Novel Erythropoiesis Stimulating Protein Research Articles

Detection of ESAs in equine urine and blood by SAR-PAGE.

AbstractErythropoiesis‐stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme‐linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel‐electrophoresis (SAR‐PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR‐PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimulating protein (NESP), continuous erythropoietin receptor activator (CERA), and fusion protein of erythropoietin with human immunoglobulin heavy chain Fc region (EPO‐Fc) in horse blood and urine. The purification technique for human blood using MAIIA kits worked well for horse samples. The major challenge was horse urine immunopurification, which proved difficult due to filter clogging, but heating and cooling of the horse urine followed by filtration in 30‐kDa molecular weight cut‐off filters solved this problem. The limits of detection (LODs) of 1.3, 1.6, 6.6, and 13.3 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, obtained in spiked urine and 40, 100, 80, and 400 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, acquired in spiked blood are lower than the LODs reported in the literature using liquid chromatography–mass spectrometry (LC–MS) methods. In addition, the presence of ESAs was detected up to 9 days after the administration of microdoses of Hemax (rHuEPO), NESP, and CERA in horse blood and urine. SAR‐PAGE may be implemented in the routine analysis of horse doping control laboratories for screening and confirmation of ESA abuse, mainly due to its high sensitivity for both matrices compared to published mass spectrometric methods.

The effects of erythropoiesis-stimulating agents on the management of chemotherapy-induced anemia and tumor growth in diffuse large B-cell lymphoma patients.

e19538Background: Erythropoiesis-stimulating agents (ESAs), such as erythropoietin (EPO) and novel erythropoiesis stimulating protein (NESP), may alleviate anemia in diffuse large B-cell lymphoma (...

EXPRESSION OF MODIFIED RECOMBINANT HUMAN ERYTHROPOIETIN IN CHO-K1 CELLS AND ITS IN VITRO PROLIFERATION ASSAY IN TF-1 CELLS

Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-α and epoetin-β, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIV-AIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-α (DARB) which possesses two additional N-linked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 N-linked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation. Key words: CHO-K1, erythropoietin, glycosylation, MTT assay and TF-1 cell line.

Partnering With The Industry: The Olympic Laboratory Model

David A Cowan and Campbell P Barker speak to Ryan De Vooght-Johnson at Bioanalysis in May 2012 about the partnership between industry and academia for the setup and running of the doping-control laboratory for the London 2012 Olympic Games. David A Cowan is Director of the London 2012 anti-doping laboratory as well as Director of the King's College London Drug Control Centre, the UK's only WADA-accredited anti-doping laboratory, and Head of the Department of Forensic Science and Drug Monitoring. Cowan co-founded the Drug Control Centre in 1978 and became its Director in 1990. He has published extensively in the field of pharmaceutical analysis, especially as it relates to detecting drug administration in sport, and was awarded a personal chair in pharmaceutical toxicology in 1996. Cowan became Head of the Department of Forensic Science and Drug Monitoring at King's College London in 2002. He has served on a number of national and international committees, including the Council of Europe Working Party Investigating Drug Abuse in Sport that led to the first World Anti-Doping Convention, the Laboratory Representative on the International Olympic Committee's Medical Commission, and WADA's Laboratory Accreditation Subcommittee. He is a member of the Crippen Club for Distinguished Toxicologists. In 1998 he was awarded the IOC Trophy for Sport Ethics by the BOA. He was a founding member of the World Association of Anti-Doping Scientists and became its first President serving on its Executive Board between 2001 and 2004. He was a Visiting Laboratory Director at the Salt Lake City Winter Olympic Games 2002, where the first novel erythropoiesis-stimulating protein (NESP) positive was discovered. He was also a senior advisory scientist at both the Turin Winter Olympic Games in 2006 and the Beijing Olympic Games in 2008. He was also a member of the IOC Medical Commission for the Sydney Olympic Games in 2000 and the Vancouver Winter Olympic Games in 2010. The Drug Control Centre undertook the sample analysis during the 2002 Manchester Commonwealth Games and Cowan was Co-Director of the laboratory for the Commonwealth Games in Kuala Lumpur in 1998. Cowan, who has directed the laboratory at King's College London for many years, was a member of the bid team making the presentation to the International Olympic Committee in February 2005. Campbell P Barker has been leading GlaxoSmithKline's delivery of the London 2012 laboratory services since September 2009. Prior to that time he was Director of Global Strategic Projects in GlaxoSmithKline's Consumer Healthcare business from 2006, and from 1994 to 2006 he worked in R&D for Procter & Gamble. Barker holds a PhD in chemistry from the University of Durham.

