Use of Recombinant Human Erythropoietin as an Antianemic and Performance Enhancing Drug

W. Jelkmann*

 

Institut für Physiologie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

*Address correspondence to this author at the Institut für Physiologie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; Tel: +49-451-500-4150; Fax: +49-451-500-4151; E-mail: Jelkmann@physio.mu-Luebeck.de

Abstract: The glycoprotein hormone erythropoietin is an essential viability and growth factor for the erythrocytic progenitors in the bone marrow. Tissue hypoxia is the main stimulus for the synthesis of the hormone in the kidneys and the liver. Endogenous erythropoietin and recombinant human erythropoietin (rHu-EPO) are similar with respect to their biological and chemical properties except for some microheterogeneities in their 4 carbohydrate chains. Generic products and alternatives to rHu-EPO are in development. Renal anemia can be corrected by rHu-EPO in a dose-dependent and predictable way without major side effects apart from a possible increase in arterial blood pressure. The optimal target hematocrit still needs to be defined. There are rare reports of antibody formation towards rHu-EPO in humans. Patients suffering from non-renal anemias may also benefit from the prescription of rHu-EPO. The drug has been approved for treatment of tumor patients with platinum-induced anemia. The cost-effectiveness and medical justification of the administration of rHu-EPO in tumor patients with respect to its positive effects on tumor oxygenation, tumor growth inhibition and support of chemo- and radiotherapy is still a matter of debate. In surgical patients, the pharmacological application of rHu-EPO can increase the yield of blood units in autologous blood donation programs and lower the severity and duration of postoperative anemia, if applicated some days prior to surgery. While rHu-EPO is a godsend in medical practice, its abuse as an performance enhancing drug by athletes in endurance sports is an unethical and potentially dangerous procedure. Unequivocal methods for detection of rHu-EPO doping still need to be established.

Introduction

     Hematocrit and the concentration of hemoglobin in blood are normally maintained constant. About l % of the red cell mass is renewed each day. Anemic persons suffer from tissue hypoxia. They present with fatigue, pallor, shortness of breath, tachycardia and angina pectoris. Severe cases can require transfusion of red cells from blood donors. Transfusion therapy with allogeneic blood components may cause immunologic reactions and infections. In addition, repeated red blood cell transfusions can lead to iron overload. Therefore, the availability of recombinant human erythropoietin (rHu-EPO) as an anti-anemic drug has been an important medical progress.

     Initial trials of the replacement therapy with rHu-EPO to restore the hematocrit in patients with end-stage renal failure were reported about 14 years ago [1,2] which then lent a new impetus to studies of the pathophysiology and pharmacology of EPO [3]. In all likelihood, rHu-EPO is today the best selling drug in the world (estimated sales 5,000 millions US $ per year).

     This review provides basic information on: (a) the role of the hormone erythropoietin (EPO) in the control of red cell production, (b) the structural and biological characteristics of rHu-EPO preparations, (c) their clinical use in the anemia associated with renal failure and inflammatory diseases, (d) the relationship between tumor oxygenation, anemia and the efficiency of anti-tumor therapy, and (e) the potential value of the administration of rHu-EPO in the perisurgical setting including its use in autologous blood transfusion programs. Finally, (f) the problem of rHu-EPO abuse by athletes in endurance sports will be stressed. It is not intended to provide a complete survey of the relevant original publications. However, specific issues that are controversial or of prospective interest will be described in more detail.

     With respect to earlier or additional information on specific topics, the reader is referred to more complete reviews on the history of EPO research [4,5], the in vivo control of EPO synthesis [5-7], the cellular and molecular mechanisms of the action of EPO [8-11] and the clinical use of rHu-EPO in the treatment of the anemia associated with renal failure [12,13] or malignancies [14-16]. Other reviews will be cited in the text.

     Erythropoiesis counterbalances the permanent destruction of aged red blood cells by macrophages in bone marrow, spleen and liver. The basal rate of the production of red cells (2-3 x 10¹¹ per day) may 10fold increase following a blood loss. Both the basal and the augmented elaboration of red cells are mediated by the hormone EPO. Experimental studies in animals have shown that almost all circulating EPO originates from peritubular interstitial cells in the cortex of the kidneys and from parenchymal cells in the liver [17,18]. In addition, some EPO mRNA has been detected in spleen, lung, testis and brain [19-21]. Tissue hypoxia is the main stimulus of EPO production [6]. EPO levels in plasma may rise to 10,000 IU/l (International Units per liter) in severe anemia, compared to the normal value of about 15 IU/l. There is an exponential increase in EPO production with decreasing blood hemoglobin concentration in persons with intact kidneys. Hence, an inverse log/linear relationship can be drawn between the concentrations of EPO and hemoglobin in blood. This relationship is lost in patients suffering from chronic renal failure, as demonstrated in Fig. (1).

