2008 ISEH - Society for Hematology and Stem Cells. Published byElsevier Inc.

Erythropoiesis, a complex physiologic process maintaininghomeostasis of oxygen (O2) levels in the body, is primarilyregulated by erythropoietin (EPO), a 30-kDa, 165daminoacid hematopoietic growth factor produced by the kidneys[1,2]. Under normal conditions, endogenous EPO levelschange with O2 tension. In the presence of EPO, bone mar-row erythroid precursor cells proliferate and differentiateinto red blood cells (RBCs). In its absence, these cellsundergo apoptosis [3,4].

The human EPO gene was cloned in 1983 [5], allowingfor clinical development of recombinant human EPO(rhEPO), a biotechnological advance that revolutionizedanemia treatment. Endogenous EPO and rhEPO share thesame amino acid sequence, with slight differences in the

mployees of Amgen. I.C.M. has received support

c, Ortho Biotech, Roche, and Affymax, and is a con-

, Ortho Biotech, Roche, and Affymax.

o: Steve Elliott, Ph.D., Amgen Inc., One Amgen

Thousand Oaks, CA 91320-1799; E-mail: selliott@

front matter. Copyright � 2008 ISEH - Society for Hemat

em.2008.08.003

sugar profile [6]. In clinical practice, rhEPO is typically ad-ministered as a bolus injection, and the dose is titrated togive the desired effect. Administration of rhEPO initiallycorresponded to clinical practice patterns, with treatmentsbeing synchronized to dialysis frequencies or chemotherapycycle schedules.

Attempts to improve or ‘‘reengineer’’ rhEPO to meet thedemands of patients and caregivers resulted in additionalerythropoiesis-stimulating agents (ESAs) with increased se-rum half-lives (compared with rhEPO), as well as differentreceptor binding properties and in vivo biological potencies[7–10]. The characteristics and properties of these newESAs allowed extension of the dosing intervals beyondthe 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 [11–15]

All ESAs share the same mechanism of action, bindingto and activating the EPO receptor (EPOR), but differencesin pharmacokinetic, pharmacodynamic, and receptor-bind-ing properties affect their clinical use. In this review, we ex-amine the biology of erythropoiesis and EPO and evaluate

ology and Stem Cells. Published by Elsevier Inc.

1574 S. Elliott et al. / Experimental Hematology 2008;36:1573–1584

the limitations and opportunities afforded by new ap-proaches to stimulating erythropoiesis through activationof the EPOR.

ErythropoiesisA primary function of RBCs is to transport O2 from thelungs to O2-dependent tissues. Changes in O2 levels neces-sitate both acute and long-term physiologic adaptations.Acute adaptations include increases in respiration and heartrate, vasoconstriction, and changes in blood volume; how-ever, these changes cannot be sustained. Erythropoiesis isa longerdterm adaptation to boost O2-carrying capacityby increasing the concentration of RBCs, and thus, hemo-globin (Hb) concentration.

RBCs are the most abundant (w99%) circulating cells inthe bloodstream, representing 40% to 45% of total bloodvolume. In a healthy human with w5 L blood, this repre-sents approximately 2.5� 1013 cells, a quantity substantialenough to provide the large O2 transport capacity needed tosupport aerobic respiration. In humans, the RBC lifespan isw100 to 120 days, with a daily loss of w0.8% to 1.0% ofcirculating RBCs. To match this loss, the body assumesa normal, prodigious production capacity of w2.5� 1011

cells/day.RBC production results from a tightly controlled

proliferation and differentiation pathway (Fig. 1). Earlyhematopoietic progenitors differentiate into burst-forming

IL-1IL-3IL-5IL-11G-CSFSCFFLT-3/FLK-2

+

TGF-MIP1-

Multipotent hematopoietic

stem cell CFU-GEMM

Cytokine receptor

–

EryReticulocyteErythrocyte

Figure 1. Erythropoiesis. BFUe 5 burst-forming unit–erythroid; CFUe 5 colon

FLT 5 fetal liver tyrosine kinase; G-CSF 5 granulocyte-colony stimulating fac

CSF 5 granulocyte macrophage CSF; IL 5 interleukin; MIP 5 macrophage inflam

tor; TPO 5 thrombopoietin.

unit–erythroid cells, in which EPORs appear for the firsttime; however, EPO is not required at this stage [16].Burst-forming unit–erythroid cells differentiate into col-ony-forming unit–erythroid cells, which are dependenton EPO for survival, and there is a corresponding risein expression of EPORs [17,18]. Continued stimulationwith EPO triggers differentiation into erythroblasts,which enucleate to form reticulocytes and after a fewdays show loss of ‘‘reticulin,’’ resulting in RBCs. Reticu-locytes and RBCs stop expressing EPOR and cease beingresponsive to EPO [18].