Current and Upcoming Erythropoiesis-Stimulating Agents, Iron Products, and Other Novel Anemia Medications

Treatment for anemia has come a long way in the last 20 years since the first recombinant human erythropoietins were licensed for the management of anemia in chronic kidney disease. The first-generation epoetins were succeeded by the development and production of a longer-acting erythropoietin (EPO) analog, darbepoetin alpha, which allowed less frequent dosing, usually once weekly or once every 2 weeks. More recently, another EPO-related molecule has been manufactured called Continuous Erythropoietin Receptor Activator with an even longer half-life, and although for patent reasons this is not available in the United States, it is licensed and is already being used in Europe. Other molecules are in development or are becoming licensed in Europe, including biosimilar epoetin products/follow-on biologics, and elsewhere in the world there are cheaper-production "copy" epoetins. Indeed, it is estimated that up to 80 such products may be sold in countries with less stringent regulatory control of pharmaceutical products. Two different biosimilar epoetins have already been licensed in Europe, one under 2 different brand names and one under 3 different brand names, and others may follow. Hematide is a synthetic peptide-based EPO receptor agonist that, interestingly, has no structural homology with EPO, and yet is still able to activate the EPO receptor and stimulate erythropoiesis. This agent is currently in phase III clinical trials. Research continues for orally active antianemic therapies, and several strategies are being investigated, although none is imminently available. Two new intravenous iron preparations have recently been developed, one in the United States (Ferumoxytol; AMAG Pharmaceuticals, Inc., Cambridge, MA) and one recently licensed in Europe (ferric carboxymaltose [Ferinject; Vifor Pharma, Zurich, Switzerland]). In conclusion, the development of effective therapies for the treatment of anemia has been a highly active field, both scientifically and economically, over the last two decades.

Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis–mass spectrometry

In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis–mass spectrometry (IA-CE–MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77–97) and NESP (77–97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE–MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81–95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81–95), only the proteotypic peptide EPO (77–97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE–MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.

Analysis of intact erythropoietin and novel erythropoiesis‐stimulating protein by capillary electrophoresis‐electrospray‐ion trap mass spectrometry

Recombinant human erythropoietin (rHuEPO) and novel erythropoiesis-stimulating protein (NESP) were analyzed by CE-ESI-MS using an IT as analyzer. The IT parameters were optimized by direct infusion of solutions of different intact proteins (myoglobin, transferrin, alpha1-acid glycoprotein and fetuin) with different degrees of glycosylation (from 0 to 35% w/w). Two physically adsorbed capillary coatings from UltraTol Pre-Coats (low normal (LN) and high reverse (HR)) were evaluated for the separation of rHuEPO and NESP glycoforms by CE-ESI-IT-MS. The results obtained with the neutral LN coating suggest that an IT mass spectrometer enables identification of the main glycoforms of a complex glycoprotein such as rHuEPO. Although LN provided acceptable glycoform resolution for rHuEPO, the separation obtained for NESP was less significant due to the higher microheterogeneity of this glycoprotein. Reproducibility studies confirmed the lack of stability and bleeding of the LN coating, which caused problems with MS detection, such as a dramatic loss of sensitivity and the presence of peaks in the mass spectra corresponding to molecular ions in the coating. In contrast, the cationic HR coating gave faster but poorer glycoform separations due to the presence of an anodal EOF. However, the positive charge of the coating provided enhanced hydrolytic stability, making it more suitable than the LN coating for the on-line MS coupling.

Angiogenic factors in preeclampsia: so complex, so simple?

