Novel Erythropoiesis-Stimulating Agents: a New Era in Anemia Management

Mini-Review

Novel Erythropoiesis-Stimulating Agents: A New Era in Anemia Management

Iain C. Macdougall Department of Renal Medicine, King’s College Hospital, London, United Kingdom

Nearly two decades ago, recombinant human erythropoietin transformed the management of chronic kidney disease anemia by allowing a more sustained increase in hemoglobin than was possible by intermittent blood transfusion. The treatment was highly effective, but because of the fairly short half-life of the molecule at approximately 6 to 8 h, injections usually had to be administered two to three times weekly. A second-generation erythropoietin analogue, darbepoetin alfa, was then created, with a longer elimination half-life in vivo that translated into less frequent dosing, usually once weekly or once every 2 wk. More recently, another erythropoietin-related molecule has been produced called Continuous Erythropoietin Receptor Activator with an even greater half-life, and other molecules are in development or are being licensed, including biosimilar epoetin products and Hematide. The latter is a synthetic peptide-based erythropoietin receptor agonist that, interestingly, has no structural homology with erythropoietin, and yet is still able to activate the erythropoietin receptor and stimulate erythropoiesis. The search goes on for orally active antianemic therapies, and several strategies are being investigated, although none is imminently available. This article reviews the latest progress with these novel erythropoietic agents in this new era in anemia management. Clin J Am Soc Nephrol 3: 200–207, 2008. doi: 10.2215/CJN.03840907

any nephrologists can still recall the days when EPO antibodies), storage and stability (must be stored at tem- large numbers of dialysis patients were transfusion peratures of approximately 4°C), and administration (all cur- M dependent, requiring repeated red cell transfusions rently licensed products are administered intravenously or every few weeks to increase the hemoglobin concentration from subcutaneously). Various strategies have been devised to cir- approximately 6 g/dl to a transient value of approximately 8 or cumvent the limitations of the currently available products 9 g/dl, before falling once again to baseline levels. The advent (Table 1), and these are discussed in this review. The strategies of recombinant human erythropoietin (EPO; epoetin) in the include the potential development of orally active ESAs, per- late-1980s transformed this desperate situation, restoring pa- haps through stabilization of hypoxia-inducible factor (HIF), tients’ ability to use their own bone marrow for red cell pro- although the HIF stabilizers have suffered a recent setback that duction, with a dramatic reduction in the number of blood has seriously jeopardized their ongoing clinical development. transfusions used in dialysis centers. Epoetin therapy was found to be highly effective in the vast majority of patients who had anemia of chronic kidney disease (CKD), and adverse Protein-Based ESA Therapy effects were uncommon or easily managed. The original recombinant human EPOs (epoetin alfa and epo- Because of the fairly short circulating half-life of plasma EPO etin beta) have now been in clinical use for nearly 20 yr. Both (approximately 6 to 8 h) (1), however, patients required two or products are synthesized in cultures of transformed Chinese three injections a week. Thus, there was a clinical need for hamster ovary (CHO) cells that carry cDNA encoding human longer acting erythropoiesis-stimulating agents (ESAs), and EPO (2). The amino acid sequence of both epoetins is therefore several of these have been developed or are under develop- identical, and the major difference between these products lies ment. To date, all ESAs licensed for clinical use are protein in their glycosylation pattern. Thus, it is recognized that human based, bearing some structural resemblance to EPO itself. Thus, EPO exists as a mixture of isoforms that differ in both glyco- for agents such as darbepoetin alfa or Continuous EPO Recep- sylation and biologic activity (3). tor Activator (CERA), modifications have been made to the Other epoetins that have recently become available or are still EPO molecule to allow it to have a longer duration of action in being developed include epoetin omega (4–6) and epoetin vivo. Protein-based therapies have a number of disadvantages, delta (7–9), as well as the copy products of epoetin alfa and notably immunogenicity (pure red cell aplasia caused by anti- other biosimilar epoetins (reviewed by Schellekens [10] in this issue of CJASN). Again, all of these products share the same

Published online ahead of print. Publication date available at www.cjasn.org. 165–amino acid sequence as for epoetin alfa and epoetin beta, as well as the endogenous hormone. The cell culture conditions, Correspondence: Dr. Iain C. Macdougall, Renal Unit, King’s College Hospital, London SE5 9RS, UK. Phone: ϩϩ44-203-299-9000, ext. 6953; Fax: ϩϩ44-207-346- however, vary. With epoetin omega, baby hamster kidney 6472; E-mail: [email protected] (BHK) cell cultures are used for the manufacture of this prod-

Table 1. Current and future erythropoietic agents for the treatment of CKD anemiaa

