Development of Recombinant Erythropoietin and Erythropoietin Analogs

C H A P T E R

13 Development of Recombinant Erythropoietin and Erythropoietin Analogs Iain C. Macdougall King’s College Hospital, London, United Kingdom

O U T L I N E 1. Introduction

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2. History of Recombinant Human Erythropoietin 218 3. Biosimilar Erythropoietins

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4. Potential Strategies for Modifying Erythropoietin to Create New Erythropoietin Analogs 220 5. Darbepoetin Alfa 221 5.1 Intravenous Administration—Hemodialysis Patients222 5.2 Subcutaneous Administration—Hemodialysis Patients223 5.3 Subcutaneous Administration—Predialysis Chronic Kidney Disease Patients 223 6. Continuous Erythropoietin Receptor Activator 224 6.1 Effects of CERA In Vitro and in Animal Models 224 6.2 Continuous Erythropoietin Receptor Activator in Healthy Subjects 225



6.3 Effects of Continuous Erythropoietin Receptor Activator in Patients With Chronic Kidney Disease Anemia 6.4 Safety and Tolerability of Continuous Erythropoietin Receptor Activator

225 226

7. Small Molecule Erythropoiesis-Stimulating Agents226 7.1 Peptide-Based Erythropoiesis-Stimulating Agents 226 7.2 Nonpeptide-Based Erythropoiesis-Stimulating Agents228 8. Other Strategies for Stimulating Erythropoiesis

228

9. Conclusions

228

References

228

Further Reading

232

1. INTRODUCTION The ability to stimulate erythropoiesis with therapeutic agents has probably had the greatest impact in the field of nephrology. Ever since it was recognized that red cell production was controlled by the hormone erythropoietin (EPO), and that this hormone was produced de novo in the kidney in response to hypoxia, there was a clear rationale for administering EPO replacement therapy.1 When this dream became a reality in the late 1980s, the true impact of this treatment was realized. Dialysis patients who were heavily transfusion-dependent and who, without regular blood transfusions could barely achieve hemoglobin (Hb) levels above 6–7 g/dL, were rendered transfusion-independent, with Hb concentrations of around 11–12 g/dL.2–4 This was truly one of the major breakthroughs in nephrology, if not in the whole of medicine, within the last two or three decades. This chapter will discuss the history of recombinant human EPO and the way this therapeutic field has evolved over the last 30 years, along with novel strategies for stimulating erythropoiesis.

Textbook of Nephro-Endocrinology, Second Edition http://dx.doi.org/10.1016/B978-0-12-803247-3.00013-1

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© 2018 Elsevier Inc. All rights reserved.

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2.  HISTORY OF RECOMBINANT HUMAN ERYTHROPOIETIN Erythropoiesis is a complex physiological process that maintains homeostasis of oxygen levels in the body. It is primarily regulated by EPO, a 30.4 kDa, 165-amino acid hematopoietic growth factor.5 In the presence of EPO, erythroid cells in the bone marrow proliferate and differentiate. In its absence, these progenitor cells undergo apoptosis. The presence of a humoral factor like EPO was first suggested by Carnot and Deflandre in Ref. 6, following a series of elegant experiments in which they injected blood from anemic rabbits into donor rabbits and observed a significant increase in red cell production. It was, however, not until the 1950s that Erslev and others conclusively demonstrated the presence of EPO7 and it took nearly another 30 years before human EPO was isolated from the urine of patients with aplastic anemia.8 The next major development was the successful isolation and cloning of the human EPO gene in 1983,9 which allowed for the development of recombinant human EPO as a clinical therapeutic. The original recombinant human EPOs (epoetin alfa and epoetin beta) were synthesized in cultures of transformed Chinese hamster ovary (CHO) cells that carry cDNA encoding human EPO. The amino acid sequence of both epoetins is therefore identical and the major difference between these products lies in their glycosylation pattern. There are also slight differences in the sugar profile between recombinant human EPO and endogenous EPO,10 but the amino acid sequence is identical. EPO exerts its mechanism of action via binding to the EPO receptor on the surface of erythroid progenitor cells. The EPO receptor undergoes a conformational change following dimerization, which involves activation of the JAK2/ STAT-5 intracellular signaling pathway.11 The metabolic fate of EPO was debated for years, but it appears that this is partly mediated via internalization of the EPO receptor complex, with subsequent lysosomal degradation.12 The latter had strong relevance for the design of new EPO analogs, since the circulating half-life of recombinant human EPO following intravenous administration is fairly short, at around 6–8 h.13 Thus, in the first clinical trials of recombinant human EPO in hemodialysis (HD) patients, the drug was administered three times weekly to coincide with the dialysis sessions.2,4 The half-life of subcutaneously administered recombinant human EPO is much longer, at around 24 h13 and this characteristic, along with the recognition that the high peak levels after intravenous administration are not necessary for its biological action, means that a lower dose of drug may be administered subcutaneously to achieve the same effect as that seen following IV administration. In a randomized controlled trial, Kaufman et al.14 demonstrated that the dosage requirements following SC administration were approximately 30% lower than those following IV administration. This was confirmed in a later meta-analysis by Besarab et al.15 In the early clinical trials, the huge benefits of EPO administration were seen. In addition to transfusion independence, patients became aware of significantly improved energy levels, greater exercise capacity and generally improved quality of life.16 Cardiac benefits, such as a reduction in left ventricular hypertrophy, were also described,3,17 as were objective measures of exercise performance.3,18 A wide range of other secondary benefits were reported (Table 13.1), including improvements in cognitive function, skeletal muscle function and immune function. Several serious adverse events were seen in the early days of EPO therapy, including severe hypertension, hypertensive encephalopathy and seizures,19 but these side effects are not commonly seen today, almost certainly due to the fact that anemia is treated earlier and with more cautious increments in Hb. The only other serious adverse effect of EPO therapy directly related to the treatment was reported in 2002 by Casadevall et al.,20 who described a case series of 13 patients who developed antibody-mediated pure red cell aplasia caused by the formation of antibodies TABLE 13.1  Benefits of Correction of Anemia in Chronic Kidney Disease (CKD) Patients ↓ Transfusions

↑ Sleep patterns

↑ Exercise capacity

↑ Sexual function

↑ Quality-of-life

↑ Endocrine function

↓ Cardiac output

↑ Immune function

↓ Angina

↑ Muscle metabolism

↓ Left ventricular hypertrophy

↓ Hospitalizations

↓ Bleeding tendency ↑ Brain/cognitive function nutrition ↓ Depression

3.  Biosimilar Erythropoietins

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TABLE 13.2  Causes of a Poor Response to ESA Therapy Major

