Development of Recombinant Erythropoietin and Erythropoietin Analogs

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. 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. 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.

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. 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. , 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 EPO and it took nearly another 30 years before human EPO was isolated from the urine of patients with aplastic anemia.

The next major development was the successful isolation and cloning of the human EPO gene in 1983, 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, 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. 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. 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. 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.

The half-life of subcutaneously administered recombinant human EPO is much longer, at around 24 h 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. 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.

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. Cardiac benefits, such as a reduction in left ventricular hypertrophy, were also described, as were objective measures of exercise performance. 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, 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., who described a case series of 13 patients who developed antibody-mediated pure red cell aplasia caused by the formation of antibodies 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. 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 factors ( 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. 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, along with a Lancet meta-analysis, 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, double-blind, 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. 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. 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. Since the manufacturing processes are different from those used by the innovator companies, there have been serious concerns about the safety, efficacy, and consistency of both biosimilar and “copy” epoetin products, 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, possibly due to tungsten exposure in the prefilled syringe.

Other epoetins that were developed around the same time include epoetin omega and epoetin delta. 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 gene-activated EPO because the expression of the native human EPO gene was activated by transformation of the cell with the cytomegalovirus promoter. 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. 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.

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 .

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 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.

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. Large EPO fusion proteins, of molecular weight 76 kDa, were designed from cDNA encoding two human EPO molecules linked by small flexible polypeptides. A single SC administration of this compound to mice increased red cell production within 7 days at a dose at which epoetin was ineffective. 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 monkeys but was later found to induce anti-EPO antibodies, causing severe anemia. Yet another approach was the genetic fusion of EPO with the Fc region of human immunoglobulin G (Fc–EPO). This molecular modification promotes recycling out of the cell upon endocytosis via the Fc recycling receptor, 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. 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.

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. In addition to the EPO derivatives administered by aerosol inhalation, other delivery systems for EPO have been investigated, including ultrasound-mediated transdermal uptake and orally via liposomes to rats. Mucoadhesive tablets containing EPO and an absorption enhancer (Labrasol) for oral administration have been studied in rats and dogs. 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.

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) ( 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. 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%.

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). 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%.

More recent studies estimated a longer half-life for subcutaneously administered darbepoetin alfa. These pharmacokinetic studies employed longer sampling periods, up to 28 days, and they suggested that the half-life of SC darbepoetin alfa may be around 70 h. Two studies by Padhi and colleagues, 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 erythropoiesis-stimulating agents (ESAs)). 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).

The half-life of darbepoetin alfa was also investigated in PD patients receiving a range of darbepoetin alfa doses. Tsubakihara and colleagues 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.

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.