     Fig. (2) illustrates the control circuit of erythropoiesis. The O₂ capacity of the blood is the primary determinant of the rate of EPO synthesis in kidneys and liver. In addition, EPO synthesis is stimulated when the arterial O₂ tension is lowered or when the O₂ affinity of the blood is increased. Changes in renal blood flow have little influence on EPO production [22]. Several other hormones interfere with the normal pO₂-dependent feedback circuit of erythropoiesis [6]. The normally higher hemoglobin and hematocrit values in males than in females are probably due to the myeloid action of androgens, which augment the effect of EPO on erythrocytic progenitors [23,24]. Thyroid hormones may stimulate EPO gene expression [25], resulting in increased circulating EPO levels in hyperthyroidism [26].

     The molecular mechanisms of O₂ sensing are beginning to be understood [27]. Possibly, extramitochondrial heme proteins function as O₂ sensor [28-31]. A recent hypothesis suggests that H₂O₂ and other reactive O₂ species that are produced by b-type cytochromes at high pO₂ act as signaling molecules which repress EPO gene expression [32,33]. A hypoxia inducible factor (HIF-1) has been identified that binds to the hypoxia-responsive enhancer in the 3'-flanking sequence of the EPO gene. HIF-1 is a dimeric protein composed of 2 different subunits, the 120 kDa HIF-1a and the 91-94 kDa HIF-1ß [34]. HIF-1 controls the expression of several genes that encode proteins which are protective against hypoxia, such as vascular endothelial growth factor and distinct glycolytic enzymes [31,35,36].

     About 3 days after an acute increase in plasma EPO reticulocytosis becomes apparent. Reticulocytes are progenies of lineage-specific progenitors originating from a small pool of stem cells in the hemopoietic organs. Fig. (3) summarizes the main stages in erythropoiesis. The functional human EPO receptor is a 484-amino acid glycoprotein and member of the class I cytokine receptor superfamily [8,10]. The number of receptors per cell decreases with differentiation beyond the colony-forming unit-erythroid (CFU-E) level. Reticulocytes and erythrocytes have no EPO receptors.

     Fig. (4) is a schematic presentation of the EPO receptor. The first step in EPO signaling is dimerization of the receptor molecules. This induces tyrosine phosphorylation of several cytoplasmic and membrane-associated proteins including the EPO receptor itself [11]. The subsequent cellular events that lead to proliferation and differentiation have not been clearly identified. Suppression of programmed cell death (apoptosis) is the likely mechanism by which EPO maintains erythropoiesis [37]. When the concentration of the hormone rises in blood, as in anemia, an increasing number of progenitor cells escape from apoptosis and proliferate. The presence of EPO is essential for the viability, proliferation and differentiation in the erythrocytic lineage. Lack of EPO leads to anemia.

     Some evidence has been provided to assume that EPO may exert a local function in the central nervous system besides its role in erythropoiesis. EPO mRNA [19,21] and EPO receptors [38-40] can be demonstrated in brain. EPO and EPO receptor are expressed by neurons and glial cells in the brain and spinal cortex of human fetuses [41]. It is speculated that EPO may act as a neurotrophic and neuroprotective factor. Indeed, EPO has been shown to alleviate the ischemia-induced place navigation disability, cortical infarction and thalamic degeneration, when applied into the cerebrovesicles of stroke-prone rats with permanent occlusion of the left middle cerebral artery [42]. The protective effect of EPO on hippocampal cells has been also demonstrated in a gerbil forebrain ischemic model [43]. The expression of EPO as well as of the EPO receptor gene increases in response to hypoxia in brain. The cellular mechanism of the neuroprotective effect of locally produced EPO is not fully understood. It has been proposed that EPO inhibits the formation of reactive O₂  species and N-methyl-D-aspartate receptor-mediated cytotoxicity [42,43].

Production and properties of recombinant DNA-derived EPO

     In a pioneering work published in 1977 Miyake et al. [44] isolated 10 mg pure human EPO of 2550 1 urine from severely anemic patients. The preparation of pure human urinary EPO enabled it to identify the amino acid sequence of a tryptic fragment of the protein and to synthesize EPO DNA probes for the isolation and cloning of the EPO gene [45,46]. Mammalian cells transfected with the EPO gene linked to an expression vector ("recombinant DNA") produce rHu-EPO in vitro. Chinese hamster ovary (CHO) cells deficient in the dihydrofolate reductase gene are most commonly used for the large-scale pharmaceutical manufacture of the drug, because in such cells EPO gene amplification can be achieved by co-selection in the presence of methotrexate [47]. The cultures are maintained in large fermenters or roller bottles. RHu-EPO is isolated from the medium by a series of chromatography steps. Care is taken that the drug is not contaminated by microorganisms, xenogenic proteins, oncogens or pyrogens.

     Human urinary EPO and rHu-EPO are identical with regard to their amino acid sequence, position of their 2 disulfide bridges and 4 glycosylation sites, and their secondary structure. The peptide consists of 165 amino acids. The molecular mass of the glycoprotein entity is 30 kDa. The carbohydrate portion (40%) is essential for molecular stability and full in vivo biological activity. There are 3 tetraantennary N-linked (Asp 24, 38 and 83) and 1 small O-linked (Ser 126) acidic oligosaccharide side chains. The molecule forms a bundle of 4 a-helices which are folded into a compact globular structure. Although the recombinant products contain no new structure elements compared to the native hormone, there are quantitative differences in glycosylation, which may also explain the fact that the specific in vivo biological activity of purified human urinary EPO is lower (70,000 IU/mg peptide) than that of the purified recombinant product (about 200,000 IU/mg peptide).