Hoechst.htm 23. Cointe D, Beliard R, Jorieux S et al. Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties. Glycobiology 2000; 10: 511–519 24. Shahrokh Z, Flatman S, Davies M, Baycroft A, Heartlein M. Erythropoietin produced by a human cell line has only trace levels of potentially immunogenic N-glycolylneuraminic acid residues. Haematologica 2006; 91 [S1]: 208 (Abstract) 25. Noguchi A, Mukuria CJ, Suzuki E et al. Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. J Biochem (Tokyo) 1995; 117: 59–62 26. Tangvoranuntakul P, Gagneux P, Diaz S et al. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc Natl Acad Sci USA 2003; 100: 12045–12050 27. Nguyen DH, Tangvoranuntakul P, Varki A. Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells. J Immunol 2005; 175: 228–236 28. Kwan JT, Pratt RD. Epoetin-d, erythropoietin produced in a human cell line, in the management of anaemia in predialysis chronic kidney disease patients. Curr Med Res Opin 2007; 23: 307–311 29. Spinowitz BS, Pratt RD. Epoetin-d is effective for the management of anaemia associated with chronic kidney disease. Curr Med Res Opin 2006; 22: 2507–2513 30. Egrie JC, Browne JK. Development and characterization of novel erythropoiesis stimulating protein (NESP). Br J Cancer 2001; 84: 3–10 31. Elliott S, Lorenzini T, Asher S et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol 2003; 21: 414–421 32. Jelkmann W. The enigma of the metabolic fate of circulating erythropoietin (Epo) in view of the pharmacokinetics of the recombinant drugs rhEpo and NESP. Eur J Haematol 2002; 69: 265–274 33. MacDougall IC. Novel erythropoiesis stimulating protein. Semin Nephrol 2000; 20: 375–381 34. Wish JB. The economic realities of erythropoiesis-stimulating agent therapy in kidney disease. Kidney Int 2006; 70: S21–S25 35. MacDougall IC. CERA (Continuous Erythropoietin Receptor Activator): a new erythropoiesis-stimulating agent for the treatment of anemia. Curr Hematol Rep 2005; 4: 436–440 36. SteadRB, Lambert J,WesselsD et al.Evaluation of the safety and pharmacodynamics of Hematide, a novel erythropoietic agent, in a phase 1, double-blind, placebo-controlled, dose-escalation study in healthy volunteers. Blood 2006; 108: 1830–1834 37. Kochendoerfer GG. Site-specific polymer modification of therapeutic proteins. Curr Opin Chem Biol 2005; 9: 555–560

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87-90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81-95), NESP (81-95), and NESP (86-104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide-OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81-95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84-106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86-91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.

Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine

IEF can be used to differentiate human urinary erythropoietin (uEPO), recombinant human erythropoietin or epoetin (rEPO) and darbepoetin (novel erythropoiesis stimulating protein (NESP)). This is the basis of the method currently used to detect misuse of rEPO and NESP by elite athletes. Recently, an unknown activity has been attributed to some urine samples (denominated 'unstable' urine by the World Anti-Doping Agency; WADA). This activity has shown to give rise to artefactual profiles for both rEPO and NESP when incubated with such urine and, thus, raised concerns with respect to doping control. We have evaluated which charges produce the characteristic IEF profiles of uEPO, rEPO and NESP and how these profiles respond to distinct enzymatic reactions. From sialidase digestions it became evident that only uEPO contains charges different from sialic acid, and a comparison of all substances after complete de-N-glycosylation localized these charges in the carbohydrate moiety. Partial desialylation, or digestion with arylsulfatase from Helix pomatia yielded profiles for recombinants species similar to those observed for unstable urine samples. The contributions from our studies to the anti-doping problem include: (i) protocols that may corroborate the potential misuse of rEPO or NESP based on the particular enzymatic activity of an arylsulfatase preparation, or a broad-specificity sialidase; (ii) assurance that the instability observed in some urine samples may only result from false-negatives, but not from false-positive testing; and (iii) a simple remedy to prevent an unstable urine from altering the IEF profile by adding selective competitive substrates.

