Development and characterization of novel erythropoiesis stimulating protein (NESP).

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP), which was engineered to contain five N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximately 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

Development and characterization of novel erythropoiesis stimulating protein (NESP).

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP, ARANESP, Amgen Inc, Thousand Oaks, CA), which was engineered to contain 5 N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients.

Novel erythropoiesis stimulating protein (NESP) is a hyperglycosylated analogue of recombinant human erythropoietin (Epoetin) which has an increased terminal half-life in animal models. The aim of this study was to extend these observations to humans. Using a double-blind, randomized, cross-over design, the single-dose pharmacokinetics of Epoetin alfa (100 U/kg) and an equivalent peptide mass of NESP were compared following intravenous bolus in 11 stable peritoneal dialysis patients. This was followed by an open-label study to determine the single-dose pharmacokinetics of an equivalent peptide mass of NESP by subcutaneous injection in six of these patients. The mean terminal half-life for intravenous NESP was threefold longer than for intravenous Epoetin (25.3 versus 8.5 h), a difference of 16.8 h (95% confidence interval, 9.4 to 24.2 h, P = 0.0008). The area under the serum concentration-time curve was significantly greater for NESP (291.0 +/- 7.6 ng x h per ml versus 131.9 +/- 8.3 ng x h per ml; mean +/- SEM; P < 0.0005), and clearance was significantly lower (1.6 +/- 0.3 ml/h per kg versus 4.0 +/- 0.3 ml/h per kg; mean +/- SEM; P < 0.0005). The volume of distribution was similar for NESP and Epoetin (52.4 +/- 2.0 ml/kg versus 48.7 +/-2.1 ml/kg; mean +/-SEM). The mean terminal half-life for subcutaneous NESP was 48.8 h. The peak concentration of subcutaneous NESP was approximately 10% of that following intravenous administration, and bioavailability was approximately 37% by the subcutaneous route. The longer half-life of NESP is likely to confer a clinical advantage over Epoetin by allowing less frequent dosing in patients treated for anemia.

Hematocrit stability following intravenous versus subcutaneous administration of epoetin alfa to dialysis patients: a post hoc analysis.

UNLABELLED: BACKGROUND, SUBJECTS AND METHODS: A previous study of epoetin alfa dose requirements [Paganini et al. 1995] among hemodialysis patients who were switched from thrice weekly intravenous (i.v.) to thrice weekly subcutaneous (s.c.) administration showed that the weekly epoetin alfa dose requirement decreased by 18.5% after 13 to 16 weeks s.c. treatment and 26.5% after 21 to 24 weeks, without significant change in hematocrit. There was patient-to-patient variation in response, however, and 39% of the patients required the same or greater doses of epoetin alfa after the change from i.v. to s.c. administration. The present study reexamines the database to compare hematocrit stability between the two routes of administration. RESULTS: During 4 weeks of i.v. epoetin alfa administration, the pooled standard deviation (SD) for the patients' (n = 72) weekly hematocrit measurements was 1.40, compared with weeks 13 to 16 of s.c. epoetin alfa administration when the SD was 1.66 (p < 0.01). Among 41 patients who completed 24 weeks of s.c. therapy, the pooled SD for the 4 weeks of i.v. treatment was 1.37 compared with 2.02 during weeks 21-24 of s.c. treatment (p < 0.01). Sixty-eight percent of patients had lower hematocrit SD during 4 weeks of i.v. therapy than during the 4 weeks of s.c. therapy (p = 0.03). CONCLUSION: These data suggest that hematocrits may be more stable when epoetin alfa is administered i.v. rather than s.c. to patients on dialysis. These results would be expected since 100% of i.v.-administered epoetin alfa reaches the systemic circulation compared with 18% to 80% bioavailability of s.c.-administered epoetin alfa. Within-patient variation in s.c. epoetin alfa absorption may be related to non-uniformity of adipose tissue, blood supply, lymphatic drainage, and other factors at sequential injection sites, and may explain the variability in hematocrit after s.c. administration.

Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.

Efficiency of signalling through cytokine receptors depends critically on receptor orientation.

Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM, 10(-2) of its Kd value for erythropoietin-EPOR binding site 1 (Kd approximately equal to nM), and 10(-5) of the Kd for erythropoietin-EPOR binding site 2 (Kd approximately equal to 1 microM). Overall half-maximal binding (IC50) of cell-surface receptors is produced with approximately 0.18 nM erythropoietin, indicating that only approximately 6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses but much less efficiently, requiring concentrations close to their Kd values (approximately 0.1 microM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 A from two crystal forms, shows that erythropoietin imposes a unique 120 degrees angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.

NMR structure of human erythropoietin and a comparison with its receptor bound conformation.

The solution structure of human erythropoietin (EPO) has been determined by nuclear magnetic resonance spectroscopy and the overall topology of the protein is revealed as a novel combination of features taken from both the long-chain and short-chain families of hematopoietic growth factors. Using the structure and data from mutagenesis studies we have elucidated the key physiochemical properties defining each of the two receptor binding sites on the EPO protein. A comparison of the NMR structure of the free EPO ligand to the receptor bound form, determined by X-ray crystallography, reveals conformational changes that may accompany receptor binding.

The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and epoetin.

BACKGROUND: In patients with end-stage renal disease, anemia develops as a result of erythropoietin deficiency, and recombinant human erythropoietin (epoetin) is prescribed to correct the anemia partially. We examined the risks and benefits of normalizing the hematocrit in patients with cardiac disease who were undergoing hemodialysis. METHODS: We studied 1233 patients with clinical evidence of congestive heart failure or ischemic heart disease who were undergoing hemodialysis: 618 patients were assigned to receive increasing doses of epoetin to achieve and maintain a hematocrit of 42 percent, and 615 were assigned to receive doses of epoetin sufficient to maintain a hematocrit of 30 percent throughout the study. The median duration of treatment was 14 months. The primary end point was the length of time to death or a first nonfatal myocardial infarction. RESULTS: After 29 months, there were 183 deaths and 19 first nonfatal myocardial infarctions among the patients in the normal-hematocrit group and 150 deaths and 14 nonfatal myocardial infarctions among those in the low-hematocrit group (risk ratio for the normal-hematocrit group as compared with the low-hematocrit group, 1.3; 95 percent confidence interval, 0.9 to 1.9). Although the difference in event-free survival between the two groups did not reach the prespecified statistical stopping boundary, the study was halted. The causes of death in the two groups were similar. The mortality rates decreased with increasing hematocrit values in both groups. The patients in the normal-hematocrit group had a decline in the adequacy of dialysis and received intravenous iron dextran more often than those in the low-hematocrit group. CONCLUSIONS: In patients with clinically evident congestive heart failure or ischemic heart disease who are receiving hemodialysis, administration of epoetin to raise their hematocrit to 42 percent is not recommended.

Use of recombinant human erythropoietin for management of anemia in dogs and cats with renal failure.

OBJECTIVE: To test efficacy and safety of recombinant human erythropoietin (r-HuEPO) administration in dogs and cats with naturally developing chronic renal failure. DESIGN: Case series. ANIMALS: 6 client-owned dogs and 11 client-owned cats with chronic renal failure. PROCEDURES: r-HuEPO was administered intravenously or subcutaneously. Erythropoietic effects were monitored by determining CBC, performing cytologic examination of bone marrow aspirates, and measuring serum iron concentration before and during treatment. Development of adverse effects was monitored by performing sequential clinical assessments, CBC, and serum biochemical tests and by measuring indirect blood pressure and anti-r-HuEPO antibody titers. RESULTS: Administration of r-HuEPO increased RBC and reticulocyte counts, hemoglobin concentration, and Hct comparably in dogs and cats. Assessments of clinical well-being, including appetite, energy, weight gain, alertness, strength, and playfulness, were improved variably. Adverse effects, including anemia, anti-r-HuEPO antibody production, seizures, systemic hypertension, and iron deficiency, were demonstrated inconsistently in dogs and cats. CLINICAL IMPLICATIONS: Anemia contributes to clinical manifestations of chronic renal failure in dogs and cats. Administration of r-HuEPO has the potential to resolve anemia and improve clinical well-being. However, its administration poses risks of antibody production and adverse effects associated with correction of RBC mass. Use of r-HuEPO in dogs and cats requires conscientious assessment of risks and benefits until homologous forms of erythropoietin are available.

Epitope mapping and immunoneutralization of recombinant human stem-cell factor.