     Like other soluble glycoproteins, native EPO exists as a pool of several isoforms that differ slightly in glycosylation. In particular, the carbohydrate side chain in position Asp 24 exhibits microheterogeneity [48]. Studies by zone electrophoresis have shown that there are intraindividual diurnal changes, interindividual differences, and abnormalities associated with inflammatory diseases with respect to the occurrence of EPO isoforms in serum [49]. Furthermore, there are differences in the electrophoretic mobility when human blood-borne, urinary and recombinant EPOs are compared [49-51]. Importantly, EPOs differing in their carbohydrate composition exhibit also differences in their biological activities and immunoreactivities [52-54]. The extent of microheterogeneity of CHO cell-expressed rHu-EPO has been studied by mass spectometry and NMR spectroscopy, as reported by Rush et al. [55].

     Two brands of CHO cell-derived EPOs, termed epoetin alfa and epoetin beta, are currently used for treatment of EPO-deficiency anemias and for support in autologous blood collection programs. Both of these types of rHu-EPO are produced in CHO cultures. In extensive studies utilising several batches of each brand Storring et al. [54] have compared epoetin alfa and epoetin beta by means of isoelectric focusing, lectin-binding, in vivo and in vitro bioassays, and immunoassays. While there were only minor inter-batch differences within the two brands, between these significant differences were apparent in the pattern of glycosylation.

Fig. (5) shows that epoetin alfa differs from epoetin beta in containing a smaller proportion of more basic isoforms. In addition, epoetin alfa possesses less N-glycans with non-sialylated outer Galb1-4GlcNAc moieties, N-glycans containing repeating Galb1-4GlcNAc sequences, tetraantennary and 2,6-branched triantennary N-glycans, and Galb1-3GalNAc containing O-glycans. Epoetin beta has a higher in-vivo: in-vitro bioactivity compared to epoetin alfa when tested in murine systems [54]. Note that this difference in biological activity cannot be explained on grounds of the degree of sialylation, because, if anything, epoetin alfa is sialylated to a greater degree than epoetin beta [54]. Clinical observations are in agreement with these results. The terminal elimination half-life of epoetin alfa is shorter than that of epoetin beta in humans following intravenous or subcutaneous administration of the drugs [56]. Still, there do not appear to be significant differences in the efficacy of the 2 brands of commercial rHu-EPO.

Attempts of developing new erythropoiesis-stimulating drugs

     According to expectations, generic rHu-EPO preparations will come to market in the near future, when the present CHO cell-derived products will no longer be protected by patent. RHu-EPO with full in vivo biological activity can be purified from the culture supernatant of other genetically engineered mammalian cells such as BHK-21 baby hamster kidney cells or C127 mouse mammary cells [57]. In addition, attempts have been successful to produce dimers and trimers of rHu-EPO which are in vivo much more efficient in stimulating erythropoiesis than the monomers [58].

     EPO gene transfer could become an alternative to the administration of rHu-EPO. Naffakh and Danos [59] have reviewed the various ex vivo and in vivo approaches for EPO gene therapy, which resulted in the production of EPO in amounts that sufficed to stimulate erythropoiesis in experimental animals. However, before clinical applications of EPO gene therapy can be explored, the stability, tissue specificity and regulatory mechanisms of the transgenes need to be more carefully investigated.

     EPO receptor agonist peptides have been isolated by random phage display peptide libraries' screening [60,61]. These EPO mimetics are cyclic 2 kDa peptides of about 20 amino acids. They show no sequence homology to EPO, but stimulate the proliferation and differentiation of erythrocytic progenitors in vitro and enhance erythropoiesis in mice in vivo. The potency of the peptides can be greatly increased through covalent dimerization [62]. The functional mimicry of a protein hormone by an unrelated small peptide is biochemically exciting. However, it is not clear whether these studies will eventually enable it to design molecules that can be administered orally or trans-dermally for stimulation of erythropoiesis in clinical practice.

Normal and abnormal levels of serum EPO

     Possible indications for assay of serum EPO in the laboratory routine include the differential diagnosis of polycythemias and anemias, the follow-up of paraneoplastic EPO production, and the election of anemic patients for rHu-EPO therapy. Radioimmunoassay or enzyme-linked immunosorbent assay procedures can be applied [63]. The sensitivity of most of the current immunoassays is still insufficient as these do not enable one to carry out valid measurements in states of low EPO concentrations, such as in polycythemia vera [64]. EPO concentrations are traditionally expressed in International Units (IU) as a measure of biological in vivo activity [65]. Reference preparations of human urinary EPO (2nd IRP) and purified DNA-derived human EPO (rDNA-derived, 130,000 IU/mg fully glycosylated protein) are available [65,66]. The concentration of serum immunoreactive EPO is 6-32 IU/1 in non-anemic individuals [63]. Interestingly, there is no significant difference detectable when the values are compared in healthy women and men, although the hemoglobin concentration is lower in the females.