Disease states and environmental conditions often alterthe tightly controlled balance between RBC productionand destruction. When RBC loss exceeds gain, anemia re-sults. Increased RBC loss can occur because of bleeding,enhanced destruction (chemically induced hematotoxicity),or reduced lifespan (sickle cell anemia). Potential causes ofinsufficient RBC production include defects in O2 sensing,excess of erythropoiesis inhibitors, and inadequate concen-trations of ESAs.

EPOErythropoiesis primarily occurs in the kidney, but other or-gans (liver, brain) also produce EPO. Interstitial fibroblastsproduce EPO in the kidney [19–21], while hepatocytes pro-duce EPO in the liver [22]. Initially, EPO is synthesized asa 193-amino-acid precursor. A 27-amino-acid signal

IL-3IL-9GM-CSFSCFTPO

+

BFUe

EPO receptor

IL-3IL-9IGF-1SCFEPO

+

EPO +

CFUethroblast

y-forming unit–erythroid; Epo 5 erythropoietin; FLK 5 fetal liver kinase;

tor; GEMM 5 granulocyte, erythrocyte, monocyte, megakaryocyte; GM-

matory protein; SCF 5 stem cell factor; TGF 5 transforming growth fac-

1575S. Elliott et al./ Experimental Hematology 2008;36:1573–1584

peptide and C-terminal arginine are removed, and carbohy-drate is added to three N-linked glycosylation sites and oneO-linked glycosylation site [23]. The secreted protein con-tains 165 amino acids and is heavily glycosylated, withw40% of its mass composed of carbohydrate. The structureof rhEPO is a compact globular bundle that contains foura helices (Fig. 2).

Generally, serum EPO concentrations of 10 to 25 mU/mL [24] maintain Hb levels within the normal range of12 to 17 g/dL [25]. The terminal half-life (t1/2) of EPO isw5 hours [26], which requires an average EPO productionrate of w2 U/kg/day. The EPO production rate per cell ap-pears constant [27], with fluctuations in EPO synthesis re-sulting from changes in the number of cells producing themolecule. In cases of severe anemia, circulating EPO levelscan increase up to 1000dfold because of a logarithmic in-crease in the number of cells producing EPO [24,27]. Otherfactors affecting EPO levels include iron availability, nutri-tional status, disease or comorbidities, environmental con-ditions, and genetic factors (congenital polycythemias).

EPO-RBC time–response relationshipA direct correlation exists between RBC production and se-rum EPO concentrations [28,29]. However, the rate oferythropoiesis change (w4dfold) [29]) is small comparedto the larger change in EPO concentrations (w1000dfold)[30]. Thus, the magnitude of increase in RBC concentrationis primarily controlled by the length of time EPO concen-trations are maintained, and not by the EPO concentrationlevel per se (Fig. 3). Increased EPO synthesis has a pro-longed effect due to the disproportionate relationship be-tween EPO t1/2 and RBC lifespan. Thirty minutes of

Figure 2. The nuclear magnetic resonance minimized average structure of

human erythropoietin.

hypoxia can result in production of EPO (t1/2 w5 hours)[26]. In turn, EPO stimulates formation of enucleated retic-ulocytes (t1/2 5 1–5 days) [31,32], which rapidly matureinto RBCs that have a long lifespan (100–120 days) [33].Thus, a short duration of EPO exposure results in a pro-longed increase in RBC concentration.

EPO and EPORThe mechanism of action by which EPO stimulates erythro-poiesis has been under extensive investigation. Early evi-dence indicated that EPO interacted with a protein on thecell surface, triggering activation of the JAK-signal trans-ducers and activators of transcription, phosphatidylinositol3 kinase, and mitogen-activated protein kinase pathways(Fig. 4), resulting in the proliferation and terminal differen-tiation of erythroid precursor cells and providing protectionfrom apoptosis [4]. The EPO-binding component on cellswas first detected by measuring physical attachment of ra-diolabeled EPO to erythroid precursor cells [18,34]. TheEPOR gene was subsequently identified by expression clon-ing and found to be a single gene with no apparent homo-logs [35,36].