Darbepoetin Alfa assessment in chemotherapy induced anemia

18617 Background: Anemia is a very common complication of cancer and its treatment. Epoetins have been proven effective in correcting anemia in cancer patients resulting to the reduction of the need for blood transfusions and having a positive impact to the quality of life (QoL). Darbepoetin alfa, is a novel erythropoiesis stimulating protein (NESP) with longer half life. Methods: This is a prospective, single-centre, clinical trial. Patients with haemoglobin < 12 g/dl, suffering from cancer (solid tumor or lymphoma), for whom chemotherapy is scheduled for at least 12 weeks and have life expectancy >6 months and performance status ECOG 0–2,are treated with Darbepoetin alfa 150 mg per week subcutaneously for at least 12 weeks. Full blood count should be tested at least every 2 weeks and biochemistry every 4 weeks. QoL was assessed before and after treatment using the FACT-An (Functional Assessment of Cancer Therapy-Anemia) scale. Primary endpoint is the correction of anemia (Hb value above 12 g/dl). Frequency of the demographic and clinical characteristics were reported for the categorical variables, while mean and standard deviation were reported for continuous variables, such as age and Hb levels. Results: Fifty-six (56) patients entered the study, 66% of them female. Median age was 64 years. The more common cancers were colorectal (26.8%) ovarian (17.9%), breast (16.1%), lung (12.5%), and bladder (8.9%). Two thirds of the patients had not received previous chemotherapy. A statistically significant increase for Hb was found between week 0 and 12 [1.48; 95% CI 1.07–1.88; p < 0.001]. The increase was significant from 3rd week onwards. There was no evidence of significant difference in any of the QoL components or for significant change in mean fatigue sub-scale (fatigue, non-fatigue), between the two assessments. Conclusions: Darbepoetin alfa given at 150 mg per week subcutaneously to cancer patients under chemotherapy appears to correct anaemia, but has not positive impact on QoL, within the limits of the sample size of this study. No significant financial relationships to disclose.

Cellular Trafficking and Degradation of Erythropoietin and Novel Erythropoiesis Stimulating Protein (NESP)

Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) = 5.0 x 10(8) m(-1) min(-1) versus 1.1 x 10(8) m(-1) min(-1)) but dissociates slower (k(off) = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level.

Characterization of N‐ and O‐glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI‐TOF and quadrupole IT/RTOF mass spectrometry

The structural characterization of the O- and N-glycan structures of three different commercially available recombinant human erythropoietins (rhEPOs) is represented by means of a microscale sample purification using ZipTip technology and MALDI-TOF and MALDI low-energy CID MS. Glycopeptides were released from rhEPO samples by a differential endoproteolytic digestion to obtain site-specific glycosylation patterns. Mass accuracies in the range of +/- 0.04% obtained by the high-resolution TOF instrument allowed an unambiguous assignment of N-glycan structures via glycan database software. Furthermore, the O-glycan structures were directly analyzed on the glycopeptide level by MS/MS experiments. Principally, site-specific glycosylation was found to be very similar for the three different rhEPOs (EPO-alpha, EPO-beta, and novel erythropoiesis stimulating protein (NESP)) but exhibiting quantitative differences in distinct O- and N-glycan moieties. Significant differences were found in the degree of sialylation and acetylation. Especially, a considerable degree of variation of the O-acetylation of sialic acid residues could be realized on the glycan structures of O- and N-glycopeptides, whereas EPO-alpha and EPO-beta could be clearly differentiated from NESP solely on the O-glycopeptide level.

Capillary electrophoresis and matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry for analysis of the novel erythropoiesis‐stimulating protein (NESP)

NESP (novel erythropoiesis-stimulating protein) is a recently approved hyperglycosylated analogue of human erythropoietin (EPO) with a long-lasting effect. In this work, the capillary electrophoresis (CE) methodology proposed by the European Pharmacopoeia for the separation of EPO glycoforms has been modified for the separation of NESP glycoforms. Optimization of pH of the separation electrolyte has been fundamental in order to achieve baseline resolution of seven peaks corresponding to NESP glycoforms. Intact NESP has also been characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). An accurate approximation to an average molecular mass of the NESP molecule has been obtained, taking into account the strong influence of laser intensity upon the MALDI-TOF mass spectra found.