     Because EPO is the only specific regulator of the growth of erythrocytic progenitors, EPO overproduction inevitably results in secondary erythrocytosis, and EPO deficiency in anemia (Table 1).

 

     Overproduction of EPO may occur as a physiological response to hypoxia, e.g. at high altitudes, or arise autonomously as a paraneoplastic syndrome. Excessive EPO production is probably the major pathogenetic factor in the development of chronic mountain sickness. Erythrocytosis increases the risk to acquire myocardial infarction and stroke. In patients suffering from secondary erythrocytosis phlebotomy treatment to hematocrit 0.52-0.50 may be beneficial to prevent hemodynamic and rheological complications such as pulmonary hypertension and peripheral thrombosis. However, repeated phlebotomies lead to iron deficiency in the long term. Unfortunately, specific erythropoiesis-inhibiting drugs have not been developed.

     Insufficient EPO production is the primary cause of the anemia in chronic renal failure [12]. The pathogenetic mechanisms may involve destruction of the EPO-producing cells, inhibition of EPO synthesis due to metabolic acidosis, accumulation of uremia toxins and increased formation of proinflammatory cytokines. Interestingly, the induction of acute renal failure in rats results in suppressed hepatic EPO gene expression [67]. Thus, the liver cannot substitute for the diseased kidneys.

     In several non-renal diseases, such as in chronic inflammation, malignancy and AIDS a relative lack of EPO contributes to the development of anemia [68,69]. Based on the results of in vitro studies, it is thought that the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-a (TNFa) suppress EPO gene expression [69]. In addition, chemotherapeutic and immunosuppressive drugs can inhibit EPO synthesis [70].

     The assessment of an "inadequate EPO response to anemia" is difficult in practice, because serum EPO levels cannot be evaluated in absolute terms but only in relation to the degree of anemia [71]. Thus, the definition of a relative deficiency of EPO relies primarily on documentation of a lowered ratio between the serum EPO level and the blood hemoglobin concentration or hematocrit in comparison with this ratio in a reference population [72]. It has been suggested to calculate the observed/predicted log [EPO] ratio (O/P ratio) for each serum sample, with the predicted level being estimated from a reference group of patients with anemia not associated with renal disease, infection, inflammation or malignancy. Such types of anemias include those of iron deficiency or hemolysis.

     Pathophysiological consequences of anemia are summarized in (Table 2).

Table 2.        Pathophysiological Consequences of Anemia

 

Pharmacokinetic of rHu-EPO

     Both intravenous and subcutaneous routes of rHu-EPO administration are effective and used in clinical practice depending on the patient's disease and accompanying medical treatment. Compared with intravenous injection, subcutaneous administration is characterized by a prolonged absorption phase (peak values reached after 12-30 h), lower peak values (about 5% of those observed after intravenous administration), lower rate of bioavailability (20-40%), but prolonged elimination half-time that has been reported in a range from 1 to 3 days [73-76]. Of note, the biological half-time of intravenously injected rHu-EPO appears to be considerably longer (4-6 h of epoetin alfa, 4-12 h of epoetin beta), when compared to the disappearance of native EPO whose half-life has been estimated to be about 2 h [77]. More detailed information on the pharmacokinetic  and pharmacodynamic of rHu-EPO is provided elsewhere [13,75,78].

RHu-EPO therapy in renal failure

     RHu-EPO as an anti-anemic drug for treatment of patients suffering from chronic renal failure was introduced 14 years ago [1,2]. Given intravenously or subcutaneously [79] it is now routinely used in patients on regular hemodialysis or continuous ambulatory peritoneal dialysis (CAPD), as well as in many predialysis patients [12,80]. At least in Europe, there appears to be a great underutilization of rHu-EPO during the predialysis period, although the correction of anemia would allow the patients to enter dialysis later than without rHu-EPO therapy and prevent left ventricular hypertrophy and congestive heart failure [81]. RHu-EPO raises hematocrit and blood hemoglobin concentration in a dose-dependent and predictable way, and it abolishes the need for red cell transfusions with its risks of incompatibility reactions, viral infections and iron overload. In previously anemic patients, rHu-EPO therapy reverses the hyperdynamic cardiac state and restores the impaired brain function. The well-being and exercise tolerance of the patients is greatly increased (Table 3).

 

 

     Eventually, rHu-EPO can correct the anemia in practically all patients with renal failure, but the dose needed is variable (Table 4). The complications responsible for rHu-EPO resistance include iron deficiency, inflammatory or infectious disease, aluminium overload, hyperpara-thyroidism, and osteitis fibrosa. In addition, the response to rHu-EPO can be improved by increasing the intensity of dialysis [82]. The main reason for rHu-EPO hyporesponsiveness in clinical practice is failure to provide sufficient iron, which is reflected by a serum ferritin concentration < 100 µg/l, a transferrin saturation < 20 %, and a proportion of hypochromic red cells > 10 % [83]. Intravenous iron is recommended for hemodialysis patients (10-20 mg/hemodialysis treatment). Oral iron supplementation is appropriate for CAPD and predialysis patients with serum ferritin > 100 µg/l [84]. The potential toxicity of chronic iron exposure has been discussed recently [85].