Protein-based ESA therapy epoetin (alfa, beta, delta, omega) biosimilar EPO darbepoetin alfa CERA SEP EPO fusion proteins EPO–EPO GM-CSF–EPO Fc-EPO CTNO 528 Small-molecule ESA Figure 1. In vivo activity of isolated recombinant human eryth- peptide-based (e.g., Hematide) ropoietin (r-HuEPO) isoforms in mice (three times per week non–peptide-based intraperitoneally). Redrawn from Egrie et al. (3). Other strategies for stimulating erythropoiesis prolyl hydroxylase inhibitors (HIF stabilizers) GATA inhibitors sis to change the amino acid sequence at sites not directly HCP inhibitors involved in binding to the EPO receptor (13,14). Thus, five– EPO gene therapy amino acid substitutions were implemented (allowing darbe- aCKD, chronic kidney disease; EPO, erythropoietin; ESA, poetin alfa to carry a maximum of 22 sialic acid residues, erythropoiesis-stimulating agent; GM-CSF, granulocyte compared with recombinant or endogenous EPO, which sup- macrophage colony-stimulating factor; HCP, hemopoietic cell port a maximum of 14 sialic acid residues). The additional phosphatase; HIF, hypoxia-inducible factor; SEP, synthetic N-linked carbohydrate chains increased the molecular weight erythropoiesis protein. of epoetin from 30.4 to 37.1 kD, and the carbohydrate contri- bution to the molecule correspondingly increased from 40% to approximately 52% (13,14). uct, which has been used clinically in some Eastern European, These molecular modifications to EPO confer a greater met- Central American, and Asian countries (4–6). abolic stability in vivo, with the elimination half-life in human Epoetin delta is another recombinant EPO that has been used after a single intravenous injection of darbepoetin alfa increas- for treating patients with CKD; it was approved by the Euro- ing three-fold (25.3 h) compared with epoetin alfa (8.5 h) (15). pean Medicines Agency in 2002 and first marketed in Germany The half-life after subcutaneous administration is doubled from in 2007 (7–9). Epoetin delta is synthesized in human fibrosar- approximately 24 h to approximately 48 h. This latter charac- coma cell cultures (line HT-1080). The product is also called teristic has allowed less frequent dosing, with most patients gene-activated EPO because the expression of the native human receiving injections once weekly or once every other week (16). EPO gene is activated by transformation of the cell with the Further extension out to once-monthly dosing with darbepoetin cytomegalovirus promoter (11). In contrast to CHO or BHK alfa may be possible in some patients, but it is not clear what cell–derived recombinant human EPO, epoetin delta does not dosage penalty this incurs. Furthermore, this is possible only in possess N-glycolylneuraminic acid (Neu5Gc) because, in con- selected patients, generally those who are clinically stable and trast to other mammals, humans are genetically unable to pro- who do not yet require dialysis. duce Neu5Gc as a result of an evolutionary mutation (12). The implications of a lack of Neu5Gc residues in synthetic recom- CERA binant EPO, if any, are not clear at present. The strategy used to synthesize CERA was to integrate a large methoxy-polyethyleneglycol polymer chain into the EPO mol- Darbepoetin alfa ecule via amide bonds between the N-terminal amino group of The development of darbepoetin alfa arose from the recogni- alanine and the ␴-amino groups of lysine (Lys45 or Lys52)by tion that the higher isoforms (those with a greater number of means of a succinimidyl butanoic acid linker (17). Because the sialic acid residues) of recombinant human EPO were more mass of the polymer chain is approximately 30 kD, this doubles potent biologically in vivo as a result of a longer circulating the molecular weight of CERA to approximately 60 kD, com- half-life than the lower isomers (those with a lower number of pared with EPO (30.4 kD). As with other pegylated therapeutic sialic acid residues) (3) (Figure 1). Because the majority of sialic proteins, the half-life of circulating CERA is considerably pro- acid residues are attached to the three N-linked glycosylation longed compared with that of epoetin: at approximately 130 h chains of the EPO molecule, attempts were made to synthesize (18). Thus, less frequent dosing regimens of once every 2 wk EPO analogues with a greater number of N-linked carbohy- and once every month have been tested in Phase II and Phase drate chains. This was achieved using site-directed mutagene- III clinical trials (19,20), and the product recently received a 202 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: 200–207, 2008 license in both the US and Europe. As with the previous eryth- with an enhanced serum half-life but no structural similarity to ropoietic agents, CERA is still administered intravenously or EPO (33). Rats that were treated with a single subcutaneous subcutaneously, and adverse events seem to be similar to those dose of CTNO 528 showed a more prolonged reticulocytosis associated with the epoetins or darbepoetin alfa. It is possible and hemoglobin rise compared with treatment with epoetin or that the metabolic fate of CERA is different from the existing darbepoetin alfa. Phase I studies in healthy volunteers showed products, with less cellular internalization after interaction with a similar effect after a single intravenous administration of the EPO receptor, but further experimental work is required to CTNO 528, with a peak reticulocyte count occurring after 8 d, confirm this. As also occurs with darbepoetin alfa, the binding and the maximum hemoglobin concentration being seen after affinity for the EPO receptor is less than for natural or recom- 22 d. None of the 24 participants in this study developed binant EPO, but the benefits of the greater stability in vivo far antibodies against the molecule (34). outweigh this minor biologic disadvantage. Interestingly, an Fc-EPO fusion protein has been successfully In addition to CERA, other pegylated molecules, including administered in a Phase I trial to human volunteers as an epoetin alfa (21) and an epoetin analogue (22), have been tested aerosol, with a demonstrable increase in EPO levels associated for their efficacy in experimental animals. These products have with an increase in reticulocyte counts (35). In addition to the not yet entered clinical trials. EPO derivatives administered by aerosol inhalation, other delivery systems for EPO have been investigated, including ul- Other Protein-Based EPO Derivatives trasound-mediated transdermal uptake (36) and orally via lipo- Several other EPO-like molecules and derivatives are in pre- somes to rats (37). Mucoadhesive tablets containing EPO and an clinical or clinical trials. A further hyperglycosylated analogue absorption enhancer (Labrasol; Gattefosse, Gennevilliers, of darbepoetin alfa was synthesized, with additional carbohy- France) for oral administration have been studied in rats and drate residues (AMG114). Although this analogue was found to dogs (38). Theoretically, this preparation is designed to allow have an even longer circulating half-life in vivo compared with the tablet to reach the small intestine intact. Preliminary exper- darbepoetin alfa, the EPO receptor binding affinity was too low iments in beagle dogs were conducted with intrajejunal admin- to develop this molecule further as a therapeutic agent, and istration of a single tablet containing 100 IU/kg recombinant clinical trials have now ceased. Another novel product is syn- human EPO, with a corresponding increase in reticulocytes 8 d thetic erythropoiesis protein (SEP), which was manufactured after administration (38). It is too early to say whether this using solid-phase peptide synthesis and branched precision strategy could have any clinical relevance in the treatment of polymer constructs. A 51-kD protein-polymer construct was anemia in patients with CKD. synthesized containing two covalently attached polymer moi- eties (23). As with darbepoetin alfa and CERA, this polymer Small-Molecule ESAs stimulates erythropoiesis through activation of the EPO recep- Peptide-Based ESAs tor, and with a longer circulating half-life than for EPO alone. Just over 10 yr ago, several small bisulfide-linked cyclic pep- The erythropoietic effect of synthetic erythropoiesis protein has tides composed of approximately 20 amino acids that were been shown to vary in experimental animals depending on the unrelated in sequence to EPO but still bound to the EPO recep- number and type of the attached polymers (24). Recombinant tor were identified by random phage display technology EPO fusion proteins that contain additional peptides at the (39,40). These small peptides were able to induce the same carboxy-terminus to increase in vivo survival have been ex- conformational change in the EPO receptor that leads to JAK2 pressed (25). Large EPO fusion proteins, of molecular weight 76 kinase/STAT-5 intracellular signaling (40), as well as other kD, have been designed from cDNA encoding two human EPO intracellular signaling mechanisms, resulting in stimulation of molecules linked by small flexible polypeptides (26,27). A sin- erythropoiesis both in vitro and in vivo. The first peptide to be gle subcutaneous administration of this compound to mice investigated (EPO-mimetic peptide-1) (40) was not potent increased red cell production within7datadosage at which enough to be considered as a potential therapeutic agent in its epoetin was ineffective (26). Another dimeric fusion protein own right, but the potency of these peptides could be greatly incorporating both EPO and granulocyte macrophage colony- enhanced by covalent peptide dimerization with a PEG linker. stimulating factor (GM-CSF) has been created, with the ratio- Thus, another EPO-mimetic peptide was selected for the devel- nale that GM-CSF is required for early erythropoiesis. This opment of Hematide (Affymax, Palo Alto, CA), a pegylated EPO–GM-CSF complex proved to be able to stimulate erythro- synthetic dimeric peptidic ESA that was found to stimulate poiesis in cynomolgus monkeys (28) but was later found to erythropoiesis in experimental animals (41). The half-life of induce anti-EPO antibodies, causing severe anemia (29). Yet Hematide in monkeys ranges from 14 to 60 h, depending on the another approach is the genetic fusion of EPO with the Fc dosage administered. Further studies in rats using quantitative region of human IgG (Fc-EPO) (30). This molecular modifica- whole-body autoradioluminography have shown that the pri- tion promotes recycling out of the cell upon endocytosis via the mary route of elimination for the peptide is the kidney (42). Fc recycling receptor (31,32), again providing an alternative A Phase I study in healthy volunteers showed that single mechanism for enhancing circulating half-life. The same effect injections of Hematide caused a dosage-dependent increase in may be achieved by fusing EPO with albumin. reticulocyte counts and hemoglobin concentrations (43). Phase Another molecule currently undergoing development is II studies have demonstrated that Hematide can correct the CTNO 528, which is an EPO-mimetic antibody fusion protein anemia associated with CKD (44), as well as maintain the Clin J Am Soc Nephrol 3: 200–207, 2008 Anemia Management with Novel Erythropoietic Agents 203 hemoglobin in dialysis patients who are already receiving con- catalytic action but also iron and 2-oxoglutarate (51). Thus, ventionals ESAs (45). Dosages in the range of 0.025 to 0.05 HIF-alfa hydroxylation can be prevented either by iron deple- mg/kg seem to be therapeutically optimal in this patient pop- tion or by the administration of 2-oxoglutarate analogues. ulation (44), and at the time of writing, four Phase III studies are These