Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow-Klinikum, Berlin, Germany
Keywords: erythropoietin therapy
Introduction
In 1985, the human erythropoietin (Epo) gene was cloned, based on limited structural information derived from a few milligrams of the hormone that were isolated from a large amount of urine. No more than two years later, the first reports about the successful correction of renal anaemia with recombinant human (rh) Epo were published. Since then, rhEpo has become (i) a standard treatment for dialysis patients, (ii) an interesting therapeutic option for several forms of non-renal anaemia and (iii), as a consequence of both, the drug with the highest annual sales worldwide. Considering this process, one might assume that we have passed the summit and that most of the excitement associated with the discovery and therapeutic application of this hormone lies in the past. On the other hand, it is this success that has set the stage for an ongoing story. Several very interesting developments regarding the when and how of Epo therapy are already foreseeable.
Renal anaemia
A symptomatic indication for Epo therapy in patients with renal anaemia has clearly been established. The most important future perspective is the increasing evidence for an additional, prognostic indication. Several studies in dialysis patients, including an analysis of the US Medicare database [1], demonstrated an inverse relationship between haematocrit and patient survival. This relationship persists after correction for demographics, comorbidity and disease severity, suggesting causality. The most likely explanation for a link between the degree of anaemia and patient prognosis is the impact of anaemia on the development and progression of left ventricular (LV) hypertrophy [2]. Prospective cohort studies identified anaemia as an independent risk factor for LV growth in the dialysis and pre-dialysis population [3,4], and partial correction of severe anaemia reduces LV mass. Several studies are underway testing the hypothesis that early and (almost) complete correction of anaemia in pre-dialysis patients prevents LV hypertrophy and improves quality of life. Although these trials have not been designed to assess effects on survival, demonstration of a reduction in LV growth would be an important argument to extend rhEpo therapy to the pre-dialysis population. According to the European Survey of Anaemia Management, so far, less than 10% of patients receive rhEpo before dialysis and the mean haemoglobin level at the onset of regular dialysis therapy is only 8.9 g/dl [5].
Non-renal indications
Potential non-renal indications for rhEpo that have been elaborated in the past include the anaemia of cancer, chronic inflammatory disease, prematurity and autologous blood donation. In most of these fields further work is required to define the value and optimal use of rhEpo. In tumour patients, studies are underway to assess the effect of anaemia correction not only on the quality of life but also on survival. An increase in tumour oxygenation, which may directly inhibit tumour growth, and an increase in the susceptibility to radio- and chemotherapy are postulated as underlying mechanisms.
At least two additional non-renal indications, in which a benefit of rhEpo appeared far less likely, have recently been addressed in first clinical trials. If the promise of these studies holds true, this will have significant implications also for nephrology.
Heart failure
A subgroup of patients with severe heart failure suffers from anaemia, but the frequency, course and origin of this phenomenon have not received much attention so far. An uncontrolled pilot study [6] and a small controlled trial by the same investigators [7] have suggested recently that in patients with severe heart failure, an increase in haemoglobin levels following combined therapy with rhEpo and iron leads to an impressive improvement of symptoms and retards disease progression. These data in patients with moderate, but not end-stage, renal failure are in sharp contrast to the results of the US normal haematocrit trial, which showed that the attempt to normalize haemoglobin levels in dialysis patients with symptomatic heart disease was not associated with prognostic benefit and might even be harmful [8]. Given the inconclusive evidence, the role of anaemia in heart failure outcome deserves further investigation. If a beneficial effect of anaemia correction can be confirmed this would considerably strengthen current concepts about the role of anaemia in the progression of LV hypertrophy in patients prior to and after initiation of dialysis.