While other components may mediate affinity or aid insignal transduction, the activation of signal transduction isinitiated by an early, direct interaction of EPO withEPOR. Activation of EPOR occurs following cross-linkingof two EPORs via one EPO ligand [37–40], which inducesa conformational change in the receptor, triggering down-stream signal transduction [41–43].

Receptor affinity and in vitro potencyThe affinity (Kd) of EPO for its receptor on human cells isw100 to 200 pM [17,44,45], which is sufficient for lowconcentrations of EPO to maintain a Hb of w14 g/dL inhealthy subjects. Normal circulating concentrations ofEPO are w2 to 5 pM [24], significantly below the EPO:E-POR Kd. At the half-maximal effective dose (ED50; w70mU [28,46]) 6.8% of the receptors are occupied [46], sug-gesting that only a fraction of the receptors need be occu-pied by EPO to achieve an adequate erythropoiesismaintenance rate.

Increased EPOR occupancy does not increase the rate ofcell division, but instead increases the rate of RBC forma-tion by recruitment and differentiation of more erythroidprecursor cells. However, the erythropoiesis rate is maxi-mized when all available erythroid progenitors are activelydividing. This was evident from phase I clinical trials withepoetin a in which the rate of hematocrit rise showed dose-dependent 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 expo-sure time and the duration of enhanced erythropoiesis.

If EPOR occupancy is inadequate, apoptosis of precur-sor cells occurs [4], with apoptosis beginning in as littleas 2 to 8 hours following removal of EPO from the culture

Reticulocyte response

B – duration of reticulocyte response

Serum level

A – duration of serum residence

Administration of ESA

Hemoglobin response

C – duration of hemoglobin response

Time axis

Baseline level (or MEC)

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.

1576 S. Elliott et al. / Experimental Hematology 2008;36:1573–1584

[3,4,47]. Formation of erythroblasts from colony-formingunit–erythroid cells can take up to a week. Thus, a singleEPO–EPOR binding event is insufficient for stimulationof complete differentiation of early erythroid precursors.Instead, adequate EPO concentrations must be presentduring the entire process to ensure survival, proliferation,and differentiation to mature RBCs. Only during the finalstages of erythropoiesis is EPO no longer required forRBC survival [46,48,49].

EPO derivatives or analogs with reduced receptor affin-ity may require higher concentrations to maintain an effec-tive number of occupied EPORs. Although low bindingaffinity can be overcome with higher dosing and the rateof erythropoiesis corresponds to the duration of EPO expo-sure [28], low receptor binding activity may be undesirablein some disease states, such as EPO resistance. In the caseof longer-acting agents with very low receptor affinity,there may be low receptor occupancy for an extendedperiod and consequently, a reduced rate of erythropoiesis,resulting in a slower rate of Hb rise.

Anemia treatment and development of rhEPOBefore development of rhEPO, blood transfusion was themost common treatment for patients with anemia. However,blood transfusions carry inherent risks, including risk oftransmission of infectious agents and iron overload. Addi-tionally, the blood supply is limited, and immune reactionsdeveloped after transfusion can make organ transplantationmore problematic [50]. Iron supplementation was largelyineffective as a stand-alone treatment for anemia. Theneed for an effective anemia treatment option was obvious,and attempts to make and test rhEPO via cloning of thehuman EPO gene began.

Successfully cloning the EPO gene was difficult, as lowcirculating 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 pu-rified human EPO became available (10 mg from 1000 Lurine from human patients with aplastic anemia), oligonu-cleotide probes for EPO were designed. Two differentprobes were used to screen a l phage library containingsheared human genomic DNA, and the human EPO genewas cloned [5].

rhEPOdThe first ESASuccessful cloning of the EPO gene in 1983 [5] allowed forthe large-scale production of rhEPO and its subsequentclinical use [29]. Epoetin alfa (Epogen; Amgen Inc., Thou-sand Oaks, CA, USA; Procrit; Ortho Biotech Products, L.P.,Bridgewater, NJ, USA) was the first rhEPO commercializedin 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 hamsterovary cells, have minor structural differences but thesame physiological effects [51]. An epoetin produced inbaby hamster kidney cells, epoetin omega, differs from pre-vious epoetins in the glycosylation profile. More recently,epoetin delta (Dynepo, Shire plc, Basingstoke, UK), pro-duced from an engineered human fibrosarcoma cell lineHT1080, has been described [52]. As patents for epoetinsa 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 Arznei-mittel Puetter GmbH & Co. KG, Iserlohn, Germany]),have been approved in Europe.