 

Table 4.           RHu-EPO Therapy in Chronic Renal Failure

 

     Infectious and inflammatory diseases may reduce responsiveness to rHu-EPO by at least two mechanisms, namely impaired availability of iron and cytokines-induced inhibition of the proliferation of erythrocytic progenitors [68,86]. Resistance to rHu-EPO in uremic patients with inflammatory disease is mediated by interferon g (IFN-g) and TNFa [87]. Goicoechea et al. [88] have recently reported a direct positive correlation between the rHu-EPO dose requirements of hemodialysis patients and the in vitro rates of TNFa and IL-6 production by peripheral blood mononuclear cells from these patients. Measurements of C-reactive protein and baseline fibrinogen concentrations [72] in serum may provide early recognition of the probability of response to rHu-EPO.

     At present, nephrologists use to set the target hematocrit value at 0.33-0.36 (hemoglobin 110-120 g/l) in patients under rHu-EPO therapy. While this level of anemia correction is clearly associated with an acceptably restored quality of life, exercise capacity, cardiac performance and cognitive function, the question has been raised recently as to whether increasing the doses of rHu-EPO to attain normal hematocrit values would benefit the patients [89-92]. Indeed, a further increase in quality of life and exercise capacity could probably be achieved in many predialysis and dialysis patients if their hematocrits were raised above 0.36. However, the benefits and risks of this procedure need to be carefully outweighed. In a recent randomized prospective long-term multicenter study 1233 patients with cardiac disease were treated with high or low dose rHu-EPO to achieve either normal (0.42) or low (0.30) hematocrit values [94]. Surprisingly, the one-year and two-year mortality rates were by 7% higher in the normal-hematocrit than in the low-hematocrit group, and the study was halted after 29 months. An explanation for the disparate outcomes could not be provided. There were no significant differences in arterial blood pressure values between the 2 groups. This observation has been confirmed in a separate report [95]. The patients in the normal-hematocrit group received intravenous iron dextran more often, which could have resulted in endothelial cell damage by reactive O₂ species or predisposed the patients to infection. Thus, despite the reduced requirement for red cell transfusion in the normal-hematocrit group, the use of rHu-EPO therapy to achieve a target hematocrit value of 0.42 was not recommended by the authors [94], at least not for hemodialysis patients with clinically evident congestive heart failure or ischemic heart disease. Further studies are required to answer the question of the optimal target hematocrit in different subgroups of patients with renal failure based on their cardiac status.

     (Table 5) summarizes the major adverse events seen in uremic patients receiving rHu-EPO. The most serious unwanted effect is the development or worsening of arterial hypertension [96]. Additional antihypertensive medication may be required. The increase in blood pressure results partly from the elevated blood viscosity and the loss of hypoxia-induced vasodilation [97]. However, other factors that are not related to the improvement of anemia contribute to the elevation of arterial blood pressure. A hypertensive effect is not observed in rHu-EPO treated anemic hemodialysis patients with cardiac disease, when hematocrit is slowly normalized [94,95]. Hemodialysis patients with a positive family history of hypertension are more susceptible to develop hypertension during rHu-EPO therapy than those with a negative family history [98]. Patients with non-renal anemia do not develop hypertension due to rHu-EPO therapy. When hematocrit is raised from 0.45 to 0.50 by rHu-EPO treatment in healthy men, their arterial blood pressure values are unchanged at rest. On submaximal exercise, however, their systolic blood pressure values are significantly higher than before rHu-EPO treatment, while their heart rates are lowered [99]. The latter finding indicates an improved physical exercise capacity at higher hematocrits.

Table 5.        Adverse Events Occurring in Uremic Patients Receiving rHu-EPO

 

     Because human native EPO and rHu-EPO are basically similar in structure, rHu-EPO is only weakly immunogenic. So far, there have been few case reports on anaphylactic reactions due to the presence of anti-rHu-EPO specific IgEs [100], on anemias due to the production of neutralising anti-rHu-EPO specific IgGs [101-105] or rHu-EPO therapy-associated vasculitis and podagra [106].

RHu-EPO therapy in non-renal anemias

     Understandingly, the number of patients and doctors who wish to avoid allogeneic blood transfusions has increased, since rHu-EPO has become available for therapy. The value of the drug has been investigated in many types of anemia [6,107]. Potential indications for rHu-EPO therapy are summarized in (Table 6). In some countries, the drug has already been approved for treatment of the anemias associated with AIDS, cancer (primarily in platinum-associated anemia), bone marrow transplantation, myelodysplastic syndromes and autoimmune diseases.