Critical illness
Anaemia is very common in the critically ill. Within a few treatment days in intensive care units (ICU), more than 90% of patients have haemoglobin levels below normal. In most hospitals, ICU patients receive the majority of all blood transfusions. Median total blood loss during an ICU treatment of >3 days was estimated at around 1 l in all patients and 3 l in those with acute renal failure [9]. Iatrogenic and gastrointestinal blood loss are significant components. In addition, a severe impairment of red cell production plays an important role in the development and maintenance of anaemia in critical illness. Inappropriately low Epo production, direct inhibitory effects of inflammatory cytokines on proliferation and maturation of red cell precursors and reduced iron availability are considered to be the main reasons. The latter two mechanisms also cause Epo resistance in dialysis patients with infections and inflammation. However, using high doses of rhEpo it seems to be possible to overcome the inflammatory block of erythropoiesis. In a prospective randomized controlled trial in ICU patients, rhEpo reduced the amount of red blood cell transfusions by 46% and increased the haematocrit significantly [10]. Future studies have to describe parameters more precisely and comorbid conditions affecting the responsiveness to rhEpo, to investigate different dosing regimens and to assess the impact of avoiding blood transfusions and/or increasing haemoglobin on outcome parameters. This appears even more significant, since accumulating evidence links blood transfusion to adverse clinical outcome [11].
Pre-clinical evidence suggests yet another indication for rhEpo with potential relevance for critical care that is independent of its erythropoietic effect, but related to a direct effect on the brain.
Brain injury
Low level expression of Epo and its receptors has since long been reported in the brain [12,13]. Although brain derived Epo is unlikely to contribute to serum Epo concentration, cerebral Epo expression is upregulated by hypoxia and in vitro rhEpo was found to protect neuronal cells against hypoxic injury [14]. Therefore, the hypothesis arose that brain Epo may serve a paracrine protective function and that exogenously added Epo could be useful to diminish brain injury. Initial experiments confirmed this concept using intrathecal administration of rhEpo [15,16]. RhEpo administration into the ventricles avoids potential uptake problems across the blood brain barrier, but this approach is not feasible in clinical medicine. It is exciting, therefore, that more recently evidence was provided for an uptake mechanism of circulating rhEpo into the brain and that systemic administration of high doses of rhEpo was found to be neuroprotective in different models of brain injury, even when the application was started several hours after the insult [17]. In view of these findings clinical trials appear warranted. It is not impossible that apart from its established use as an anti-anaemic drug, Epo may, in addition, become an emergency-medication in patients with stroke or traumatic brain injury.
Novel treatment approaches
Irrespective of how different potential future indications for rhEpo will be, they will altogether increase the total need of the hormone, in particular, since several indications may require doses much higher than those currently used in the dialysis population. The future of Epo will, therefore, very much depend on its price. Expiration of current patents is foreseeable and will probably have a significant impact. Other developments are underway, which are likely to increase the number of providers, may result in alternative and less expensive methods of administration or will even lead to new therapeutic agents capable of mimicking and amplifying the action of Epo.
Gene activated Epo (gaEpo)
Currently approved preparations of rhEpo are obtained by expressing a human Epo gene introduced into rodent cell lines (Chinese hamster ovary (CHO) cells). Meanwhile, investigators have also succeeded to produce Epo by a process called gene activation in which protein production results from the upregulation of a quiescent endogenous gene in human cells. While the protein backbone of both preparations is identical, the carbohydrate structure may be slightly different since it is influenced by the enzymatic repertoire in the Golgi apparatus of each cell. However, since no relevant problems of antigenicity have been observed with CHO-derived Epo, the advantage of a carbohydrate moiety synthesized by a human cell appears somewhat theoretical. For several years patent lawyers have tried to unravel whether the production of gaEpo is covered by patents for rhEpo. In the most recent legal decision, the position was taken that this is the case.
Novel erythropoiesis-stimulating protein (NESP)
NESP is an Epo derivative, which results from mutations that have been intentionally introduced into the gene to exchange two out of the 165 amino acids. This change is of functional relevance since it creates two additional recognition sites for the attachment of N-linked carbohydrates. As compared to rhEpo, NESP has 5 instead of 3 N-linked sugar side chains in addition to the 1 O-linked chain. Since the carbohydrate moiety of Epo critically determines the rate of metabolism, plasma half-life time of NESP is prolonged [18]. This implies the clinical advantage of less frequent dosing and patients in clinical trials were successfully switched from 23 times weekly rhEpo to once weekly NESP or once weekly rhEpo to NESP every other week [19,20]. The launch of NESP will, therefore, probably reinitiate a discussion about optimal dosing strategies. Interestingly, in a recent issue of this journal a randomized controlled trial was published, indicating that once weekly s.c. dosing of conventional rhEpo can be as efficient as the application of the same dose in 23 injections/week [21].