Minor

Iron deficiency

Blood loss

Infection/inflammation

Hyperparathyroidism

Underdialysis

Aluminum toxicity   B12/folate deficiency  Hemolysis   Marrow disorders, e.g., MDS  Hemoglobinopathies   Angiotensin-converting enzyme (ACE) inhibitors   Carnitine deficiency   Obesity (SC EPO)   Anti-EPO antibodies (PRCA)

against recombinant human EPO. The mechanism behind this effect has been debated, but factors such as inadequate cold storage facilities, SC route of administration, and leachates from the rubber plungers of the syringes (acting as immune adjuvants) may all have contributed.21 Although this side effect was devastating, it is extremely rare and, worldwide, only approximately a few hundred cases have been seen. Most of these were with a particular formulation of epoetin alfa manufactured outside the United States, under the trade name of Eprex (Erypo in Germany). The majority of chronic kidney disease (CKD) patients respond to EPO therapy, although a minority show a more sluggish response, which may be due to iron insufficiency, inflammation, or a number of more minor factors22 (Table 13.2). The ability to boost Hb levels without blood transfusions also generated considerable debate over the optimal target Hb for patients receiving EPO therapy (see Chapter 5). Anemia guidelines discussing this issue were first published in 1997 and suggested that a target Hb range of 11–12 g/dL was appropriate.23 A series of other anemia guidelines from Europe, Canada, Australia, and the UK then followed and some of these were revised. This recommendation was driven by the results from three large, randomized, controlled trials,24–26 along with a Lancet metaanalysis,27 which suggested that there was likely harm in targeting Hb levels above 13 g/dL, due to an increased risk of cardiovascular events. Then followed an even more definitive study (TREAT), which was a randomized, doubleblind, placebo-controlled trial confirming harm in targeting Hb levels of 13 g/dL, with a doubling of the risk of stroke and venous thromboembolism, as well as a 10-fold increased risk of cancer-related death in patients with a previous malignancy. Thus, the latest anemia guidelines KDIGO, European Renal Best Practice, and UK NICE all endorse a target Hb somewhere in the range of 10–12 g/dL.

3.  BIOSIMILAR ERYTHROPOIETINS Since the patents for epoetin alfa and epoetin beta have now expired in several countries, and because the market for recombinant human EPO is so lucrative, copies of the established EPO preparations are now beginning to appear on the market. These products are named “biosimilars” in the European Union and “follow on biologics” in the United States.28 Biosimilar EPOs, by definition, are those that have been through the EU regulatory process. In addition, outside the EU and the United States, “copy” epoetins are already produced by companies other than the innovators and used clinically as antianemic drugs. For example, a CHO cell-derived recombinant human EPO produced in Havana, Cuba, was one of the earliest to be shown to have therapeutic efficacy.29 All recombinant proteins are, however, associated with a number of issues that distinguish them from traditional drugs and their generics. Recombinant proteins are highly complex at the molecular level, and biological manufacturing processes are highly elaborate, involving cloning, selection of a suitable cell line, fermentation, purification, and formulation. In addition, the therapeutic properties of recombinant proteins are highly dependent on each step of the manufacturing process. Despite this, many biosimilar and “copy” epoetin products have been produced around the world.30 Since the manufacturing processes are different from those used by the innovator companies, there have been serious concerns

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about the safety, efficacy, and consistency of both biosimilar and “copy” epoetin products,28 particularly in relation to their potential to produce antibody-mediated pure red cell aplasia. Indeed, in a clinical trial of just over 300 patients, one particular biosimilar epoetin produced two possible cases of Ab-mediated pure red cell aplasia,31 possibly due to tungsten exposure in the prefilled syringe. Other epoetins that were developed around the same time include epoetin omega32–34 and epoetin delta.35–37 As with all recombinant human EPOs, these products share the same amino acid sequence as for epoetin alfa and epoetin beta, as well as the endogenous hormone. The cell culture conditions, however, vary. With epoetin omega, baby hamster kidney (BHK) cell cultures were used for the manufacture of the product, which has been used clinically in some Eastern European, Central American, and Asian countries. Epoetin delta is another recombinant EPO that was previously used for treating patients with CKD in Europe, but not in the United States where patent issues prevented its introduction into the anemia marketplace. Epoetin delta (Dynepo) was approved by the European Medicines Agency (EMEA) in 2002 and first marketed in Germany in 2007. Epoetin delta was synthesized in human fibrosarcoma cell cultures (line HT-1080). The product was also called geneactivated EPO because the expression of the native human EPO gene was activated by transformation of the cell with the cytomegalovirus promoter.38 In contrast to CHO or BHK cell-derived recombinant human EPO, epoetin delta did not possess N-glycolylneuraminic acid (Neu5Gc) since, in contrast to other mammals including great apes, humans are genetically unable to produce Neu5Gc due to an evolutionary mutation.39 The implications of a lack of Neu5Gc residues in synthetic recombinant EPO, however, were not clear and were certainly not strong enough to support its ongoing manufacture. Indeed, in early 2009, the manufacturer (Shire Pharmaceuticals) voluntarily withdrew epoetin delta from the market since it was no longer commercially viable.40

4.  POTENTIAL STRATEGIES FOR MODIFYING ERYTHROPOIETIN TO CREATE NEW ERYTHROPOIETIN ANALOGS The major limitation of recombinant human EPO is its short duration of action and thus the patient needs to receive 1–3 injections per week. Given the lucrative nature of the anemia market, several companies investigated means of modifying the EPO molecule to create longer-acting EPO receptor agonists. Some of the strategies that have been employed in this process are summarized in Table 13.3.41 The first strategy to be investigated was the creation of a hyperglycosylated analog of EPO. The rationale for this is described in more detail below (see Darbepoetin alfa), but the addition of extra sialic acid residues to the EPO molecule was found to confer greater metabolic stability in vivo. Another strategy that has been used for prolonging the duration of action of other therapeutic proteins such as G-CSF and interferon-alfa, is pegylation of the protein. This is the strategy that was adopted in the creation of CERA (see below) and the circulating half-life of this molecule is considerably enhanced compared to native or recombinant EPO. Solid phase peptide synthesis and branched TABLE 13.3  EPO-Receptor Agonists PROTEIN-BASED ESA THERAPY Epoetin (alfa, beta, delta, omega) Biosimilar EPOs (epoetin zeta) Darbepoetin alfa CERA (methoxy polyethylene glycol epoetin beta) Synthetic erythropoiesis protein (SEP) EPO fusion proteins • EPO–EPO • GM-CSF–EPO • Fc–EPO • CTNO 528 SMALL MOLECULE ESAS Peptide based (e.g., peginesatide; Hematide) Nonpeptide based