Table 6.        Possible Indications for the Administration of rHu-EPO*

 

*References are minireviews or representative original articles

     In contrast with the high response rate in renal anemia, rHu-EPO resistance (hemoglobin increase < 10 g/1 in 4 weeks) is often seen in this diverse population of patients [107]. In addition, the rHu-EPO doses (3 x > 150 IU/kg and week) required for correction of anemia are relatively high. An algorithm for rHu-EPO therapy in anemic cancer patients undergoing platinum-chemotherapy is given in Fig. (6). In patients with myelodysplastic syndromes, the treatment with a combination of rHu-EPO with rHu-G-CSF (granulocyte colony-stimulating factor) may be more effective than treatment with either of these hemopoietic growth factors alone [108,109].

     Because the drug is expensive, it must be administered most economicly. Attempts have been undertaken to determine predictors of the response to rHu-EPO in non-renal applications [72,110]. The combination of low base-line endogenous serum EPO concentrations, a low observed/predicted (O/P) ratio of serum log EPO values, and serum ferritin < 400 µg/l seems to provide some evidence for a positive response. For identification of patients at high risk for chemotherapy-induced severe anemia requiring red blood cell transfusion Ray-Coquard et al. [111] have recently calculated a risk model involving blood hemoglobin concentration, lymphocyte count and performance status just before chemotherapy was initiated.

     Cost-benefit calculations are difficult to carry out so far with respect to rHu-Epo therapy in non-renal anemia. Dosage and administration rules still need to be further defined. Also, there are major variances between countries both in regard to the national health-care economics and the safety and costs of blood transfusions.

Anemia and anti-tumor therapy

     Anemia develops in the majority of patients with malignant tumors. Its causative mechanisms are summarized in (Table 7). Depending on the type and stage of the malignant disease, direct effects of the tumor (bone marrow invasion, hemolysis, bleeding), inflammatory reactions ("anemia of chronic disease") and anti-tumor therapy (chemotherapy, radiation, surgery) will be involved. As reviewed by Nowrousian [15], among patients with solid tumors those with lung and ovarian cancer have the highest incidence of anemia and the highest rate of red blood cell transfusion requirements (about 25%). Patients with a low level of hemoglobin (120-110 g/l) at the start of chemotherapy are particularly at risk of developing severe anemia.

Table 7.        Causes of Anemia in Malignancies

 

     The anemia in tumor patients is generally normochromic and normocytic. Iron stores are normal, but the concentration of circulating iron is often low due to the reduced iron mobilization from the storage sites. Although red cell survival may be shortened, the anemia is primarily of the hypoproliferative type. Cytokines such as IL-1, TNFa and interferons inhibit the proliferation of erythrocytic progenitors [68]. In addition, IL-1 and TNFa suppress EPO gene expression [69]. The concentration of serum EPO is indeed relatively low in many adult cancer patients, when related to the blood hemoglobin concentration [112]. The anemia in children with solid tumors is reportedly not related to defective EPO production [113].

     The cost-consequences of red blood cell transfusion versus rHu-EPO treatment in tumor- and chemotherapy-associated anemia are difficult to evaluate at present [114]. In addition, the objectives of blood transfusion and rHu-EPO therapy differ. Blood transfusion is merely practiced to avoid or abolish troublesome symptoms of severe anemia. RHu-EPO therapy aims at maintaining the patients' hemoglobin values above the transfusion trigger, increasing their exercise tolerance and improving quality-of-life parameters [111,115-118].

     As reviewed by others [14-16] several randomized and nonrandomized studies have been carried out to evaluate the efficacy of rHu-EPO treatment of the anemia associated with malignancy and chemotherapy in patients with solid tumors and primary hematological disorders. The overall response rates have ranged from 40% to 85%. Moreover, 10% to 30% of the patients still had to be transfused despite rHu-EPO therapy [117]. In view of the relatively large number of nonresponders, there is an implicit need to control spending rHu-EPO [117]. Hence, investigators have attempted to identify parameters that allow the selection of tumor patients who are likely to respond to rHu-EPO [119]. In hematological malignancies an inverse correlation has been noted between the baseline endogenous EPO level or the observed/predicted log [EPO] ratio and the response rate [110,120]. This correlation could not be confirmed in patients with solid tumors in general [15]. A small prospective phase IV clinical trial of our own has shown that the response rate to rHu-EPO is 100% in patients with moderate renal failure (increased serum creatinine and low EPO concentration) and solid tumors [121]. On the other hand, none of the following singular baseline parameters is of sufficient prognostic power in patients with solid tumors: circulating hemoglobin, iron, ferritin, transferrin or soluble transferrin receptor [122]. A significant increase in hemoglobin levels (³ 10 g/l) and reticulocytes (³ 40 x 10⁹/l) after 4 weeks of rHu-EPO treatment is considered a reliable indicator of response, as applied in Fig. (6). However, as pointed out by Beguin [119] considering the rate of response as a parameter of response prediction is trivial.