latter molecules have recently been termed HIF stabiliz- about to be initiated. Hematide may be administered either ers, and these compounds have been shown to promote EPO intravenously or subcutaneously, and dosing once a month is expression in cell cultures, as well as in animals and humans. effective (44). Interestingly, these compounds were originally developed The potential advantages of this new agent are greater sta- for their inhibitory action on collagen prolyl 4-hydroxylases, bility at room temperature, lower immunogenicity compared which also need 2-oxoglutarate as a co-factor. The primary aim with conventional ESAs, and a much simpler (and cheaper) of the initial studies was to develop drugs for the treatment of manufacturing process, avoiding the need for cell lines and fibrotic diseases (52,53); however, the 2-oxoglutarate analogues genetic engineering techniques. Antibodies against Hematide were found to stimulate erythropoiesis in vivo (54). HIF stabi- do not cross-react with EPO, and similarly anti-EPO antibodies lizers have already been administered to healthy control sub- do not cross-react with Hematide (46). This has two major jects (55) and to patients with CKD (56) in clinical trials inves- implications: First, even if a patient does develop anti-Hema- tigating novel strategies for the treatment of anemia. After their tide antibodies, these should not neutralize the patient’s own administration, increases in plasma EPO levels were found, endogenous EPO, and the patient should not develop pure red with a concomitant increase in reticulocyte count. Phase II cell aplasia. Second, patients with antibody-mediated pure red studies of the first candidate molecule, FG-2216, demonstrated cell aplasia should be able to respond to Hematide therapy by correction of anemia in patients with CKD, in contrast to pla- an increase in their hemoglobin concentration, because Hema- cebo (56). These agents have the advantage of being orally tide is not neutralized by anti-EPO antibodies. This latter hy- active, and they also seem to upregulate other genes involved pothesis has already been confirmed in animals (46). Rats that in the process of erythropoiesis, notably those that improve received regular injections of recombinant human EPO were iron utilization. shown to develop anti-EPO antibodies. Injections of Hematide Unfortunately, several concerns have seriously jeopardized were able to “rescue” these animals and restore their hemoglo- the future application of these orally active agents in the treat- bin concentration, in contrast to the vehicle-treated group (46). ment of anemia in CKD. First, at least 100 other genes are A clinical trial examining this issue in patients with antibody- upregulated by inhibition of the prolyl hydroxylases, not only mediated pure red cell aplasia was also recently performed genes that promote erythropoiesis but also other hypoxia-sen- (47). sitive genes, such as vascular endothelial growth factor (57). Other peptide-based ESAs are in preclinical development. A Although there may be attempts to create HIF stabilizers that compound made by AplaGen (Baesweiler, Germany) has upregulate only erythropoiesis genes and not other HIF-sensi- linked a peptide to a starch residue, again demonstrating pro- tive genes, it will take some time to persuade clinicians that longation of the circulating half-life of the molecule (48). In- there is no risk for potentiation of tumor growth as well as deed, altering the molecular weight of the starch moiety has other unwanted adverse effects arising from such ubiquitous been shown to alter the pharmacologic properties of the com- gene upregulation. pound. In mid-2007, there arose another serious barrier to further development of these agents in the treatment of anemia. During Non–peptide-Based ESAs one of the Phase II clinical trials of FG-2216, a female patient Several nonpeptide molecules that are capable of mimicking developed fatal hepatic necrosis that was temporally related to the effects of EPO have also been identified, after screens of the introduction of this compound (58). Although investiga- small-molecule nonpeptide libraries for molecules with EPO tions regarding causality are ongoing, the Food and Drug Ad- receptor–binding activity (49,50). One such compound was ministration has for now suspended any further clinical trials found, but this bound to only a single chain of the EPO receptor with HIF stabilizers. and was not biologically active. The compound was ligated to enable it to interact with both domains of the EPO receptor, and GATA Inhibition this second molecule was shown to stimulate erythropoiesis The GATA family consists of six transcription factors, GATA 1 (49). Further development of nonpeptide EPO mimetics could through 6. Dame et al. (59) reported that GATA-4 is critically lead to the production of an orally active ESA in the future. involved in EPO gene expression and may be responsible for the switch in the site of EPO production from the fetal liver to Other Strategies for Stimulating the adult kidney. In addition, GATA-2 inhibits EPO gene tran- Erythropoiesis scription by binding to the GATA sequence on the EPO pro- Prolyl Hydroxylase Inhibition (HIF Stabilizers) moter, thereby leading to downregulation of EPO mRNA ex- Under normoxic conditions, EPO gene expression is sup- pression and subsequent EPO synthesis (60). GATA-2 therefore pressed physiologically as a result of inactivation of the hy- acts as a negative regulatory molecule of EPO gene expression. poxia-inducible transcription factors (HIF). This inactivation is Disrupting this negative signal is therefore a potential future mediated by HIF-alfa