5.  Darbepoetin Alfa

221

precision polymer constructs were used to create Synthetic Erythropoiesis Protein, the erythropoietic effect of which was shown to vary in experimental animals depending on the number and type of the attached polymers.42 Another strategy that was adopted was the fusion of EPO with other proteins. These recombinant EPO fusion proteins contain additional peptides at the carboxy-terminus to increase the in vivo survival of the molecule.43 Large EPO fusion proteins, of molecular weight 76 kDa, were designed from cDNA encoding two human EPO molecules linked by small flexible polypeptides.44,45 A single SC administration of this compound to mice increased red cell production within 7 days at a dose at which epoetin was ineffective.45 Another dimeric fusion protein incorporating both EPO and granulocyte-macrophage colony-stimulating factor (GM-CSF) was created, with the rationale that GM-CSF is required for early erythropoiesis. This EPO–GM-CSF complex proved to be able to stimulate erythropoiesis in cynomolgus monkeys46 but was later found to induce anti-EPO antibodies, causing severe anemia.47 Yet another approach was the genetic fusion of EPO with the Fc region of human immunoglobulin G (Fc–EPO).48 This molecular modification promotes recycling out of the cell upon endocytosis via the Fc recycling receptor,49,50 again providing an alternative mechanism for enhancing circulating half-life. The same effect may be achieved by fusing EPO with albumin. Another molecule being developed is CNTO 528, which is an EPO–mimetic antibody fusion protein with an enhanced serum half-life but no structural similarity to EPO.51 Rats treated with a single SC dose of CTNO 528 showed a more prolonged reticulocytosis and Hb rise compared to treatment with epoetin or darbepoetin alfa. Phase I studies in healthy volunteers showed a similar effect following a single intravenous administration of CNTO 528, with a peak reticulocyte count occurring after 8 days and the maximum Hb concentration being seen after 22 days. None of the 24 subjects in this study developed antibodies against the molecule.52 Interestingly, an Fc–EPO fusion protein was successfully administered in a phase I trial to human volunteers as an aerosol, with a demonstrable increase in EPO levels associated with an increase in reticulocyte counts.53 In addition to the EPO derivatives administered by aerosol inhalation, other delivery systems for EPO have been investigated, including ultrasound-mediated transdermal uptake54 and orally via liposomes to rats.55 Mucoadhesive tablets containing EPO and an absorption enhancer (Labrasol) for oral administration have been studied in rats and dogs.56 Theoretically, this preparation was designed to allow the tablet to reach the small intestine intact. Experiments in beagle dogs were conducted with intrajejunal administration of a single tablet containing 100 IU/kg of recombinant human EPO, with a corresponding increase in reticulocytes 8 days after administration.56

5.  DARBEPOETIN ALFA Darbepoetin alfa, initially termed novel erythropoiesis stimulating protein (NESP), and now marketed under the trade name of Aranesp, is a second-generation EPO analog. Its development arose from the recognition that the higher isoforms (those with a greater number of sialic acid residues) of recombinant human EPO were more potent biologically in vivo due to a longer circulating half-life than the lower isomers (those with a lower number of sialic acid residues)57 (Fig 13.1). Since the majority of sialic acid residues are attached to the three N-linked glycosylation chains of the EPO molecule, attempts were made to synthesize EPO analogs with a greater number of N-linked carbohydrate chains. This was achieved using site-directed mutagenesis, to change the amino acid sequence at sites not directly involved in binding to the EPO receptor.58,59 Thus, five amino acid substitutions were implemented (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr), allowing darbepoetin alfa to carry a maximum of 22 sialic acid residues, compared with recombinant or endogenous EPO which support a maximum of 14 sialic acid residues. The additional N-linked carbohydrate chains increased the molecular weight of epoetin from 30.4 to 37.1 kDa and the carbohydrate contribution to the molecule correspondingly increased from 40% to around 52%.58,59 These molecular modifications to EPO conferred a greater metabolic stability in vivo and this was confirmed in a single-dose pharmacokinetic study performed in EPO-naïve patients undergoing continuous ambulatory peritoneal dialysis (PD).60 Following a single IV injection of darbepoetin alfa, the mean terminal half-life was approximately threefold longer compared to a single IV injection of epoetin alfa (25.3 vs. 8.5 h, respectively) and the AUC was more than twofold greater (291 ± 8 vs. 138 ± 8 ng h/mL), as well as a threefold lower clearance (1.6 ± 0.3 vs. 4.0 ± 0.3 mL/h per kg), which was biphasic. The volume of distribution was similar for the two molecules (52.4 ± 2.0 and 48.7 ± 2.1 mL/ kg, respectively). The mean terminal half-life in patients given darbepoetin alfa subcutaneously was approximately 49 h, which is around twice that following IV administration and the mean bioavailability was 37%.60 More recent studies estimated a longer half-life for subcutaneously administered darbepoetin alfa.61 These pharmacokinetic studies employed longer sampling periods, up to 28 days, and they suggested that the half-life of SC

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13.  DEVELOPMENT OF RECOMBINANT ERYTHROPOIETIN AND ERYTHROPOIETIN ANALOGS

75

Isoform 14 Isoform 13 r-HuEPO (9–14) Isoform 12 Isoform 11 Isoform 10 Isoform 9 Isoform 8

Hematocrit (%)

70 65 60 55 50 45

Placebo

40 0

5

10

15

20

25

30

Day of study

FIGURE 13.1  In vivo activity of isolated recombinant human erythropoietin isoforms in mice injected three times per week intraperitoneally. Redrawn from Egrie JC, Grant JR, Gillies DK, Aoki KH, Strickland TW. The role of carbohydrate on the biological activity of erythropoietin. Glycoconj J 1993;10:263.