     In patients with solid tumors the correction of anemia by rHu-EPO may not merely be a palliative intervention. Another aspect of major actual interest is the impact of the blood hemoglobin concentration on the tumor growth per se, as well as on the outcome of anti-tumor therapy. Solid tumors are characterized by regions where the cells are hypoxic due to the long O₂ diffusion distance between the incomplete vasculature and the mass of O₂-consuming cells [123,124]. Studies in animal models have shown that the intratumoral pO₂ increases when the O₂ capacity of the blood is enhanced by hemoglobin infusion [123] or rHu-EPO treatment [125]. The finding that tumor oxygenation is influenced by the O₂ capacity of the blood is important, because hypoxia induces transcription of a number of genes encoding proteins which control tumor growth [124]. Among these proteins are the vascular endothelial growth factor (VEGF), which is necessary for tumor angiogenesis [126,127] and the p53 protein, which is a key regulator of growth arrest and apoptosis [128,129]. Gagic et al. [130] have shown that the administration of rHu-EPO in combination with clenbuterol, a b₂-adrenergic agonist, attenuates both the cancer-associated anemia and the growth of carcinosarcomas in rats. In contrast, Kelleher et al. [125] reported a slower tumor growth in anemic than in non-anemic rats. Neither did these authors observe an effect of rHu-EPO treatment on tumor growth [125]. Thus, with a view to the clinical use of rHu-EPO additional studies are required to clarify the direct relationship between blood O₂ capacity and tumor growth.

     Another point of interest is the relationship between the degree of hypoxia and the therapeutic responsiveness of tumors. DNA damage by sparsely ionizing radiation (g-radiation and X-ray) is caused by reactive O₂ species. The effect of such radiation is up to 3-fold stronger in normoxic tissue (pO₂ > 15 mm Hg) than in hypoxic tissue (so-called "oxygen enhancement ratio", OER). Similarly, the cytotoxicity of a number of chemotherapeutics (f. e. carboplatin, cyclophosphamide, actinomycin D, 5-fluorouracil) is greater in normoxic than in hypoxic cells [131]. The differences are related directly to the reduced formation of reactive O₂ species and indirectly to the slowed cell cycling in hypoxia [132]. Most of the studies on the relationship between hemoglobin concentration and radiation efficiency have revealed a poorer tumor control in anemic compared to nonanemic patients. This has been shown for cancers of the head, neck, lung, uterine cervix and bladder [133,134]. In a randomized trial in patients with cervical cancer mortality was reduced in cases of allogeneic blood transfusion prior to radiation therapy [135]. However, there is also fear of increased cancer recurrence, because allogeneic blood transfusions are suspected to exert an immunosuppressive effect [132,136,137]. Some studies have provided evidence that rHu-EPO is safe and effective in raising hemoglobin levels during radiotherapy with or without concurrent chemotherapy of anemic patients with cancers of the head, neck or thorax [138], uterine cervix [139], and lung, breast or prostate [140]. Thews et al. [141] have recently reported that the correction of carboplatin-induced anemia by rHu-EPO results in an increased radiosensitivity of experimental tumors in rats. Here, clinical trials are warranted to investigate whether the administration of rHu-EPO indeed improves the efficacy of radio- and chemotherapy.

RHu-EPO in the perisurgical setting

     Surgeons and anesthesiologists take care of maintaining hemodynamics and peripheral O₂ supply within normal limits during and after surgical interventions. The decision when to transfuse blood in surgical patients is difficult, as there is no clear threshold hemoglobin value for intervention. The critical level can best be determined by measurements of physiological parameters such as mixed venous O₂ saturation or O₂ extraction ratio [142]. In practice the decision to transfuse will be based on clinical parameters such as symptoms of hypoxia, age and comorbidity. Because the transfusion of allogeneic blood components is associated with a – statistically small – risk of noninfectious or infectious complications, three strategies have been employed to use rHu-EPO for blood conservation in the surgical setting: (a) the preoperative stimulation of erythropoiesis in aggressive phlebotomy programs for autologous re-donation, (b) the correction of preoperative anemia, and (c) the postoperative acceleration of the recovery of red blood cell mass.

     Sufficient autologous blood donation can prevent patients from allogeneic red blood cell transfusion in elective orthopedic and heart surgery. RHu-EPO has been used as adjunctive therapy to accelerate erythropoiesis preoperatively so to facilitate the collection of blood units [143,144]. Universally accepted guidelines regarding the route, dose and frequency of rHu-EPO therapy still need to be established both in orthopedic [145-147] and cardiac surgery [148-150]. Clearly, relatively high doses of rHu-EPO are required. It is preferable to perform the blood collection and rHu-EPO treatment on an ambulant basis. The effect of rHu-EPO is supported by intravenous iron therapy [151].