The epitope regions of three anti-[stem-cell factor (SCF)]g have been mapped by characterization of immunoreactivities against truncated forms of SCF in immunoblots and against synthetic peptides in solution-phase competition ELISA. Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61-95 and 95-110, respectively. The epitope of pAb 1337 has been mapped to residues 21-31. The ability of the anti-SCF Ig to recognize E. coli-derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied, mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured, mAb 7H6 was found to neutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61-65 and 91-95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implications for the structure of SCF.

Isolation and characterization of conformation sensitive antierythropoietin monoclonal antibodies: effect of disulfide bonds and carbohydrate on recombinant human erythropoietin structure.

We have isolated and characterized three anti-recombinant human erythropoietin (rHuEPO) monoclonal antibodies (MoAbs) that recognize nonoverlapping epitopes on rHuEPO. Anti-EPO MoAb D11 neutralizes rHuEPO activity whereas MoAbs F12 and 9G8A do not. This suggests that D11 may bind to the rHuEPO active site. MoAbs F12 and D11 recognize conformation dependent epitopes whereas 9G8A does not. Immunoassays were developed for each monoclonal. The 9G8A immunoassay was novel and useful because immunoreactivity increased when rHuEPO was denatured. Disruption of disulfide bonds or removal of carbohydrate increased 9G8A immunoreactivity, which suggests that these elements are important for rHuEPO structure or stability.

Intravenous versus subcutaneous dosing of epoetin alfa in hemodialysis patients.

Hemodialysis patients were studied to determine whether the dose of recombinant human erythropoietin (Epoetin alfa; Amgen Inc, Thousand Oaks, CA) required to maintain a therapeutic hematocrit level changed when the route of administration was switched from intravenously (IV) three times per week to subcutaneously (SC) three times per week. Thirteen to 16 weeks after patients were changed from IV three times per week to SC three times per week treatment, the Epoetin alfa requirement was reduced by 18.5% +/- 3.8% (P < 0.001; n = 72), and after 21 to 24 weeks of SC treatment the mean dosage had decreased from the IV dose by 26.5% +/- 4.2% (P < 0.001; n = 41). Sixty-one percent (44 of 72) of patients experienced maintenance-dose reductions over 13 to 16 weeks of treatment and 80% (33 of 41) were maintained on lower weekly doses after 21 to 24 weeks of treatment than at baseline (IV). There was interpatient variability, however: 26% of the patients required greater doses SC than IV following 13 to 16 weeks of SC treatment, and 15% required greater doses SC than IV following 21 to 24 weeks. On completing the initial SC three-times-per-week stage of the study, patients were randomized to one of three SC dosing strategies for an additional 12 weeks: (1) once per week, (2) three times per week Epoetin alfa diluted 1:2 with bacteriostatic saline to mitigate stinging at the injection site, or (3) continued three times per week with undiluted Epoetin alfa. Patients who were switched to administration of SC once per week undiluted Epoetin alfa (n = 20) had their total weekly dose lowered by 18.0% +/- 9.4% (P > 0.05), but the mean hematocrit for this cohort also decreased, from 34.3% +/- 3.0% to 32.4% +/- 3.9% (P > 0.05), rendering dose comparison between the two schedules ambiguous. The maintenance dose for patients who received Epoetin alfa diluted 1:2 with bacteriostatic saline (n = 23) did not differ from the undiluted three times per week dose at the end of stage 1. The third cohort of patients (n = 24), who continued to receive undiluted Epoetin alfa on the same SC three-times-per-week schedule, did not require a significant change in dosage over the ensuing 12 weeks. Comparison of SC three times per week mean dosage after an average of 32 weeks following the switch from IV three times per week for this latter cohort revealed a decrease of 23.5% +/- 6.5% (P < 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of recombinant human erythropoietin and interleukin-3 on erythropoietic recovery from acute anemia.