darbepoetin alfa may be around 70 h. Two studies by Padhi and colleagues,62,63 conducted in patients with chronic renal insufficiency (CRI) used sampling times of 648–672 h to estimate the SC half-life. The first study, a pilot, was conducted in a subset of five patients from an open-label, multicenter investigation of QM SC administration of darbepoetin alfa. These patients had been receiving darbepoetin alfa Q2W and had stable Hb levels between 10.0 and 12.0 g/dL. They were switched to QM darbepoetin alfa at a dose equal to the total dose received in the previous month. Pharmacokinetic analysis was performed between 6 and 672 h after administration of the first QM darbepoetin alfa dose. Absorption after SC injection was slow in all patients, with peak concentrations of 0.75–6.29 ng/mL reached at 34–58 h postdose, respectively, followed by a generally monophasic decline. The mean terminal half-life of darbepoetin alfa was 73 h (range 39.9–115.0 h, consistent with the variability range expected for all erythropoiesisstimulating agents (ESAs)).62 The second study was a single-dose, open-label study of SC darbepoetin alfa in 20 adult patients with CRI. The extended sampling period was 672 h. Peak concentrations of darbepoetin alfa were reached in a median of 36.0 h (range 12.0–72.0 h), with a mean terminal half-life of 69.6 h (95% CI, 54.9–84.4 h).63 The half-life of darbepoetin alfa was also investigated in PD patients receiving a range of darbepoetin alfa doses. Tsubakihara and colleagues64 performed pharmacokinetic analyses in patients receiving PD and patients with CRI following single doses of SC darbepoetin alfa. Darbepoetin alfa was administered to 32 PD patients at 20, 40, 90, or 180 μg (8 patients per treatment group) and to 32 patients with CRI (same dose groups and patient allocation). Serum darbepoetin alfa concentrations were followed for 336 h for patients receiving the 20, 40, or 90 μg doses, or 672 h for patients receiving the 180 μg dose. The mean terminal half-life in the different dose groups ranged from 64.7 to 91.4 h in the PD patients and from 73.6 to 104.9 h in CRI patients but was not dose dependent. This study also showed that there was no effect of differing levels of renal function on the half-life of darbepoetin alfa.64 The more prolonged half-life of darbepoetin alfa compared to either epoetin alfa or epoetin beta has translated into less frequent dosing, with most patients receiving injections once-weekly or once-every-other-week.

5.1 Intravenous Administration—Hemodialysis Patients Results from two studies support the conclusion that IV darbepoetin alfa is clinically efficacious in maintaining Hb levels without a need to increase the dose in HD patients when administered at longer intervals compared with epoetin alfa. In a 28-week, randomized study, Nissenson et al.65 assigned HD patients receiving stable therapy with IV epoetin alfa TIW to continue treatment or to switch to IV darbepoetin alfa QW. There was no statistically or clinically significant change in mean Hb levels from baseline to the evaluation period (the final 8 weeks of the study). During the evaluation period, 49% of patients in the epoetin alfa group versus 44% of patients in the darbepoetin alfa group required a dose change to maintain Hb levels within the 9–13 g/dL target range. The mean dose during the evaluation period did not differ statistically from baseline values in either treatment group. Safety profiles were comparable between the two treatments, with similar rates of adverse events.65 Locatelli and colleagues showed that Hb

5.  Darbepoetin Alfa

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levels were maintained in HD patients over 30 weeks of treatment with QW or Q2W darbepoetin alfa. Importantly, this study also demonstrated that there was no significant dose increase with the extension of the darbepoetin alfa interval out to Q2W and that the treatment was well tolerated at both dosing schedules.66 A prospective, multicenter, 24-week study determined the bioequivalent dose of darbepoetin alfa given IV QW in stable HD patients who had previously received epoetin alfa SC or IV and who had Hb levels between 10.8 and 13 g/ dL.67 Using the European label-recommended conversion ratio (1 μg darbepoetin alfa to 200 IU epoetin alfa), subjects previously stable on epoetin alfa BIW or TIW were converted to darbepoetin alfa QW, and subjects previously stable on epoetin alfa QW were converted to darbepoetin alfa Q2W. The dose of darbepoetin alfa was subsequently adjusted to maintain Hb levels within ±1 g/dL of the baseline value. In the 100 study completers, Hb was well maintained. The dose of darbepoetin alfa was 45.6 and 25.8 μg at baseline for the QW and Q2W groups, respectively, and 31.5 and 21.4 μg at the end of the study, respectively.67 Although PD patients are more likely to receive ESA therapy via the SC route, the efficacy and safety of IV darbepoetin alfa at various dosing frequencies was also investigated in these patients. In one study, PD patients either naïve to ESAs or previously receiving epoetin (alfa or beta not specified) were treated with darbepoetin alfa Q2W. Once stable, patients could extend the dosing interval out to QM. All patients received darbepoetin alfa for up to 28 weeks to achieve and maintain Hb levels between 11 and 13 g/dL. Hb in ESA-naïve patients increased from 8.15 to 11 g/dL over the first 10 weeks of darbepoetin alfa therapy, and all patients’ Hb levels were successfully maintained within the target range regardless of whether darbepoetin alfa was dosed Q2W or QM.68 It should be noted, however, that only stable patients were included in this study and QM administration may not be appropriate for an unselected dialysis population.

5.2 Subcutaneous Administration—Hemodialysis Patients A number of studies support the efficacy of SC darbepoetin alfa administered to HD and PD patients who were switched from more frequent epoetin therapy (alfa or beta).69–73 In these studies, the patients receiving SC epoetin therapy were switched to darbepoetin alfa: epoetin BIW/TIW to darbepoetin alfa QW, and epoetin QW to darbepoetin alfa Q2W. Hb levels were successfully maintained within the target range (for the majority of studies, 10–13 g/ dL) and without the need for darbepoetin alfa dose increases.69–73 Administration of darbepoetin alfa SC was also shown to be effective in PD patients not previously treated with an ESA. As part of a larger study, Macdougall and colleagues administered a range of darbepoetin alfa doses TIW or QW to 47 PD patients.74 The investigators found that overall, both 0.45 and 0.75 μg/kg per week doses increased mean Hb levels ≥ 1 g/dL per 4 weeks and that there was no apparent difference in efficacy between the TIW and QW regimens. The individual patients who achieved a Hb rate of rise ≥1 g/dL per 4 weeks continued darbepoetin alfa treatment for up to 52 weeks with Hb levels being maintained between 10 and 13 g/dL.74 The safety profile of darbepoetin alfa was similar to that expected for this patient population, thus demonstrating that PD patients can safely achieve a Hb response within a month of initiating darbepoetin alfa therapy.