Exercise performance in hypoxia after novel erythropoiesis stimulating protein treatment

Maximal aerobic power at high altitude (<4000 m) does not increase with altitude acclimatization. In order to investigate the isolated effects of increased arterial oxygen content (CaO2) on maximal oxygen uptake (VO2max) in hypoxia, we studied 10 subjects during exercise in acute exposure to 12.6% O2 before and after novel erythropoiesis stimulating protein (NESP) induced increases in CaO2. Over a period of 1 month, weekly NESP treatment increased resting hemoglobin (Hb) from 13.8+/-0.9 to 16.2+/-0.5 g/dL, hematocrit from 42.1+/-0.6% to 49.0+/-1.5%, and CaO2 from 189.7+/-3.0 to 218.6+/-5.7 mL/L. At maximal exercise CaO2 was increased from 172.3+/-3.7 to 191.5+/-3.8 mL/L with NESP treatment, and although maximal heart rate was similar in both conditions (178.4+/-2.6 and 180.9+/-2.5 b.p.m.) VO2max remained unaltered, the values being 3.12+/-2.0 and 3.12+/-2.0, before and after NESP treatment, respectively. NESP-injections in human subjects causes Hb and accordingly CaO2 to increase both in normoxia and hypoxia. Despite increases in CaO2 at maximal exercise in hypoxia VO2max is not increased.

Management of Anemia: Erythropoietin Therapy or Red Blood Cell Transfusion?

SUMMARYErythropoietin therapy was approved for use as a blood conservation intervention beginning in 1989 for patients with medical anemia and in 1997 for surgical patients. The adoption of this strategy has been rapid in some settings (e.g., renal failure patients), progressive in others (e.g., cancer patients), and slow in others (e.g., surgery patients). At the same time, the risks of blood transfusion have declined substantially whereas the costs of blood transfusion have increased significantly. Issues regarding the risks and costs associated with blood transfusion have led to a reevaluation of clinical transfusion practices. The evolution of new techniques such as acute normovolemic hemodilution (ANH) and the novel erythropoiesis‐stimulating protein (NESP) bring new options to allogeneic blood transfusion. Erythropoietin therapy, with or without autologous blood procurement, is undergoing new scrutiny as an alternative to blood transfusion. This is not only because of traditional concerns regarding blood risks but because of new blood inventory and cost considerations.

Development of surgery during the last 50 years

Doping with darbepoetin-α, also termed novel erythropoiesis stimulating protein (NESP), a hypersialylated, very effective analogue of erythropoietin, is a serious threat in sport. We report here on a new principle for the detection of darbepoetin-α in serum based upon increase in immunoactivity after desialylation with neuraminidase. The method is evaluated on sera from patients taken 2-14 days after last injection of darbepoetin-α.Thirty-two venous blood samples and 3 capillary samples taken from finger tips were obtained from 13 patients with end stage renal disease treated with intravenous or subcutaneous injections of Aranesp, 0.45 to 2.60 μg*kg-1. Blood samples from 37 individuals with endogenous erythropoietin were used as controls. The sera were diluted 1:2 with acetate buffer pH 5.6 with or without neuraminidase and incubated at 37°C for 1 or 24 h before immunoassay. The erythropoietin immunoactivity in serum volumes of 12.5-50 μL was measured with ELISA-kits from R&D Systems Inc and medac GmbH.The relative increase in immunoactivity after desialylation was in all cases higher for the darbepoetin-α samples than for any of the control samples assayed in parallel, varying incubation time with the enzyme, serum volumes and batches of both ELISA-kits. The mean relative increase in immunoactivity of endogenous erythropoietin after neuraminidase was 42% with the medac-kit and 117% with R&D-kit while the corresponding figures for darbepoetin-α were 282% and 231% with 1 h and 299% and 256% with 24 h enzyme incubation, respectively. Endogenous and recombinant human erythropoietin showed similar relative increase after desialylation.The method to detect darbepoetin-α in serum is simple to perform, robust, sensitive and requires a small amount of blood. The drug was detected in all patient sera taken 2-14 days after last injection. We suggest that the method should be evaluated for the detection of darbepoetin-α doping in sport.

The enigma of the metabolic fate of circulating erythropoietin (Epo) in view of the pharmacokinetics of the recombinant drugs rhEpo and NESP.