Epo-mimetics and modulation of receptor activity
The Epo receptor consists of an extracellular ligand binding domain, a single-pass transmembrane region and an intracellular signalling domain. Its activation involves binding and homodimerization of two receptor monomers by a single Epo molecule [22]. It was considered as a major breakthrough in biotechnology when investigators succeeded several years ago to synthesize small peptide molecules (20 amino acids) without sequence homology with Epo, which bound to the receptor and possessed Epo mimetic activity both in vitro and in vivo [23]. Activity of such Epo-mimetics has meanwhile been improved and nonpeptidic mimetics of Epo have also been discovered [24]. Although many open questions and technical hurdles remain, these developments may eventually lead to the availability of orally active drugs that activate the Epo receptor [25].
A further potential target for therapeutic intervention is the intracellular signal transduction cascade. It involves phosphorylation of a Janus kinase (JAK) linked to the cytoplasmic domain of the receptor molecule. A haematopoietic cell phosphatase has been identified, which binds to a site near the C-terminal, cytoplasmic end of the receptor chain. It dephosphorylates JAK and, thus, functions as a negative regulator of the signalling cascade [26]. The functional significance of this enzyme is exemplified by the observation that a loss of its binding site in individuals carrying a truncated Epo receptor causes benign erythrocytosis [27]. Pharmacological inhibitors of this phosphatase may therefore be efficient tools to amplify the response to Epo.
Epo gene therapy
Given the necessary frequency of treatments, the costs of current therapy and the ease to measure the biological effect of Epo, a gene therapy protocol for the treatment of anaemia could be a great advantage. In over five years, several reports have been published demonstrating the feasibility of Epo-gene transfer in rodents and non-human primates [e.g. 28,29]. The two principal approaches are either direct gene transfer in vivo or ex vivo gene transfer into isolated cells, which are then transplanted into the recipient organism. A sustained elevation of haemoglobin levels can be achieved with both strategies. For several reasons the former is preferable for consideration in humans. Its applicability in patients will depend on safety issues related to gene transfer in general and reliable techniques to control Epo secretion rate in vivo.
By transfecting cells with a vector containing the human Epo cDNA driven by a hypoxia responsive promoter, it has been possible to stimulate transgene expression by hypoxia in experimental settings in vitro and in vivo [30]. However, considering the complexity and sensitivity of the physiological oxygen-dependent control of Epo production in the kidneys, it will be extremely difficult to achieve a similar regulation through autoregulated gene therapy. Alternatively, systems of ligand-dependent transgene regulation could be used, which involve specific activation of a transgene through different drugs. The most important of the currently developed systems are those driven by tetracycline, a synthetic antiprogestin or chemical dimerizers such as rapamycin. With all three systems, iterative regulation of Epo secretion and haematocrit has been demonstrated in experimental animals depending on the systemic application of the respective ligand [3133]. If and at which speed these developments will lead to clinical applications, remains to be seen.
Conclusions
Considering the adverse consequences of anaemia in association with different diseases and the technical and market developments, which are likely to make erythropoietic therapy cheaper, there is great potential for an increase of the number of patients treated and the benefit obtained from this type of therapy. However, it will be necessary to define precisely the cost/benefit relationship for different indications as well as the optimal ways to use Epo and related therapies. So far only 3 out of the 18 European best practice guidelines for the treatment of renal anaemia are, at least in part, supported by level A evidence [34]. Given the enormous interest in this therapeutic approach, the relative scarcity of strong data has remained a somewhat disappointing aspect of Epo therapy during the last fifteen years and strongly calls for continued research.
Notes
Correspondence and offprint requests to: Prof. K.-U. Eckardt MD, Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
References
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