5.3 Subcutaneous Administration—Predialysis Chronic Kidney Disease Patients In addition to dialysis patients, studies have also investigated the efficacy and safety of Q2W and QM dosing with darbepoetin alfa in CRI patients.75–80 Two large, 24-week studies by Suranyi et al. and Toto et al. examined the administration of darbepoetin alfa in CRI patients not previously receiving an ESA. Within approximately 5 weeks of initiating SC, de novo Q2W darbepoetin alfa, 95%–97% of these patients achieved Hb levels between 11 and 13 g/ dL. These results were supported by a chart review that compared de novo Q2W or QM epoetin alfa therapy with de novo Q2W or QM darbepoetin alfa. The proportion of patients achieving a mean Hb level ≥11 g/dL within 100 days was recorded. Of the patients dosed with Q2W or QM darbepoetin alfa, 66.7% and 80.0%, respectively, achieved the Hb target. In comparison, of the patients with Q2W or QM epoetin alfa, 53.8% and 50.0%, respectively, achieved the Hb target.78 Three further studies examined the efficacy of SC QM darbepoetin alfa in maintaining Hb levels in CRI patients following extension of the dosing interval from previous Q2W darbepoetin alfa. In the study by Disney and colleagues, 83% of patients who received at least one QM dose of darbepoetin alfa (the modified intent-to-treat population; mITT) and 95% of the patients who completed the study achieved a target Hb level of ≥10 g/dL.76 Likewise, Ling et al. reported that the Hb target of 10–12 g/dL was achieved in 79% of the mITT population and in 85% of those patients completing the study following extension of the darbepoetin alfa dosing interval to QM.77 Finally, the study

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by Agarwal et al. further confirmed the efficacy of QM darbepoetin alfa by showing that following a switch from Q2W darbepoetin alfa dosing, Hb levels could be maintained ≥11 g/dL in 76% of the mITT population and in 85% of patients who completed the study.75 In a setting representative of current nephrology practice, nephrologists at centers participating in the Aranesp Registry Group in the Netherlands enrolled patients in a registry to investigate the feasibility of administering darbepoetin alfa QM.81 Nephrologists were first informed of the possibility of dosing darbepoetin alfa QM and then their patients’ treatments were monitored for a 12-month period. The patients had CRI, were not receiving renal replacement therapy, and were currently receiving or about to initiate QM darbepoetin alfa therapy. Of 108 patients completing the 12-month follow-up period, 66% were found to be receiving QM darbepoetin alfa, and mean Hb levels were maintained at approximately 12 g/dL throughout the study. Fifty-nine percent of the patients who had ever received a QM dose of darbepoetin alfa preferred this regimen over any other and 31% had no preference. These results were interesting, but clearly the potential for selection bias in this study was considerable, since nephrologists were far more likely to select patients for QM dosing if they were stable and likely to manage with less frequent dosing.81 Unfortunately, all of these studies of QM darbepoetin alfa were nonrandomized and uncontrolled, and thus QM dosing frequency for darbepoetin alfa should be used largely in highly selected stable patients.

6.  CONTINUOUS ERYTHROPOIETIN RECEPTOR ACTIVATOR The next erythropoiesis-stimulating agent to be developed was Continuous Erythropoietin Receptor Activator (CERA), which was created by integrating a large polymer chain into EPO, thus increasing the molecular weight to twice that of epoetin at approximately 60 kDa.82 This methoxy-polyethylene glycol polymer chain was integrated via amide bonds between the N-terminal amino group or the ε-amino group of lysine (predominantly lysine-52 or lysine-45), using a single succinimidyl butanoic acid linker. CERA has very different receptor binding characteristics and pharmacokinetic properties compared with both epoetin and darbepoetin alfa.83 It has a much lower affinity for the EPO receptor compared with the natural ligand, leading to a reduced specific activity in vitro. However, since the elimination half-life is so prolonged, CERA has increased erythropoietic activity in vivo. Preclinical and then clinical studies of CERA will be discussed in turn.

6.1 Effects of CERA In Vitro and in Animal Models The erythropoietic activities of CERA and epoetin were compared in vitro by measuring their effect on the proliferation of a human acute myeloid leukemia cell line (UT-7) that expresses the EPO receptor. Across the dose range 0.003–3 U/mL, epoetin stimulated greater proliferation of UT-7 cells compared with CERA.84 However, in vivo studies in normothycemic mice comparing identical amounts of protein across the dose range 60–1000 ng protein/animal have shown that CERA was more effective than epoetin at stimulating bone marrow precursor cells and increasing reticulocyte count.84 At a dose of 1000 ng, CERA increased the mean reticulocyte count by 14%, compared with 9% for epoetin. Preclinical studies in various animal models have investigated the pharmacodynamic and pharmacokinetic properties of IV and SC CERA administered in both single and multiple doses, across the dose range 0.75–20 μg/kg. In mice, a single SC injection of CERA 20 μg/kg increased the mean reticulocyte count by 13%, compared with 7.8% in response to a comparable dose of epoetin beta.85 The median duration of the response was approximately 3 days longer with CERA compared with epoetin. In addition, a single SC or IV administration of CERA 2.5 μg/kg in mice elicited a greater reticulocyte response than multiple doses of epoetin 2.5 μg/kg, in terms of the magnitude and duration of response. Further studies in mice showed that SC administration of CERA once weekly (1.25 and 5 μg/ kg) or once every 2 weeks (5 μg/kg) produced a greater reticulocyte response compared with epoetin (1.25 μg/kg) three times weekly.85 Moreover, approximately equal numbers of reticulocytes were produced with CERA 1.25 μg/ kg administered once every 2 weeks as with epoetin 1.25 μg/kg administered three times weekly. Pharmacokinetic studies in animals showed that CERA has a longer half-life and lower systemic clearance than epoetin.83 From the results of these preclinical studies, it appears that CERA has receptor binding and pharmacokinetic properties that give rise to more potent stimulation of erythropoiesis in vivo than epoetin, with regard to both the magnitude and duration of response. These findings suggested the potential for CERA to be administered at extended dosing intervals.

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6.2 Continuous Erythropoietin Receptor Activator in Healthy Subjects Four phase I studies were conducted in healthy subjects to investigate the pharmacokinetic and pharmacodynamic properties of CERA. In two single ascending dose (SAD) studies, subjects were randomized to receive single IV doses of CERA (0.4–3.2 μg/kg) or placebo [n = 38] or single SC doses of CERA (0.1–3.2 μg/kg) or placebo [n = 70]. In two multiple ascending dose (MAD) studies, subjects were randomized to receive three IV doses of CERA (0.4– 3.2 μg/kg) or placebo [n = 61] once every 3 or 4 weeks SC doses of CERA (0.4–3.2 μg/kg) or placebo [n = 48] once every 2 weeks. The half-life of CERA,86 administered IV or SC, was observed to be considerably longer than that previously reported for epoetin (alfa or beta)87 or darbepoetin alfa60 (Fig. 13.2). The pharmacokinetics of CERA were apparently unaffected by repeated dosing. In the MAD studies, the clearance of both IV and SC CERA was low (IV 27.6–44.6 mL/h, SC 97–347 mL/h).88 No accumulation was observed when steady state was achieved with the different frequencies tested. The prolonged half-life and low clearance seen with both IV and SC CERA in healthy subjects supported the data from animal studies, suggesting that it might be possible to administer CERA at extended dosing intervals.