PharmacokineticsIn healthy volunteers, the t1/2 of intravenous (IV) rhEPOranged from 5 to 11 hours [53], similar to that of endoge-nous EPO (average t1/2 5 5.2 hours) [26]. The volume of

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 ac-

tivation of signaling molecules. Phosphorylation of signal transducers and activators of transcription (STAT) 5 transcription factor (TF) causes homodime-

rization, 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) phosphor-

ylates 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 (apo-

ptosis protease-activating factor-1 [Apaf-1], apoptosis-inducing factor [AIF]).

1577S. Elliott et al./ Experimental Hematology 2008;36:1573–1584

distribution was generally similar to the plasma volume(40–60 mL/kg), indicating limited extravascular distribu-tion. Subcutaneous (SC) administration resulted in slowerabsorption, leading to lower peak plasma levels (5–10%of those seen with IV administration) and an apparent ex-tended t1/2 (w20–25 hours) [53]. Peak plasma levels arereached in most studies between 15 and 29 hours.

Bioavailability estimates for SC rhEPO range fromabout 20% to 40%, suggesting a substantial loss of materialduring transport from the interstitial space to the lymphatic

system and blood [53]. The pharmacokinetic characteristicsof rhEPO in healthy volunteers appear similar or compara-ble to those in several other populations, including chronickidney disease (CKD), liver cirrhosis, and myelodysplasticsyndrome patients [53].

ClearanceThe mechanism of rhEPO clearance and the site of degra-dation still are not definitively characterized. The observa-tion that rhEPO clearance is dose-dependent and saturable

1578 S. Elliott et al. / Experimental Hematology 2008;36:1573–1584

is consistent with at least two clearance pathways [54–57],and emerging data suggest that multiple pathways playsome role in clearance [58].

Clearance was first thought to be mediated primarilythrough the liver and kidney [59] or via the EPOR on recep-tor-expressing cells. However, subsequent research indi-cated neither the liver [59,60] nor the kidney [61–63]plays a major role in EPO clearance.

Binding of EPO to the EPOR can lead to cellular inter-nalization, during which the ligand may be degraded[34,64]. Chemotherapy, which reduces the number ofEPOR-bearing cells, reduces EPO clearance [54,65–67].Thus, one clearance pathway may involve uptake and deg-radation of EPO via the EPOR-expressing cells, but it is un-likely to be the only one and may not necessarily be themajor pathway. Bone marrow cells can deplete ESAsthrough nondEPOR pathways in vitro, and absent or re-duced binding activity of some rhEPO analogs with theEPOR resulted in only modest reductions in clearance [58].

In healthy men, only a small amount of intact radiola-beled epoetin b (!5% of the dose) was found to be ex-creted in the urine, suggesting that rhEPO is degradedelsewhere in the body [62]. Therefore, one important path-way may be degradation or metabolism in the interstitium,possibly via cells in the reticuloendothelial scavengingpathway or lymphatic system [68]. Consistent with thishypothesis, the lymphatic system is believed to play an im-portant role in the reduced bioavailability after SC admin-istration of proteins [69]. In addition, only small peptidesor free 125I, and not intact material, are detected in tissuesfollowing IV administration of 125Iddarbepoetin a, sug-gesting that degradation may occur in tissue [70].

SC vs IV treatment efficiencyWhile potency and dose of rhEPO are drug-related consid-erations in treatment choice, other considerations arise asa result of practice patterns and the patient population.SC administration requires additional needle-sticks and isassociated with an increased risk of immunogenicity [71].IV administration may be preferred in hemodialysis patientswho already have IV access enabled.

Development of additional ESAsIntroduction of epoetins into clinical practice representeda milestone in anemia treatment, yet opportunities for fur-ther improvement in certain patient populations and clinicalsettings remained. The initial TIW dosing regimen for he-modialysis patients proved to be burdensome and inconve-nient in other clinical settings such as CKD and oncology.Thus, research and development of additional treatmentopportunities continued.