prolyl- and asparaginyl-hydroxylation. strategy in the management of renal anemia. Several molecules The HIF-alfa hydroxylases require not only oxygen for their are under investigation, including K-11706, which has been 204 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: 200–207, 2008 shown to enhance EPO production both in vitro and in vivo. expressed in a specific B cell clone (71). Interestingly, animal Oral administration of K-11706 restored the hemoglobin con- experiments have shown that linking the EPO transgene to a centrations, reticulocyte counts, EPO levels, and numbers of hypoxia-responsive DNA element (the HIF binding site) can CFU-E induced by IL-1beta or TNF-alfa in a mouse model of establish an oxygen-dependent feedback regulation of the anemia (61). These results raise the possibility of using orally transgene, similar to that of the endogenous EPO gene (72). administered K-11706 in the treatment of renal anemia, but clinical trials are not yet under way. Conclusions As the molecular mechanisms that control red cell production Hemopoietic Cell Phosphatase Inhibition have been elucidated, so, too, have new targets and strategies Another strategy with the potential for enhancing erythropoi- been developed for stimulating erythropoiesis and treating esis is targeting the src homology domain 2–containing tyrosine anemia. After the introduction of recombinant human EPO, phosphatase-1 (SHP-1), also known as hemopoietic cell phos- attempts were made to modify the molecule and produce phatase (HCP) (62). This protein tyrosine phosphatase is lo- longer acting erythropoietic agents, such as darbepoetin alfa cated in the cytoplasm of hemopoietic cells and was originally and CERA. Other modifications to the molecule, such as the identified in human breast carcinoma cDNA (63). SHP-1 binds production of fusion proteins, are being explored, as is the to the negative regulatory domain of the EPO receptor via its potential for EPO gene therapy. The concept that smaller mol- src-homology 2 domains and causes dephosphorylation of ecules such as peptides or even nonpeptides may be able to JAK-2, thereby functioning as a negative regulator of EPO bind to and activate the EPO receptor is also being investigated, intracellular signal transduction (64). The potential importance and the first such molecule (Hematide) is currently in Phase III of this molecule in mediating responsiveness to EPO therapy ϩ of its clinical development program. Other strategies, attempt- was studied in CD34 cells derived from a population of he- ing to create orally active agents, such as inhibition of prolyl modialysis patients who were responding poorly to EPO (65). ϩ hydroxylase, GATA, or HCP, remain in the laboratory but may Compared with an EPO-responsive group, CD34 cells from yet translate into future therapeutic agents for the management EPO-hyporesponsive patients showed increased mRNA and of CKD anemia. protein expression of SHP-1. Furthermore, treatment of the ϩ CD34 cells from EPO-hyporesponsive patients with an SHP-1 antisense oligonucleotide decreased SHP-1 protein expression Disclosures and upregulated STAT-5, resulting in the partial recovery of Dr. Macdougall has received consulting fees, lecture fees, and grant erythroid colony formation (65). The gene for SHP-1 has been support from Amgen, Ortho Biotech, Roche, Shire, and Affymax. cloned, and SHP-1 inhibitors have been identified. In vitro inhibition of SHP-1 resulted in a dosage-dependent erythroid proliferation (65). As with the GATA inhibitors, the HCP in- References hibitors have not yet been tested in humans, and it is therefore 1. Macdougall IC, Roberts DE, Coles GA, Williams JD: Clin- not clear whether they would have any role in the management ical pharmacokinetics of epoetin (recombinant human of CKD anemia. These orally active agents, however, could erythropoietin). Clin Pharmacokinet 20: 99–113, 1991 potentially be used as adjuvant therapy to enhance the re- 2. Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, sponse to other ESAs or even to enhance the patient’s own Chen KK, Fox GM, Martin F, Stabinsky Z, et al.: Cloning endogenous EPO. and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A 82: 7580–7584, 1985 3. Egrie JC, Grant JR, Gillies DK, Aoki KH, Strickland TW: EPO Gene Therapy The role of carbohydrate on the biological activity of eryth- With increasing concern that high dosages of erythropoietic ropoietin. Glycoconj J 10: 263, 1993 products may be harmful, the ability to generate lower but 4. Acharya VN, Sinha DK, Almeida AF, Pathare AV: Effect of continuous levels of EPO as a result of gene therapy is a low dose recombinant human omega erythropoietin potentially attractive area of research. It does not seem to (rHuEPO) on anaemia in patients on hemodialysis. J Assoc matter by which cells and at which site EPO is released into the Physicians India 43: 539–542, 1995 circulation, and a number of delivery systems have been inves- 5. Bren A, Kandus A, Varl J, Buturovic J, Ponikvar R, Kveder tigated, such as injection of naked DNA (66), adenovirus trans- R, Primozic S, Ivanovich P: A comparison between epoetin fection (67), use of artificial human chromosomes (68), and omega and epoetin alfa in the correction of anemia in transplantation of autologous or allogeneic cells manipulated hemodialysis patients: A prospective, controlled crossover ex vivo (69,70). study. Artif Organs 26: 91–97, 2002 6. Sikole A, Spasovski G, Zafirov D, Polenakovic M: Epoetin As with all gene therapy, there are many hurdles to over- omega for treatment of anemia in maintenance hemodial- come before this could be used in humans. Not only would ysis patients. Clin Nephrol 57: 237–245, 2002 there need to be reassurance regarding the absence of oncoge- 7. Spinowitz BS, Pratt RD: Epoetin delta is effective for the nicity, but it would also be imperative to show that tight control management of anaemia associated with chronic kidney of the activity of the transferred gene can be achieved. This may disease. Curr Med Res Opin 22: 2507–2513, 2006 be possible using a number of pharmacologic strategies or 8. Kwan JT, Pratt RD: Epoetin delta, erythropoietin produced potentially by exposure to a rare antigen, when the transgene is in a human cell line, in the management of anaemia in Clin J Am Soc Nephrol 3: 200–207, 2008 Anemia Management with Novel Erythropoietic Agents 205