6.3 Effects of Continuous Erythropoietin Receptor Activator in Patients With Chronic Kidney Disease Anemia Four phase II dose-finding studies investigated the feasibility of CERA for the correction of anemia and maintenance of Hb levels at extended administration intervals in more than 350 patients with CKD. In two studies, one in CKD patients receiving dialysis [n = 61]89 and one in CKD patients not yet receiving dialysis [n = 65],90 SC CERA was administered for the correction of anemia. All patients were aged ≥18 years, with Hb 8–11 g/dL and were ESA naïve. The first study, conducted in patients receiving dialysis treatment, examined escalating doses of CERA in three patient groups.89 After a 4-week run-in period, patients were randomized to receive one of three CERA doses (0.15, 0.30 or 0.45 μg/kg per week). Once-weekly, once every 2 weeks, and once every 3 weeks administration schedules were assessed in each dose group. The second study, conducted in patients not receiving dialysis, also investigated escalating doses of CERA in three patient groups.90 After a 2-week run-in period, patients were randomized to receive one of three CERA doses (0.15, 0.30, or 0.60 μg/kg per week). Again, once-weekly, once every 2 weeks, and once every 3 weeks administration schedules were assessed in each dose group. For both studies, individual dose adjustments were permitted according to defined Hb criteria after the initial 6-week period. Patients were followed for a total of 12 weeks (patients receiving dialysis) or 18 weeks (patients not receiving dialysis), respectively. In these studies, there was a statistically significant dose response to CERA treatment and the Hb response was independent of the frequency of administration. These results suggested that CERA was capable of correcting anemia when administered to ESA-naïve CKD patients at extended dosing intervals. Two phase II, multicenter, dose-finding studies were conducted to determine the efficacy of CERA for the maintenance of Hb levels in adult patients with renal anemia (Hb 10–13 g/dL) and receiving dialysis treatment. In one study, 91 patients previously maintained on three times weekly IV epoetin alfa were switched to IV CERA.91 After a 2-week run-in period, patients were randomized to one of three CERA doses based on their previous epoetin dose and data on exposure to CERA in healthy subjects. Once-weekly and once every 2 weeks administration schedules were assessed in each dose group. Patients were followed for a total of 19 weeks.

FIGURE 13.2  IV half-lives of ESA therapy.

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A statistically significant (P < .0001) dose-dependent Hb response was observed. At the highest dose studied, IV CERA maintained stable Hb levels within ±1.5 g/dL from baseline in the highest percentage of patients when administered once every 2 weeks. These results suggested that IV CERA administered once every 2 weeks may maintain stable Hb levels in dialysis patients. In the second maintenance study, 137 patients previously maintained on three-times weekly SC epoetin treatment were switched to SC CERA.92 After a 2-week run-in period, patients were randomized to one of three CERA doses based on their previous epoetin dose and data on exposure to CERA from healthy subjects. Once-weekly, once every 3 weeks, and once every 4 weeks administration schedules were assessed in each dose group. Patients were followed for a total of 19 weeks; those in the once every 4 weeks group were followed for 21 weeks. There was a statistically significant (P < .001) dose-dependent response to CERA in the three treatment groups which was independent of the frequency of administration (up to once every 4 weeks). The results suggested that SC CERA administered once every 4 weeks may maintain stable Hb levels in dialysis patients. Six phase III studies of CERA were conducted to further investigate the reduced dosing frequency of this new agent.93–98 Two of these (AMICUS, ARCTOS) investigated the correction of anemia in ESA-naïve patients, with an initial dosing frequency of once every 2 weeks, with a second phase randomization to either continue once every 2-week dosing or switch to once every 4-week administration. The AMICUS study94 investigated intravenously administered CERA in HD patients, while the ARCTOS study96 investigated subcutaneously administered CERA in predialysis CKD patients. Both studies were randomized and controlled, using a comparator drug (epoetin alfa for AMICUS, and darbepoetin alfa for ARCTOS). The four maintenance phase studies were MAXIMA, PROTOS, STRIATA, and RUBRA. MAXIMA compared once every 2-week administration of CERA with injections once every 4 weeks in HD patients95 while the PROTOS study had an almost identical protocol but with subcutaneously administered CERA.98 STRIATA also utilized once every 2-week dosing of CERA administered intravenously in HD patients93 and RUBRA recruited patients on dialysis to receive either intravenous or SC CERA, once every 2 weeks, in dialysis patients using prefilled syringes.97 All the phase III studies met their primary noninferiority end point with the comparator drug, confirming the hypothesis that CERA may be administered less frequently to achieve either correction of anemia or maintenance of the target Hb in both predialysis and dialysis-dependent CKD patients. Since the marketing authorization and launch of CERA as a therapeutic agent outside the United States (patent regulations prevented the launch of CERA in the US), a further important randomized controlled trial has been published. This study (PATRONUS) compared once-monthly CERA with darbepoetin alfa in 490 HD patients previously stable on once-weekly intravenous darbepoetin alfa. Dose adjustments were made to keep the individual Hb target level between 11 and 13 g/dL. The primary end point of the study was the proportion of patients who maintained an average Hb ≥10.5 g/dL, with a decrease from baseline of less than 1 g/dL, in weeks 50–53. The secondary end point was dose change over time: 64.1% of patients on CERA and 40.4% of patients on darbepoetin alfa met the primary end point (P < .0001). Doses increased by 6.8% with CERA and by 58.8% with darbepoetin alfa during the once-monthly maintenance phase.

6.4 Safety and Tolerability of Continuous Erythropoietin Receptor Activator In the studies in CKD patients reported to date, CERA has generally been well tolerated with no unexpected safety concerns, and the side-effect profile of this agent is comparable to both epoetin and darbepoetin alfa.