Design considerationsWhile all ESAs have the same mechanism regarding EPORactivation, they differ in molecular structure, receptor

binding affinity, serum t1/2, clearance, bioavailability, andin vivo potency. Together, these characteristics shape theclinical efficacy and safety of these agents, as well as theirversatility, especially in terms of dosing schedules.

The clinical advantages of extended dosing regimens ledto development of newer ESAs with a longer duration ofaction, but comparable clinical benefit. For example, ex-tending the t1/2 may allow for extended-dosing schedulesthat offer patients and caregivers, especially those withCKD not on dialysis and cancer patients, the convenienceof less frequent visits. Increased molecular stability mayalso decrease degradation products and the risk of immuno-genicity, as well as allow for alternate storage opportunitiesor delivery systems (e.g., oral administration or slow deliv-ery 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 cy-cling, while too short a t1/2 may limit the range of extendeddosing regimens. Too high an affinity for the EPOR may af-fect the dose-response relationship, while insufficient bind-ing may result in a slower or incomplete response.Additionally, differences in IV vs SC potency may resultin the need for an increased drug dose with the less-efficientroute. These factors must be considered when developingnew ESAs and will vary with patient needs, indication, re-gion, and clinical practice. Ultimately, these newer mole-cules should offer patients efficacy and safety at leastcomparable with the original ESAs.

Potential strategiesResearchers have taken several approaches to reengineerand thereby extend treatment options with original ESAs.Prolonging the time of erythropoietic stimulation can in-crease response, which may allow for extending the dosinginterval. Increasing the dose of fasterdclearing moleculesis one strategy, but this can be inefficient because of thehigh doses required. A more dose-conserving strategy isto increase serum t1/2 [7,9,72]. Administration of a long-acting ESA may extend serum residence time, resulting inan increased relative biological response over time [28].

Early attempts to improve rhEPO included enhancingmolecular stability or increasing affinity for EPOR [73].However, increased affinity did not necessarily increase invivo biological potency, because serum t1/2 was the primarydriver of the degree of response [9,28,74]. The exceptionwas modifications that yielded molecules with an affinitythat was too low, resulting in ineffective EPOR dimeriza-tion and activation.

Many different approaches to extend the t1/2 have beenconsidered, including addition of polyethylene glycol(PEG), hyperglycosylation, dimerization, and physical fu-sion 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 thewell-established safety and efficacy profile of rhEPO by

1579S. Elliott et al./ Experimental Hematology 2008;36:1573–1584

modifying the ‘‘nonfunctional’’ regions without loss ofstructure or substantial reductions in affinity for EPOR.Any structural loss is of particular concern because of theincreased potential for degradation or molecular instabilityof the drug with long-term storage, and hence, reducedpotency and potentially increased risk of immunogenicity.Reduced affinity for EPOR may occur due to sterichindrance from the attachments (e.g., PEG [77,78]) orchanges in electrostatic properties of the molecule (hyper-glycosylation) [28,79], which directly or indirectly inhibitbinding.

HyperglycosylationBoth endogenous and recombinant EPO have microhetero-geneity in carbohydrate structures with variation in sialicacid contentdup to 14 attached sialic acid residues[74,80–82]. The importance of sialic acid content for clear-ance was noted in experiments with rhEPO isoforms, re-vealing a direct and positive relationship between sialicacid content and in vivo potency [74]. The moleculeswith increased sialic acid content had reduced affinity forthe EPOR and increased serum t1/2, suggesting that a longert1/2 was a stronger determinant of potency than was recep-tor affinity. Consequently, the mixture of glycoforms wasimportant in defining the biologic properties of the particu-lar rhEPO products. This observation also led to the hypoth-esis that ‘‘reengineering’’ of the EPO molecule, by addingmore carbohydrate chains and increasing the sialic acidcontent (O14 residues), might prolong the serum t1/2 andenhance potency compared with rhEPO [7].

Darbepoetin a

Darbepoetin a is a hyperglycosylated analog that containstwo additional N-linked carbohydrate chains at positions30 and 88, as a result of five amino acid substitutions(Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr)

Figure 5. Molecular structures of rhEPO (A) and darbepoetin alfa (B). Reprinted

2003. rhEPO 5 recombinant human erythropoietin.