pre-dialysis chronic kidney disease patients. Curr Med Res MS, Christensen KR, Edwards DP, Chlipala EA, Cox GN: Opin 23: 307–311, 2007 Design of homogeneous, monopegylated erythropoietin 9. Martin KJ, on behalf of the Epoetin Delta 3001 Study analogs with preserved in vitro bioactivity. Exp Hematol 34: Group: Epoetin delta in the management of renal anaemia: 697–704, 2006 Results of a 6-month study. Nephrol Dial Transplant 22: 23. Kochendoerfer GG, Chen SY, Mao F, Cressman S, Traviglia 3052–3054, 2007 S, Shao H, Hunter CL, Low DW, Cagle EN, Carnevali M, 10. Schellekens H: The first biosimilar epoetin: But how similar Gueriguian V, Keogh PJ, Porter H, Stratton SM, Wiedeke is it? Clin J Am Soc Nephrol 3: 174–178, 2008 MC, Wilken J, Tang J, Levy JJ, Miranda LP, Crnogorac MM, 11. The Court Service—Court of Appeal—Civil—Judgement: Kalbag S, Botti P, Schindler-Horvat J, Savatski L, Adamson In TKT’s technology, those cells were designated as R223 JW, Kung A, Kent SB, Bradburne JA: Design and chemical cells. Neutral Citation Number: [2002] EWCA Civ. 1096, synthesis of a homogeneous polymer-modified erythropoi- 2002. Available at: http://www.hmcourts-service.gov.uk/ esis protein. Science 299: 884–887, 2003 judgmentsfiles/j1329/Kirin_v_Hoechst.htm 24. Chen SY, Cressman S: Mao F, Shao H, Low DW, Beilan HS, 12. Tangvoranuntakul P, Gagneux P, Diaz S, Bardor M, Varki Cagle EN, Carnevali M, Gueriguian V, Keogh PJ, Porter H, N, Varki A, Muchmore E: Human uptake and incorpora- Stratton SM, Wiedeke MC, Savatski L, Adamson JW, tion of an immunogenic nonhuman dietary sialic acid. Proc Bozzini CE, Kung A, Kent SB, Bradburne JA, Kochendoer- Natl Acad Sci U S A 100: 12045–12050, 2003 fer GG: Synthetic erythropoietic proteins: Tuning biologi- 13. Egrie JC, Browne JK: Development and characterization of cal performance by site-specific polymer attachment. Chem novel erythropoiesis stimulating protein (NESP). Br J Can- Biol 12: 371–383, 2005 cer 84: 3–10, 2001 25. Lee DE, Son W, Ha BJ, Oh MS, Yoo OJ: The prolonged 14. Elliott S, Lorenzini T, Asher S, Aoki K, Brankow D, Buck L, half-lives of new erythropoietin derivatives via peptide Busse L, Chang D, Fuller J, Grant J, Hernday N, Hokum M, addition. Biochem Biophys Res Commun 339: 380–385, 2006 Hu S, Knudten A, Levin N, Komorowski R, Martin F, 26. Sytkowski AJ, Lunn ED, Risinger MA, Davis KL: An eryth- Navarro R, Osslund T, Rogers G, Rogers N, Trail G, Egrie ropoietin fusion protein comprised of identical repeating J: Enhancement of therapeutic protein in vivo activities domains exhibits enhanced biological properties. J Biol through glycoengineering. Nat Biotechnol 21: 414–421, 2003 Chem 274: 24773–24778, 1999 15. Macdougall IC, Gray SJ, Elston O, Breen C, Jenkins B, 27. Dalle B, Henri A, Rouyer-Fessard P, Bettan M, Scherman Browne J, Egrie J: Pharmacokinetics of novel erythropoiesis D, Beuzard Y, Payen E: Dimeric erythropoietin fusion pro- stimulating protein compared with epoetin alfa in dialysis tein with enhanced erythropoietic activity in vitro and in patients. J Am Soc Nephrol 10: 2392–2395, 1999 vivo. Blood 97: 3776–3782, 2001 16. Macdougall IC, Padhi D, Jang G: Pharmacology of darbe- 28. Coscarella A, Liddi R, Bach S, Zappitelli S, Urso R, Mele A, poetin alfa. Nephrol Dial Transplant 22[Suppl 4]: iv2–iv9, De Santis R: Pharmacokinetic and immunogenic behavior 2007 of three recombinant human GM-CSF-EPO hybrid proteins 17. Macdougall IC: CERA (Continuous Erythropoietin Recep- in cynomolgus monkeys. Mol Biotechnol 10: 115–122, 1998 tor Activator): a new erythropoiesis-stimulating agent for 29. Coscarella A, Liddi R, Di Loreto M, Bach S, Faiella A, van the treatment of anemia. Curr Hematol Rep 4: 436–440, 2005 der Meide PH, Mele A, De Santis R: The rhGM-CSF-EPO 18. Macdougall IC, Robson R, Opatrna S, Liogier X, Pannier A, hybrid protein MEN 11300 induces anti-EPO antibodies Jordan P, Dougherty FC, Reigner B: Pharmacokinetics and and severe anaemia in rhesus monkeys. Cytokine 10: 964– pharmacodynamics of intravenous and subcutaneous con- 969, 1998 tinuous erythropoietin receptor activator (C.E.R.A.) in pa- 30. Way JC, Lauder S, Brunkhorst B, Kong SM, Qi A, Webster tients with chronic kidney disease. Clin J Am Soc Nephrol 1: G, Campbell I, McKenzie S, Lan Y, Marelli B, Nguyen LA, 1211–1215, 2006 Degon S, Lo KM, Gillies SD: Improvement of Fc-erythro- 19. Provenzano R, Besarab A, Macdougall IC, Ellison DH, poietin structure and pharmacokinetics by modification at Maxwell AP, Sulowicz W, Klinger M, Rutkowski B, Cor- a disulfide bond. Protein Eng Des Sel 18: 111–118, 2005 rea-Rotter R, Dougherty FC, BA 16528 Study Investigators: 31. Capon DJ, Chamow SM, Mordenti J, Marsters SA, Gregory The continuous erythropoietin receptor activator T, Mitsuya H, Byrn RA, Lucas C, Wurm FM, Groopman JE, (C.E.R.A.) corrects anemia at extended administration in- et al.: Designing CD4 immunoadhesins for AIDS therapy. tervals in patients with chronic kidney disease not on Nature 337: 525–531, 1989 