7.  SMALL MOLECULE ERYTHROPOIESIS-STIMULATING AGENTS 7.1 Peptide-Based Erythropoiesis-Stimulating Agents In the 1990s, several small bisulfide-linked cyclic peptides composed of around 20 amino acids were identified by random phage display technology that were unrelated in sequence to EPO but still bound to the EPO receptor.99,100 These small peptides were able to induce the same conformational change in the EPO receptor that leads to JAK2 kinase/STAT-5 intracellular signaling,100 as well as other intracellular signaling mechanisms, resulting in stimulation of erythropoiesis both in vitro and in vivo. The first peptide to be investigated (EPO-mimetic peptide-1; EMP-1)100 was not potent enough to be considered as a potential therapeutic agent in its own right, but the potency of these peptides could be greatly enhanced by covalent peptide dimerization with a PEG linker. Thus, another EMP was selected for the development of Hematide, a pegylated synthetic dimeric peptidic ESA, that was found to stimulate

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erythropoiesis in experimental animals.101 The half-life of Hematide in monkeys ranged from 14 to 60 h depending on the dose administered. Further studies in rats using quantitative whole-body auto radioluminography showed that the primary route of elimination for the peptide is the kidney.102 A phase I study in healthy volunteers showed that single injections of Hematide caused a dose-dependent increase in reticulocyte counts and Hb concentrations.103 Phase II studies demonstrated that Hematide can correct the anemia associated with CKD,103a as well as maintaining the Hb in dialysis patients who are already receiving conventional ESAs.104 Doses in the range of 0.025–0.05 mg/kg appeared to be therapeutically optimal in this patient population103a and a comprehensive phase III program was undertaken, involving four large randomized controlled trials, two in HD patients (EMERALD), and two in nondialysis CKD patients (PEARL). In the two PEARL studies, 983 patients received Hematide (now named “peginesatide”) once a month, at a starting dose of 0.025 mg or 0.04 mg/kg, or darbepoetin alfa once every 2 weeks at a starting dose of 0.75 μg/kg. Doses of both drugs were adjusted to achieve and maintain Hb levels between 11 and 12 g/dL for 52 weeks or more. The primary efficacy end point was the mean change from baseline Hb level to the mean level during the evaluation period, and cardiovascular safety was evaluated on the basis of an adjudicated composite endpoint. In both the PEARL studies, peginesatide was noninferior to darbepoetin alfa in increasing and maintaining Hb levels. The mean differences in the Hb level with peginesatide as compared with darbepoetin alfa in PEARL 1 were 0.03 g/dL for the lower starting dose and 0.26 g/dL for the higher starting dose, and in PEARL 2 they were 0.14 and 0.31 g/dL, respectively. However, there was a concerning and somewhat unexpected issue regarding the safety of peginesatide in the pooled data from the PEARL studies: the hazard ratio for the cardiovascular safety end point was 1.32 (95% CI, 0.97–1.81) for peginesatide relative to darbepoetin alfa, with higher incidences of death, unstable angina, and cardiac arrhythmia with peginesatide. In the EMERALD studies, the efficacy and safety of peginesatide in HD patients were assessed. Cardiovascular safety was evaluated by analysis of an adjudicated composite safety end point from any cause, stroke, myocardial infarction, or serious adverse events of congestive heart failure, unstable angina, or arrhythmia: 1608 patients received peginesatide once-monthly or continued to receive epoetin one or three times a week, with doses adjusted as necessary to maintain a Hb level between 10.0 and 12.0 g/dL for 52 weeks or more. Again, the primary efficacy end point was the mean change from baseline Hb level to the mean level during the evaluation period. In an analysis involving the 693 patients from EMERALD 1 and the 725 patients from EMERALD 2, peginesatide was noninferior to epoetin in maintaining Hb levels (mean between-group difference −0.15 g/dL). The hazard ratio for the composite safety end point in the EMERALD studies was 0.95 (95% CI, 0.77–1.17). In the pooled data from all four studies in the phase III program, totaling 2591 patients, the hazard ratio for the composite safety end point was 1.06 (95% CI 0.89–1.26) with peginesatide relative to the comparator ESA. Thus, overall, the cardiovascular safety of peginesatide was similar to that of the comparator ESA in the pooled cohort. Because of the safety signal in the nondialysis CKD patients with peginesatide (PEARL studies), marketing authorization was only granted by the FDA for use in HD patients, and the product was launched in the United States in April 2012 as Omontys. Unfortunately, following its introduction, cases of severe and immediate hypersensitivity reactions began to be reported in relation to the intravenous administration, and by February 2013, the FDA had received 19 reports of severe anaphylactic reactions occurring within 30 min of a patient’s first dose. Indeed, among 19,428 patients who received intravenous peginesatide in a postapproval study, there were seven deaths due to anaphylaxis. The cause of this was not clear, but the preparation used in preapproval clinical trials was a singledose vial, whereas the product used in dialysis units in the United States was a multidose vial with preservatives. Regardless, the manufacturer voluntarily withdrew peginesatide from further clinical use, and this drug is no longer available. The potential advantages of this new agent had been greater stability at room temperature and a much simpler (and cheaper) manufacturing process, avoiding the need for cell lines and genetic engineering techniques. Furthermore, antibodies against peginesatide do not cross-react with EPO and, similarly, anti-EPO antibodies do not cross-react with peginesatide.105 This has two major implications: first, even if a patient were to have developed antipeginesatide antibodies, these should not neutralize the patient’s own endogenous EPO, and the patient should not develop pure red cell aplasia. Secondly, patients with antibody-mediated pure red cell aplasia should be able to respond to peginesatide therapy by an increase in their Hb concentration, since peginesatide is not neutralized by anti-EPO antibodies. This latter hypothesis was confirmed in an animal model.105 Thus, rats receiving regular injections of recombinant human EPO were shown to develop anti-EPO antibodies. Injections of peginesatide were able to “rescue” these animals and restore their Hb concentration, in contrast to the vehicletreated group.105 A clinical trial examining this issue in patients with antibody-mediated pure red cell aplasia was also conducted in the middle of peginesatide’s clinical development program, enrolling patients from the UK,

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France, and Germany.106 Peginesatide was administered by SC injection at an initial dose of 0.05 mg/kg every 4 weeks, and the primary end point was a Hb concentration above 11 g/dL without the need for transfusions: 14 patients were treated for a median of 28 months, and the median Hb concentration increased from 9.0 g/dL (with transfusion support in the case of 12 patients) before treatment to 11.4 g/dL; transfusion requirements diminished within 12 weeks after the first dose, after which 13 of the 14 patients no longer required regular transfusions. Peak reticulocyte counts increased from a median of 10 × 109/L before treatment to peak counts of greater than 100 × 109/L. The level of anti-EPO antibodies declined over the course of the study and became undetectable in six patients. Other peptide-based ESAs have also been developed. A compound made by AplaGen in Germany linked a peptide to a starch residue, again demonstrating prolongation of the circulating half-life of the peptide.107 Indeed, altering the molecular weight of the starch moiety has been shown to alter the pharmacological properties of the compound.