The risks inherent in the use of homologous blood products have increased efforts toward identifying alternatives to transfusion. We have previously shown that the administration of recombinant human erythropoietin (rhEpo) enhances the erythropoietic response to acute blood loss. Recombinant human interleukin-3 (rh-IL-3) is a hematopoietic growth factor that has been shown to act synergistically with rhEpo in accelerating erythropoiesis in vitro. The purpose of this study in a primate model was to determine if the administration of rhIL-3 in combination with rhEpo could augment the erythropoietic response to acute blood loss more than rhEpo therapy alone. Twenty-four adult male baboons were randomized into four groups. The induction of acute normovolemic anemia to a hematocrit of 20% was accomplished via exchange-transfusion with 6% hetastarch. The groups were then treated for 7 consecutive days with the following growth factors: group I (n = 7), no growth factors; group II (n = 5), rhIL-3 alone (100 micrograms/kg/d); group III (n = 6), rhEpo alone (1000 U/kg/d); group IV (n = 6), rhEpo (1000 U/kg/d) plus rhIL-3 (100 micrograms/kg/d). All animals received folate, vitamin B12, and intravenous iron-dextran immediately following the exchange-transfusion. Response to therapy was monitored for 35 days. There were no adverse reactions following growth factor administration. The analysis of erythropoietic rates between study days 1 through 11, as determined via linear regression analysis, revealed that hematocrits increased significantly faster in the groups receiving rhEpo compared to controls. The administration of rhIL-3, however, did not increase the rate of erythropoiesis when compared to controls, nor did it augment response when added to the rhEpo regimen. The results of this study demonstrate that the administration of rhIL-3 alone had no significant effect on erythropoiesis in this setting of acute blood loss. Further, despite promising in vitro data, rhIL-3 provided no additional stimulation of erythropoiesis in animals receiving rhEpo. Nevertheless, the study confirms that the pharmacologic acceleration of erythropoiesis by rhEpo alone remains an attractive alternative to homologous transfusion.

A comparison of the responses to recombinant human erythropoietin in normal and uremic subjects.

The erythropoietic response to graded doses of recombinant human erythropoietin (epoetin alfa) was assessed in 24 hemodialysis patients by quantitative ferrokinetic studies, and measurement of the reticulocyte count and plasma levels of transferrin receptor protein. These responses were compared to those of 22 normal subjects. Epoetin alfa was given intravenously at 15, 50 or 150 U/kg every other day for four injections. Three patients with chronic renal failure were restudied after renal function was restored following renal transplantation. The results of these three different measurements of erythroid function showed that the acute response to recombinant human erythropoietin was similar in normal subjects and patients with renal failure. We conclude that chronic uremia does not alter the responsiveness to erythropoietin in vivo.

Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor.

Stem cell factor (SCF) acts in concert with lineage-specific growth factors to stimulate the growth of hematopoietic colonies. To determine if neoplastic human hematopoietic cells would also respond to SCF, we cultured marrow mononuclear cells from 20 patients with newly diagnosed acute myelogenous leukemia (AML) and two normal donors with SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or combinations of growth factors in semisolid medium, and assessed colony growth. SCF receptors (c-kit receptors) were quantitated by equilibrium binding studies with 125I-SCF, and binding parameters were estimated using the ligand program. The cellular distribution of c-kit receptors was determined by autoradiography. Our results show that SCF alone or in combination with IL-3 or GM-CSF increased both the number and size of colonies in 10 of the patients. Receptors for SCF were identified on the blasts from all 20 AML patients. The number of receptors ranged from 600 to 29,000 per cell. In the majority of patients, both high- and low-affinity binding sites were identified. Neither the number of receptors per cell nor the finding of one or two classes of receptors correlated with growth response to SCF. Autoradiographic analysis of 125I-SCF binding to normal marrow mononuclear cells revealed grains associated with blasts and megakaryocytes. Grain counts on blasts from 10 AML patients and on normal marrow blasts suggested that high-affinity c-kit receptor expression on AML blasts is lower than or similar to that of normal blasts. These results identify c-kit receptors on human AML blasts, and indicate that SCF acts synergistically with IL-3 or GM-CSF to stimulate colony growth from the marrow cells of a portion of patients with AML.

Erythropoietin deficiency after coronary artery bypass procedures.

Erythropoietin is the primary regulator of erythropoiesis. Erythropoietin has been shown to increase exponentially in response to linear decreases in hematocrit in normal, unstressed animals. However, the effect of operation, with its attendant stress, on erythropoietin levels is unknown. The purpose of this study is to evaluate the effect of surgical stress on erythropoietin. Twenty otherwise healthy patients scheduled for elective surgical procedures were studied. The cholecystectomy group included 10 patients who underwent cholecystectomy for documented stone disease. Ten patients who underwent coronary artery bypass procedures constituted the coronary artery bypass grafting group. Patients were studied preoperatively as well as on the first and second postoperative days. The hematocrit and erythropoietin levels were similar in both groups preoperatively. The hematocrit in the coronary artery bypass grafting group was lower than that of the cholecystectomy group on postoperative day 1 (0.31 versus 0.36; p less than 0.003) and postoperative day 2 (0.30 versus 0.36; p less than 0.001). During the first two postoperative days the erythropoietin levels were similar between groups. The data show that postoperative erythropoietin levels are similar after coronary artery bypass grafting, despite more severe anemia, when compared with cholecystectomy. This suggests that after coronary artery bypass grafting there is a relative deficiency of erythropoietin. Administration of recombinant human erythropoietin to patients undergoing surgical procedures could correct the erythropoietin deficiency and accelerate postoperative erythropoiesis.