dialysis: Results of a phase II study. Clin Nephrol 67: 306– 32. Yeh P, Landais D, Lemaitre M, Maury I, Crenne JY, Bec- 317, 2007 quart J, Murry-Brelier A, Boucher F, Montay G, Fleer R, et 20. Locatelli F, Sulowicz W, Harris K, Selgas R, Kaufman J, al.: Design of yeast-secreted albumin derivatives for hu- Klinger M, Malberti F, Dougherty FC: SC C.E.R.A. (contin- man therapy: Biological and antiviral properties of a serum uous erythropoietin receptor activator) once every 2 weeks albumin-CD4 genetic conjugate. Proc Natl Acad Sci U S A or once monthly maintains stable Hb levels after convert- 89: 1904–1908, 1992 ing directly from SC epoetin 1–3 times per week in patients 33. Bugelski P, Nesspor BT, Spinka-Doms T, Markopoulos D, with CKD on dialysis [Abstract]. J Am Soc Nephrol 17: 619A, Eirikis E, Fisher J, Volk A, Shamberger K, James I, Fisher P, 2006 Pool C, Jang H, Miller B, Huang C, Heavner G, Knight D, 21. Jolling K, Perez Ruixo JJ, Hemeryck A, Vermeulen A, Gre- Ghrayeb J, Scallon B: Pharmacokinetics and pharmacody- way T: Mixed-effects modelling of the interspecies phar- namics of CTNO528, a novel erythropoiesis receptor ago- macokinetic scaling of pegylated human erythropoietin. nist in normal and anemic rats. Blood 106: 146b, 2005 Eur J Pharm Sci 24: 465–475, 2005 34. Franson KL, Burggraaf, Bouman-Trio EA, Cohen AF, 22. Long DL, Doherty DH, Eisenberg SP, Smith DJ, Rosendahl Miller BE, Jang H, Marciniak SL, van de Ketterij EPS, 206 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: 200–207, 2008

Frederick B, Jiao Q, Getsy J, Bald EH, Walker H, Schantz A, ropoiesis stimulating agent, maintains hemoglobin in he- Ford JA, Mascioli K, Kowalchick JB: A phase I, single and modialysis patients previously treated epoetin alfa [Ab- fractionated, ascending dose study evaluating the safety, stract 99]. Presented at the National Kidney Foundation pharmacokinetics, pharmacodynamics, and immunogenic- Spring Clinical Meeting; April 10–14, 2007; Orlando, FL ity of an erythropoietic mimetic antibody fusion protein, 46. Woodburn KW, Fan Q, Winslow S, Chen MJ, Mortensen CTNO528 in healthy male subjects. Blood 106: 146b, 2005 RB, Casadevall N, Stead RB, Schatz PJ: Hematide is immu- 35. Dumont JA, Bitonti AJ, Clark D, Evans S, Pickford M, nologically distinct from erythropoietin and corrects ane- Newman SP: Delivery of an erythropoietin-Fc fusion pro- mia induced by antierythropoietin antibodies in a rat pure tein by inhalation in humans through an immunoglobulin red cell aplasia model. Exp Hematol 35: 1201–1208, 2007 transport pathway. J Aerosol Med 18: 294–303, 2005 47. Macdougall IC, Rossert J, Casadevall N, Stead RB, Duliege 36. Mitragotri S, Blankschtein D, Langer R: Ultrasound-medi- A-M, Eckardt K-U: Treatment of anti-erythropoietin anti- ated transdermal protein delivery. Science 269: 850–853, body-mediated pure red cell aplasia with Hematide. 1995 N Engl J Med 2008, in press 37. Maitani Y, Moriya H, Shimoda N, Takayama K, Nagai T: 48. Po¨tgens A, Haberl U, Rybka A, Knorr K, Emgenbroich M, Distribution characteristics of entrapped recombinant hu- Casaretto M, Breuer B, Greindl A, Kessler C, Kromminga man erythropoietin in liposomes and its intestinal absorp- A, Frank H-G: An optimized supravalent EPO mimetic tion in rats. Int J Pharm 185: 13–22, 1999 peptide with unprecedented efficacy. Ann Hematol 85: 643, 38. Venkatesan N, Yoshimitsu J, Ohashi Y, Ito Y, Sugioka N, 2006 Shibata N, Takada K: Pharmacokinetic and pharmacody- 49. Qureshi SA, Kim RM, Konteatis Z, Biazzo DE, Motamedi namic studies following oral administration of erythropoi- H, Rodrigues R, Boice JA, Calaycay JR, Bednarek MA, etin mucoadhesive tablets to beagle dogs. Int J Pharm 310: Griffin P, Gao YD, Chapman K, Mark DF: Mimicry of 46–52, 2006 erythropoietin by a nonpeptide molecule. Proc Natl Acad 39. Livnah O, Stura EA, Johnson DL, Middleton SA, Mulcahy Sci U S A 96: 12156–12161, 1999 LS, Wrighton NC, Dower WJ, Jolliffe LK, Wilson IA: Func- 50. Goldberg J, Jin Q, Ambroise Y, Satoh S, Desharnais J, tional mimicry of a protein hormone by a peptide agonist: Capps K, Boger DL: Erythropoietin mimetics derived from The EPO receptor complex at 2.8 A. Science 273: 464–471, solution phase combinatorial libraries. J Am Chem Soc 124: 1996 544–555, 2002 40. Wrighton NC, Farrell FX, Chang R, Kashyap AK, Barbone 51. Bruegge K, Jelkmann W, Metzen E: Hydroxylation of hy- FP, Mulcahy LS, Johnson DL, Barrett RW, Jolliffe LK, poxia-inducible transcription factors and chemical com- Dower WJ: Small peptides as potent mimetics of the pro- pounds targeting the HIF-hydroxylases. Curr Med Chem 14: tein hormone erythropoietin. Science 273: 458–464, 1996 103–112, 2007 41. Fan Q, Leuther KK, Holmes CP, Fong KL, Zhang J, Velk- 52. Kivirikko KI, Myllyla R, Pihlajaniemi T: Protein hydroxy- ovska S, Chen MJ, Mortensen RB, Leu K, Green JM, Schatz lation: Prolyl 4-hydroxylase, an enzyme with four cosub- PJ, Woodburn KW: Preclinical evaluation of Hematide, a strates and a multifunctional subunit. FASEB J 3: 1609– novel erythropoiesis stimulating agent, for the treatment of 1617, 1989 anemia. Exp Hematol 34: 1303–1311, 2006 53. Baader E, Tschank G, Baringhaus KH, Burghard H, Gun- 42. Woodburn KW, Leuther K, Fan Q, Holmes C, Frederick B, zler V: Inhibition of prolyl 4-hydroxylase by oxalyl amino Xu C, Wehrman T, Press R: Renal excretion is the primary acid derivatives in vitro, in isolated microsomes and in route of elimination for the erythropoiesis stimulating embryonic chicken tissues. Biochem J 300: 525–530, 1994 agent Hematide as assessed by quantitative whole-body 54. Safran M, Kim WY, O’Connell F, Flippin L, Gunzler V, autoradioluminography (QWBA) in Sprague Dawley rats