7.2 Nonpeptide-Based Erythropoiesis-Stimulating Agents Several nonpeptide molecules capable of mimicking the effects of EPO have also been identified following screens of small molecule nonpeptide libraries for molecules with EPO receptor-binding activity.108 One such compound was found, but this bound to only a single chain of the EPO receptor and was not biologically active. The compound was ligated to enable it to interact with both domains of the EPO receptor and this second molecule was shown to stimulate erythropoiesis. Further development of nonpeptide EPO mimetics could lead to the production of an orally active ESA in the future.

8.  OTHER STRATEGIES FOR STIMULATING ERYTHROPOIESIS The focus of this chapter is agents that act via stimulation of the EPO receptor, inducing a conformational change and homodimerization of the receptor, followed by activation of the JAK2/STAT5 intracellular signaling pathway. Other strategies to enhance erythropoiesis are described in Chapter 6, although at the time of writing, these remain experimental drugs in clinical trials.

9. CONCLUSIONS As the molecular mechanisms controlling red cell production have been elucidated, so too have new targets and strategies been developed for stimulating erythropoiesis and treating anemia. Following the introduction of recombinant human EPO in the late 1980s, attempts were made to modify the molecule and produce longer-acting erythropoietic agents such as darbepoetin alfa and CERA. Other modifications to the molecule, such as the production of fusion proteins, have been explored, as is the concept that smaller molecules such as peptides, or even nonpeptides, may be able to bind to and activate the EPO receptor. Unfortunately, the first such molecule (peginesatide) to enter the therapeutic arena was withdrawn within a year of launch due to the development of life-threatening anaphylactic reactions. All of these molecules are EPO receptor agonists, acting via activation of the EPO receptor. Other strategies for anemia are under investigation, such as inhibition of HIF prolyl hydroxylase, and these are described in Chapter 6.

References

















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Further Reading 1. Dougherty FC, Beyer U. Safety and tolerability profile of Continuous Erythropoietin Receptor Activator (CERA) with extended dosing intervals in patients with chronic kidney disease on dialysis. Nephrology 2005;10:A313. 2. National Kidney Foundation. KDOQI clinical practice guideline and clinical practice recommendations for anemia in chronic kidney disease – 2007 update of hemoglobin target. Am J Kidney Dis 2007;50:471–530. 3. Macdougall IC, Provenzano R, Sharma A, Spinowitz BS, Schmidt RJ, Pergola PE, Zabaneh RI, Tong-Starksen S, Mayo MR, Tang H, Polu KR, Duliege AM, Fishbane S, PEARL Study Groups. Peginesatide for anemia in patients with chronic kidney disease not receiving dialysis. N Engl J Med 2013;368:320–32. 4. Fishbane S, Schiller B, Locatelli F, Covic AC, Provenzano R, Wiecek A, Levin NW, Kaplan M, Macdougall IC, Francisco C, Mayo MR, Polu KR, Duliege AM, Besarab A, EMERALD Study Groups. Peginesatide in patients with anemia undergoing hemodialysis. N Engl J Med 2013;368:307–19. 5. Bennett CL, Jacob S, Hymes J, Usvyat LA, Maddux FW. Anaphylaxis and hypotension after administration of peginesatide. N Engl J Med 2014;370:2055–6. 6. Bennett CL, Spiegel DM, Macdougall IC, Norris L, Qureshi ZP, Sartor O, Lai SY, Tallman MS, Raisch DW, Smith SW, Silver S, Murday AS, Armitage JO, Goldsmith D. A review of safety, efficacy, and utilization of erythropoietin, darbepoetin, and peginesatide for patients with cancer or chronic kidney disease: a report from the Southern Network on Adverse Reactions (SONAR). Semin Thromb Hemost 2012;38:783–96. 7. Pfeffer MA, Burdmann EA, Chen CY, Cooper ME, de Zeeuw D, Eckardt KU, Feyzi JM, Ivanovich P, Kewalramani R, Levey AS, Lewis EF, McGill JB, McMurray JJ, Parfrey P, Parving HH, Remuzzi G, Singh AK, Solomon SD, Toto R, TREAT Investigators. A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease. N Engl J Med 2009;361:2019–32. 8. Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO clinical practice guideline for anemia in chronic kidney disease. Kidney Int Suppl 2012;2:279–335. 9. Locatelli F, Bárány P, Covic A, De Francisco A, Del Vecchio L, Goldsmith D, Hörl W, London G, Vanholder R, Van Biesen W, ERA-EDTA ERBP Advisory Board. Kidney disease: improving global outcomes guidelines on anaemia management in chronic kidney disease: a European renal best practice position statement. Nephrol Dial Transplant 2013;28:1346–59. 10. National Institute for Health and Care Excellence. Anaemia management in people with chronic kidney disease. https://www.nice.org.uk/ guidance/cg114. 11. Praditpornsilpa K, Tiranathanagul K, Kupatawintu P, Jootar S, Intragumtornchai T, Tungsanga K, Teerapornlertratt T, Lumlertkul D, Townamchai N, Susantitaphong P, Katavetin P, Kanjanabuch T, Avihingsanon Y, Eiam-Ong S. Biosimilar recombinant human erythropoietin induces the production of neutralizing antibodies. Kidney Int 2011;80:88–92. 12. Seidl A, Hainzl O, Richter M, Fischer R, Böhm S, Deutel B, Hartinger M, Windisch J, Casadevall N, London GM, Macdougall I. Tungsteninduced denaturation and aggregation of epoetin alfa during primary packaging as a cause of immunogenicity. Pharm Res 2012;29:1454–67. 13. Carrera F, Lok CE, de Francisco A, Locatelli F, Mann JF, Canaud B, Kerr PG, Macdougall IC, Besarab A, Villa G, Kazes I, Van Vlem B, Jolly S, Beyer U, Dougherty FC, PATRONUS Investigators. Maintenance treatment of renal anaemia in haemodialysis patients with methoxy polyethylene glycol-epoetin beta versus darbepoetin alfa administered monthly: a randomized comparative trial. Nephrol Dial Transplant 2010;25:4009–17.