     The beneficial effect of rHu-EPO in the treatment of preoperative or postoperative anemia has been noted in several case reports, mostly on Jehovah's Witnesses [152]. A large, placebo-controlled, and double-blind multicentre trial has shown that the administration of rHu-EPO (300 IU/kg subcutaneously, daily from 10 days pre- through to 3 days postsurgery) reduces the red cell transfusion requirements of patients undergoing elective hip replacement [153]. Similarly, the allogeneic blood requirement was reduced by preoperative administration of rHu-EPO (5 x 500 IU/kg intravenously, started 2 weeks before surgery) in a controlled trial in patients undergoing elective open-heart surgery [154]. In contrast, preoperative rHu-EPO treatment failed to reduce the transfusion frequency in anemic patients undergoing colorectal cancer surgery [155,156].

     Preoperative rHu-EPO treatment may be indicated particularly in patients with hypoproliferative anemia, i.e. the anemia of chronic disorder, if these are unable or unwilling to receive autologous or allogeneic blood components. A mathematical evaluation has shown that the efficacy of preoperative rHu-EPO therapy on the postoperative hematocrit is related to the patient's blood volume and to the volume of blood lost during surgery [157]. According to this study rHu-EPO is most effectively used in patients with mild anemia who are undergoing routine surgical procedures that otherwise require red cell transfusion. Maximum benefit of rHu-EPO therapy can be achieved if it is combined with acute normovolemic hemodilution [157].

RHu-EPO abuse in sports

     The Medical Commission of the International Olympic Committee (IOC) has established rules to forbid unethical and potentially hazardous chemical, pharmacological and physical manipulations. The banned groups of drugs include stimulants, narcotics, anabolic steroids, diuretics and peptide hormones such as EPO. Recently, some sports federations have decided to include blood sampling in addition to urine collection in doping controls [158].

     In endurance sports – such as long-distance running, cycling and skiing – performance relies on an adequate O₂ supply of heart and skeletal muscles. Hence, the rate of maximal O₂ uptake (O₂max) is an important determinant of aerobic physical power. O₂max correlates with the O₂ carrying capacity of the blood. A legal method to improve performance is training at altitude, which may result in a moderate stimulation of erythropoiesis, a lowered O₂ affinity of red blood cells due to an increase in intraerythrocytic 2,3-bisphosphoglycerate levels, and an improved oxidative capacity of muscles [159-161]. A way outside of ethical medical practice is the transfusion of blood to improve endurance performance of athletes during training and competition [162].

     Since rHu-EPO became available as an erythropoiesis-stimulating drug, it has been imputed to be abused by athletes in aerobic sports [163,164]. The performance enhancing (ergogenic) effect of this application is documented [165].

     There is suspicion that rHu-EPO-induced erythrocytosis caused the deaths of 18 world-class Dutch and Belgian cyclists [164], although it was never proven that any of these received rHu-EPO. If hematocrit exceeds 0.50, blood viscosity and, hence, cardiac afterload increases significantly. In microvessels blood stasis may occur. The main risks of erythrocytosis with hematocrits > 0.55 include heart failure, myocardial infarction, seizures, peripheral thromboembolic events and pulmonary embolism. Endurance athletes are at increased risk during the competition, when blood viscosity increases further due to the great loss of fluid associated with sweating.

     (Table 8) summarizes the procedures that have been proposed for detection of abuse of rHu-EPO. In this author's mind, the demonstration of EPO with the typical physicochemical characteristics of the recombinant DNA-derived drug in urinary or blood specimens of athletes is the most appealing approach [51]. Samples should be collected on a random un-announced basis both in and out competition. Other blood variables which have been applied for monitoring rHu-EPO doping include serum EPO concentrations > 40 U/l in absence of anemia [166], increased serum soluble transferrin receptor levels [165,167], hematocrits ³ 0.48 in females and ³ 0.52 in males [168], > 5 % hypochromic red cells in absence of iron deficiency [169], and > 150 x 10⁹ reticulocytes per l blood [166]. The diagnostic value of these parameters is limited, however, if the drug is administered at repeated low doses and associated with an iron supplement [170]. Note, that the increase in urinary fibrin degradation products following rHu-EPO therapy [171] is a very unspecific indicator, because the pharmacological mechanism of this reaction is not yet known. The exclusion from competition of athletes with abnormally high hematocrit values (e.g. > 0.50; "Tour de France", 1999) may be justified on medical prophylactic grounds. By no means, however, it is proof for rHu-EPO doping, because hematocrits can clearly exceed this limit in unmanipulated male subjects [172].

 

Table 8.  Suggested Screening Methods for rHu-EPO Doping

 

Acknowledgement

     Thanks are due to Ms Beate Nürnberg and Ms Lisa Zieske for their diligent secretarial work. The author is supported by the Deutsche Forschungsgemeinschaft (SFB 367-C8).

Abbreviations

CAPD      =          Chronic ambulatory peritoneal dialysis

CHO        =          Chinese hamster ovary

EPO         =          Erythropoietin

EPOR      =          EPO receptor

G-CSF     =          Granulocyte colony-stimulating factor

HIF-1       =          Hypoxia inducible factor

IFN-g      =          Interferon g

IG            =          Immunoglobulin

IL             =          Interleukin

IU            =          International unit

O/P          =          Observed/predicted

rHu          =          Recombinant human

TNFa      =          Tumor necrosis factor a

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