Horner JW, Depinho RA, Kaelin WG Jr: Mouse model for [Abstract SaP333]. Presented at the ERA-EDTA Congress; noninvasive imaging of HIF prolyl hydroxylase activity: June 21–24, 2007; Barcelona, Spain Assessment of an oral agent that stimulates erythropoietin 43. Stead RB, Lambert J, Wessels D, Iwashita JS, Leuther KK, production. Proc Natl Acad Sci U S A 103: 105–110, 2006 Woodburn KW, Schatz PJ, Okamoto DM, Naso R, Duliege 55. Urquilla P, Fong A, Oksanen S, Leigh S, Turtle E, Flippin L, AM: Evaluation of the safety and pharmacodynamics of Brenner M, Muthukrishnan E, Fourney P, Lin A, Yeowell Hematide, a novel erythropoietic agent, in a phase 1, dou- D, Molineaux C: Upregulation of endogenous EPO in ble-blind placebo-controlled, dose-escalation study in healthy subjects by inhibition of HIF-PH [Abstract]. JAm healthy volunteers. Blood 108: 1830–1834, 2006 Soc Nephrol 15: 546A, 2004 44. Macdougall IC, Tucker B, Yaqoob M, Mikhail A, Nowicki 56. Wiecek A, Piecha G, Ignacy W, Schmidt R, Neumayer HH, M, MacPhee I, Mysliwiec M, Sulowicz W, Zamauskaite A, Scigalla P, Urquilla P: Pharmacological stabilization of HIF Leong R, Iwashita J, Duliege AM, Wiecek A: Hematide, a increases hemoglobin concentration in anemic patients synthetic peptide-based erythropoiesis stimulating agent, with chronic kidney disease [Abstract]. Nephrol Dial Trans- achieves correction of anemia and maintains hemoglobin plant 20[Suppl 5]: v195, 2005 in patients with chronic kidney disease not on dialysis 57. Maxwell P: HIF-1: An oxygen response system with special [Abstract F-FC079]. Presented at the annual meeting of the relevance to the kidney. J Am Soc Nephrol 14: 2712–2722, American Society of Nephrology; November 14–19, 2006; 2003 San Diego, CA 58. Astellas Pharma Inc: Adverse Event of FG-2216 for the 45. Besarab A, Zeig S, Geronemus R, Pergola P, Whittier F, Treatment of Anemia [News release], May 7, 2007. Avail- Zabaneh R, Schiller B, Kaplan M, Levin N, Wright S, Swan able at: http://http://www.astellas.com/global/about/ S, Wintz R, Wombolt D, Leong R, Lang W, Franco M, news/2007/pdf/070507_eg.pdf Duliege AM: Hematide™, a synthetic peptide-based eryth- 59. Dame C, Sola MC, Lim K-C, Leach KM, Fandrey J, Ma Y, Clin J Am Soc Nephrol 3: 200–207, 2008 Anemia Management with Novel Erythropoietic Agents 207

Kno¨pfle G, Engel JD, Bungert J: Hepatic erythropoietin erto G, La Monica N: Gene electro-transfer on an improved gene regulation by GATA-4. J Biol Chem 279: 2955–2961, erythropoietin plasmid in mice and non-human primates. 2004 J Gene Med 7: 228–236, 2005 60. Imagawa S, Yamamoto M, Miura Y: Negative regulation of 67. Rivera VM, Gao GP, Grant RL, Schnell MA, Zoltick PW, the erythropoietin gene expression by the GATA transcrip- Rozamus LW, Clackson T, Wilson JM: Long-term pharma- tion factors. Blood 89: 1430–1439, 1997 cologically regulated expression of erythropoietin in pri- 61. Nakano Y, Imagawa S, Matsumoto K, Stockmann C, Obara mates following AAV-mediated gene transfer. Blood 105: N, Suzuki N, Doi T, Kodama T, Takahashi S, Nagasawa T, 1424–1430, 2005 Yamamoto M: Oral administration of K-11706 inhibits 68. Kakeda M, Hiratsuka M, Nagata K, Kuroiwa Y, Kakitani GATA binding activity, enhances hypoxia-inducible factor M, Katoh M, Oshimura M, Tomizuka K: Human artificial 1 binding activity, and restores indicators in an in vivo chromosome (HAC) vector provides long-term therapeutic mouse model of anemia of chronic disease. Blood 104: transgene expression in normal human primary fibro- 4300–4307, 2004 blasts. Gene Ther 12: 852–856, 2005 62. Barbone FP, Johnson DL, Farrell FX, Collins A, Middleton 69. Schwenter F, Schneider BL, Pralong WF, Deglon N, Aebi- SA, McMahon FJ, Tullai J, Jolliffe LK: New epoetin mole- scher P: Survival of encapsulated human primary fibro- cules and novel therapeutic approaches. Nephrol Dial blasts and erythropoietin expression under xenogeneic Transplant 14[Suppl 2]: 80–84, 1999 conditions. Hum Gene Ther 15: 669–680, 2004 63. Shen SH, Bastien L, Posner BI, Chretien P: A protein- 70. Lippin Y, Dranitzki-Elhalel M, Brill-Almon E, Mei-Zahav tyrosine phosphatase with sequence similarity to the SH2 C, Mizrachi S, Liberman Y, Iaina A, Kaplan E, Podjarny E, domain of the protein-tyrosine kinases. Nature 352: 736– Zeira E, Harati M, Casadevall N, Shani N, Galun E: Human 739, 1991 erythropoietin gene therapy for patients with chronic renal 64. Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF: failure. Blood 106: 2280–2286, 2005 Specific recruitment of SH-PTP1 to the erythropoietin re- 71. Taka´cs K, Du Roure C, Nabarro S, Dillon N, McVey JH, ceptor causes inactivation of JAK2 and termination of pro- Webster Z, Macneil A, Barto´k I, Higgins C, Gray D, liferative signals. Cell 80: 729–738, 1995 Merkenschlager M, Fisher AG: The regulated long-term 65. Akagi S, Ichikawa H, Okada T, Sarai A, Sugimoto T, Mo- delivery of therapeutic proteins by using antigen-specific B rimoto H, Kihara T, Yano A, Nakao K, Nagake Y, Wada J, lymphocytes. Proc Natl Acad Sci U S A 101: 16298–16303, Makino H: The critical role of SRC homology domain 2004 2-containing tyrosine phosphatase-1 in recombinant hu- 72. Binley K, Askham Z, Iqball S, Spearman H, Martin L, de man erythropoietin hyporesponsive anemia in chronic he- Alwis M, Thrasher AJ, Ali RR, Maxwell PH, Kingsman S, modialysis patients. J Am Soc Nephrol 15: 3215–3224, 2004 Naylor S: Long-term reversal of chronic anemia using a 66. Fattori E, Cappelletti M, Zampaglione I, Mennuni C, Cal- hypoxia-regulated erythropoietin gene therapy. Blood 100: varuso F, Arcuri M, Rizzuto G, Costa P, Perretta G, Cilib- 2406–2413, 2002

See related editorial, “Anemia of Chronic Kidney Disease” on pages 3–6.