(Fig. 5) [7]. The new carbohydrates did not directly interferewith receptor binding or disrupt the structure or stability ofthe molecule. The carbohydrate portion increased from40% to 51%, and the approximate molecular weight in-creased from 30 kDa to 37 kDa. The theoretical maximumnumber of sialic acid residues increased from 14 to 22.

Compared with epoetin a, darbepoetin a had a threefoldlonger serum t1/2, a lower receptor-binding affinity, and en-hanced in vivo bioactivity in multiple species [7,28,72,74].In a phase I trial in rhEPO-naıve patients receiving perito-neal dialysis [83], darbepoetin a administered IV had an ap-proximately threefold longer mean terminal t1/2 comparedwith rhEPO (25.3 vs 8.5 hours, respectively), a more thantwofold greater area under the curve (291 6 8 vs132 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 distribu-tion was similar for the two molecules (52.4 6 2.0 and48.7 6 2.1 mL/kg, respectively). The mean terminal t1/2

for darbepoetin a SC was longer than that for IV adminis-tration (w49 hours); Cmax averaged about 10% of the IVvalue, Tmax averaged 54 6 5 hours, and mean bioavailabil-ity was 37%.

Relative potency of darbepoetin a compared with rhEPOincreased when the dosing interval was extended. At TIWdosing, three times more rhEPO was needed than darbepoe-tin a to maintain or elicit a similar erythropoietic responsein normal mice [9,72]. With QW administration, the differ-ence increased 13-fold. With a single injection, the rhEPOdose needed to be 30 to 40 times higher to match the effectof the lower dose of darbepoetin a. In humans receivingrhEPO or darbepoetin a QW, the dose of rhEPO neededto be 45% higher than that of darbepoetin a to maintainHb levels in CKD patients [84]. The differences in relativepotencies likely resulted from the threefold longer serumt1/2 of darbepoetin a, allowing for a prolonged period oftime for erythroid cell exposure to the ESA [28].

by permission from Macmillan Publishers Ltd: Nature Biotechnology [7],

1580 S. Elliott et al. / Experimental Hematology 2008;36:1573–1584

The apparent paradoxdthat darbepoetin a has lower re-ceptor-binding affinity but increased in vivo activitydwasexplained by the counteracting effects of sialic acid–con-taining carbohydrate on clearance [9,28]. At extendedtime intervals, the darbepoetin a relative concentrationgreatly exceeded that of rhEPO. Thus, prolonged exposuremore than compensated for the reduced receptor-bindingaffinity, resulting in increased in vivo activity.

Unlike rhEPO, studies with darbepoetin a in humansdemonstrated that the dose efficiency after SC and IV ad-ministration was approximately equivalent, with similardoses providing similar efficacy (i.e., similar Hb increases)[85]. The apparent t1/2 of darbepoetin a administered SC isextended approximately twofold relative to IV administra-tion, which compensates for decreased bioavailability [83].

PEGylationPEGylation, the addition of PEG to reactive regions of pro-teins or carbohydrates by either solution or solid-phasechemistry, has been used successfully to extend the serumt1/2 of many different recombinant proteins [86]. PEGylatedmolecules have an increased hydrodynamic size becausea ‘‘water shell’’ surrounds the molecule. The increased hy-drodynamic size can result in reduced clearance because ofa reduced rate of translocation from blood to extravasculartissues [86]. PEG is thought to be relatively inert and non-immunogenic and, thus, to be a suitable starting materialfor protein conjugate therapeutics. However, PEGylationof a molecule does not guarantee protection against animmune response [87].

One issue with drugs made by solution or solid-phasechemistry is poor specificity of conjugation in the chemicalreaction, with generation of undesirable byproducts. Cur-rent chemistries typically target the reactive amino groupson lysine or the amino terminal amine [88]. Consequently,it is difficult to target specific reactive amine groups. PEGy-lated rhEPO molecules typically contain mixtures of mole-cules with PEG attached to different reactive amines, eachof which may have differential effects on activity and pro-tein folding. For example, if lysines are proximal to activesites involved in receptor binding, PEGylation can reducebinding activity and, consequently, in vitro activity, eitherby altering essential amino acids important in EPOR bind-ing or by steric hindrance [89–91].