Recombinant human erythropoietin (rhEpo) is a mainstay in the treatment of anaemia, primarily in renal failure. Because the half-life of circulating rhEpo is relatively short (4-8 h), the drug is usually administered 2-3 times weekly. Recently, a novel erythropoiesis-stimulating protein (NESP) with a longer half-life (24-26 h) has been approved. NESP possesses two additional N-glycans compared to endogenous Epo or rhEpo. The pharmacokinetics of rhEpo and NESP in humans have been investigated in detail. The composition of the N-glycans is clearly important in determining the biological activity and the velocity of the degradation of Epo and its analogues. However, due to the lack of knowledge of the main site and mechanism of the removal of Epo from circulation, the difference in survival of rhEpo and NESP has remained phenomenological. Investigators have implicated the liver, kidneys, and bone marrow as possible sites of the catabolism of Epo. However, while hepatocytes take up desialylated Epo, the liver does not appear to play a major role in the degradation of intact Epo. Likewise, renal Epo clearance is apparently of secondary importance. Studies showing non-linear pharmacokinetics of Epo suggest that Epo is eliminated by saturable mechanisms. The hormone, as well as the recombinant drugs, can be incorporated by erythrocytic progenitors and other tissues expressing the Epo receptor. The affinity of the Epo receptor for rhEpo is 4.3-fold higher than for NESP. Taken together, it seems most likely that native Epo, rhEpo and NESP are degraded following Epo receptor-mediated uptake, mainly in bone marrow.

Management of Anemia: Erythropoietin Therapy or Red Blood Cell Transfusion?

SUMMARY Erythropoietin therapy was approved for use as a blood conservation intervention beginning in 1989 for patients with medical anemia and in 1997 for surgical patients. The adoption of this strategy has been rapid in some settings (e.g., renal failure patients), progressive in others (e.g., cancer patients), and slow in others (e.g., surgery patients). At the same time, the risks of blood transfusion have declined substantially whereas the costs of blood transfusion have increased significantly. Issues regarding the risks and costs associated with blood transfusion have led to a reevaluation of clinical transfusion practices. The evolution of new techniques such as acute normovolemic hemodilution (ANH) and the novel erythropoiesis-stimulating protein (NESP) bring new options to allogeneic blood transfusion. Erythropoietin therapy, with or without autologous blood procurement, is undergoing new scrutiny as an alternative to blood transfusion. This is not only because of traditional concerns regarding blood risks but because of new blood inventory and cost considerations.

Novel erythropoiesis stimulating protein for treatment of anemia in chronic renal insufficiency

Novel erythropoiesis stimulating protein (NESP) is a glycoprotein with a threefold longer terminal half-life than recombinant human erythropoietin (rHuEPO) in humans. The aim of this study was to determine whether NESP is effective for the treatment of anemia at a reduced dosing frequency relative to rHuEPO in patients with chronic renal failure not yet on dialysis [chronic renal insufficiency (CRI)]. This was a multicenter, randomized, open-label study. A total of 166 rHuEPO-naive patients with CRI were randomized in a 3:1 ratio to receive NESP (0.45 microg/kg once weekly) or rHuEPO (50 U/kg twice weekly) administered subcutaneously for up to 24 weeks. Dose adjustments were made as necessary to achieve a hemoglobin response, defined as an increase > or =1.0 g/dL from baseline and a concentration > or = 11.0 g/dL. During the 24-week treatment period, 93% (95% CI, 87 to 97%) of patients receiving NESP and 92% (95% CI, 78 to 98%) of patients receiving rHuEPO achieved a hemoglobin response. The median time to response was seven weeks (range of 3 to 25 weeks) in both groups. After correction of anemia, mean hemoglobin concentrations were maintained within the target range of 11.0 to 13.0 g/dL for the remainder of the 24-week treatment period. The safety profiles of NESP and rHuEPO were similar, and no antibodies were detected to either drug. These results demonstrate that NESP safely and effectively corrects and maintains hemoglobin concentrations at a reduced dosing frequency relative to rHuEPO in patients with CRI, providing a potential benefit to patients and health care providers.