Low affinity for EPOR associated with amine chemistrymay be overcome via other PEGylation strategies. Cysteinesubstitutions at targeted ‘‘nonfunctional’’ regions can allowaddition of the conjugate with high specificity to the sulfhy-dryl group with retention of in vitro activity [89,92,93]. An-other strategy is to produce PEGylated EPO synthetically.During synthesis, a PEGdconjugated amino acid is intro-duced in place of the unconjugated amino acid, allowingtargeting of particular amino acid positions for PEG attach-ment, such as the glycosylation sites, and reducing the po-tential for loss of in vitro activity [77,94]. Both strategies

may allow for extended serum t1/2. However, it is unclearif these particular molecules will have similar stability, invivo activity, and lack of immunogenicity to their glycosy-lated counterparts.

Development of PEGylatederythropoiesis-stimulating agentsPEGylated EPO molecules with potential clinical utilityhave been considered [89,90,95]. Methoxy polyethyleneglycol-epoetin b (PEG-epoetin b; Mircera; F. Hoffmann-La Roche Ltd., Basel, Switzerland), recently approved byregulatory agencies in the United States and Europe, isa PEGylated form of epoetin b. Pharmacokinetic parame-ters of PEG-epoetin b were measured in patients receivingperitoneal dialysis [10]. Mean t1/2 was 134 hours whenPEG-epoetin b was administered IV and 139 hours whenadministered SC. Clearance was 0.49 and 0.90 mL/h/kg,respectively, and SC bioavailability was 52%. Another ver-sion of PEG-rhEPO was examined in rats, with similar con-clusions [95]. PEGdepoetin b did not display ‘‘flip-flop’’kinetics (absorption constant much slower than eliminationconstant) after SC administration because of the signifi-cantly decreased systemic clearance and correspondingincrease in t1/2.

Other PEGylated ESAs include PEGylated darbepoetina [58] and Hematide (Affymax Inc., Palo Alto, CA,USA) [96]. Mean t1/2 in rats of a PEG-darbepoetin wasreported to be 24.3 hours compared with 17.5 hours for dar-bepoetin a [58]. Hematide is a synthetic PEGylated dimericpeptide that binds to and activates EPOR. Because of therapid clearance of the peptides, Hematide was PEGylatedto extend the serum t1/2, which was found to be 21 to30 hours in rats [97].

Impact of PEG ylation andhyperglycosylation on clearanceBoth hyperglycosylation and PEGylation of ESAs can re-duce receptor-binding affinity, although the reduction issubstantially greater for PEGylated vs hyperglycosylatedrhEPO (50- to 100-fold vs 5-fold) [7,9,28,78,90,91]. Thereduced EPOR binding activity results in corresponding de-creases in in vitro potency with these molecules. It has beensuggested that the reduced clearance of PEGylated andhyperglycosylated ESAs is due to their effect on receptor-mediated endocytosis and degradation, which has beenreported in vitro [64,91]. However, clearance studies withrhEPO analogs with altered receptor-binding characteristicssuggested PEGylated and hyperglycosylated ESAs havereduced clearance due to their impact on non-EPOR–mediated clearance pathways [58,89,94].

Hyperglycosylated or PEGylated rhEPO and analogshave increased hydrodynamic size due to attached hydratedcarbohydrate or PEG [86]. Thus, the reduced clearance ofthese molecules may be partially explained by steric fac-tors. For example, the transport of ESAs from the blood

1581S. Elliott et al./ Experimental Hematology 2008;36:1573–1584

to the clearing organs may be reduced as the size of themolecule increases, as was reported for unconjugatedPEG [86].

ESA use in clinical practiceESAs have been used successfully to treat anemia in manydifferent patient populations, helping to prevent or mini-mize use of blood transfusions. ESAs have been usedprimarily to treat anemia associated with kidney failureand chemotherapy-induced anemia. Additional indicationshave been considered, including anemia associated withcancer (including myelodysplasia), anemia of chronic dis-ease, anemia associated with AIDS treatment, and treat-ment in perisurgery indications. In some disease settings(e.g., end-stage renal disease and chronic kidney diseasewithout dialysis), there is a consistent relationship betweenESA treatment, increased hemoglobin, and improved qual-ity of life [98–103]. This is particularly true for measuresrelated to energy, physical function, and exercise. While re-sults are mixed in the oncology setting, some studies sug-gest aspects of quality of life, such as fatigue, may beimproved by the resultant increases in Hb levels [104].

Practice patterns vary with the indication and geograph-ical region. Dosing frequency is often associated with visitsto health care professionals and the duration of action of theESA. When rhEPO first became available, it was used totreat anemia in patients on hemodialysis. Such patientsmay attend either a dialysis unit or a hospital twice or thriceweekly for hemodialysis, and frequent administration ofESAs that coincided with hemodialysis sessions wasdeemed practical. For CKD patients who are not on dialysisand for cancer patients receiving chemotherapy, extendeddosing regimens may be more convenient and, in somecases, more cost efficient. Thus, subsequent clinical studieshave investigated different dosing schedules (QW, Q2 W,Q3 W, and QM) across different clinical settings [11–14,105,106]. Current practice patterns, even when limitedto the nephrology setting, vary considerably. Data fromthe Dialysis Outcomes Practice Patterns trial show a broad

Table 1. Hemoglobin and erythropoiesis-stimulating agent (ESA) therapy in pre

Patterns Studya

Hemoglobin (median) ESA therapy (mean 6

Country n Hb (g/dL) n

% of Patients

treated SC D

France 521 11.1 581 75 6 1.8

Germany 565 10.9 586 77 6 1.73

Italy 556 10.8 595 86 6 1.44

Spain 508 11.6 551 91 6 1.25

UK 440 11.0 454 92 6 1.3

Total 2590 11.1 2767 84 6 0.71

ESA 5 erythropoiesis-stimulating agent; Hb, hemoglobin; SC 5 subcutaneous; SaModified from Locatelli et al. [107].

spectrum of ESA dosing patterns and hemoglobin outcomesin different countries (Table 1) [107].

In the oncology setting, patients were initially dosedtwice or thrice weekly primarily because of the short serumt1/2 of rhEPO and the common use of these regimens in thenephrology setting. However, dose frequencies were subse-quently extended to QW to minimize needle-sticks and un-necessary visits to health care professionals. Less frequentadministration of longer-lived ESAs (e.g., darbepoetin a)that coincide with QW, Q2 W, and Q3 W chemotherapycycles have also been explored.

ESA therapy has transformed anemia management inboth the renal and oncology settings. The major advantagesof ESAs have been the treatment of anemia and avoidanceor minimization of blood transfusions, as well as possibleimprovements in quality of life associated with higher Hblevels. However, there may be an Hb limit beyond whichthe risk-to-benefit ratio is no longer favorable for the pa-tient. The frequency of dosing of the various ESAs is drivenby a number of factors, including practice patterns and theduration of action of the ESA. While extending the dosinginterval of ESA therapy is often possible, the major consid-erations from the clinical and pharmacoeconomicviewpoints are whether Hb levels can be effectivelymaintained within a target range, e.g., 11 to 12 g/dL, andwhether there is any dose penalty (i.e., need for dose in-creases greater than those calculated by simple multiplica-tion of time intervals) for extending the dosing-frequencyschedule. We have expanded our understanding of theways in which molecules stimulate erythropoiesis, includ-ing the interaction of EPO with its receptor and the effecton in vitro and in vivo activity. Further refinement of thisunderstanding to probe the trade-off between the receptoraffinity of ESAs and the circulating t1/2 of the molecule in-dicated that there is a balance between these biologic prop-erties that translates into the clinical arena. It is to be hopedthat greater appreciation of this phenomenon will fosterhigh-quality randomized controlled trials to determine theoptimal use for each ESA.

valent hemodialysis patients from the Dialysis Outcomes and Practice

SEM) Doses per week (% of patients)

ose (units/kg/week) n 3� 2� 1�

102 6 4.81 442 47 38 14

86 6 2.92 467 71 17 11

132 6 4.04 527 50 30 18

114 6 3.7 510 49 36 15

107 6 3.6 422 60 31 8

109 6 1.76 2368 55 30 14

EM, standard error of the mean.

1582 S. Elliott et al. / Experimental Hematology 2008;36:1573–1584

AcknowledgmentsThe authors wish to thank Michael Raffin and Beatrice Benoit ofNexus Communications, Inc. for their editorial assistance on thismanuscript.

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