The erythropoietic effects of interleukin 6 and erythropoietin in vivo.

The purpose of this investigation was to compare the erythropoietic effects of recombinant interleukin 6 (IL-6) and recombinant erythropoietin (EPO) on the marrow and peripheral blood in vivo. IL-6 administered to rats as a single i.v. injection induces a selective erythroid hyperplasia of the marrow's late normoblasts at 12 and 24 h with a return to preinjection numbers of normoblasts at 48 and 72 h. The hyperplasia of late normoblasts in the marrow is accompanied by a left-shifted peripheral reticulocytosis. Daily injection of IL-6 does not induce any effects on the erythroid population of the marrow or circulation beyond those of a single injection. After daily administration of IL-6 for 4 or 7 days, the erythroid differential in the marrow and the peripheral reticulocyte count are equal to negative control values, indicating a rapid tachyphylaxis to the erythropoietic effect of IL-6. In contrast to IL-6, EPO administered as a single i.v. injection induces a panerythroid marrow hyperplasia with sequential peak increases in pronormoblasts and early normoblasts at 24 h, intermediate normoblasts at 24-48 h, and late normoblasts at 72 h. The peripheral reticulocyte count mirrors the development of erythroid precursors in the marrow by demonstrating an increasing left-shifted reticulocytosis between 24 and 72 h. Daily injection of EPO for 7 days induces a striking erythroid hyperplasia and a myeloid hypoplasia in the marrow. In summary, IL-6 in vivo is a differentiation factor that rapidly induces tolerance to its own effect, whereas EPO in vivo affects all stages of erythropoiesis and sustains erythropoiesis indefinitely. IL-6 may be one of the non-EPO factors in pokeweed mitogen spleen cell-conditioned medium that has been reported by previous investigators to enhance erythropoiesis, although many of those factors were thought to act upon an earlier stage of erythropoiesis. IL-6 is unlikely to exert an indirect erythropoietic effect in vivo via the induction of EPO because the sera of IL-6-treated rats did not contain elevated levels of EPO and because the effects of exogenously administered IL-6 and EPO are so different.

Recombinant human erythroid potentiating activity enhances the effect of erythropoietin in mice.

Endotoxin-free purified recombinant human erythroid potentiating activity (EPA) was administered to mice either alone or concomitantly with recombinant human erythropoietin (Epo). Epo treatment alone caused an increased hematocrit and reticulocyte count in the peripheral blood. In the spleen, the number of morphologically recognizable erythroid cells, CFU-E and BFU-E was also increased. Concomitant administration of EPA with Epo further enhanced erythropoiesis as judged by the same parameters. No significant effect on granulopoiesis was observed in association with Epo or combined Epo and EPA treatment. Our data indicates that EPA is active in vivo in augmenting the erythropoietic response to Epo.

Recombinant human granulocyte colony-stimulating factor increases circulating burst forming unit-erythron and red blood cell production in patients with severe human immunodeficiency virus infection.

Erythropoietin (EPO) is a major regulatory factor controlling red blood cell (RBC) production in humans. Although other humoral factors can alter the proliferation of committed early erythroid progenitors in vitro, no factor other than EPO has been clearly shown to induce proliferation of these cells in vivo. In a clinical trail of recombinant granulocyte colony-stimulating factor (G-CSF) and recombinant EPO in patients with advanced human immunodeficiency virus (HIV) infection, we noted reticulocytosis and increases in hemoglobin when G-CSF was administered before the administration of EPO. Subsequent studies demonstrated a significant increase in circulating burst forming unit-erythron (BFU-E) during daily recombinant G-CSF therapy. This increase was both time- and dose-dependent. The magnitude of increase in BFU-E correlated with the magnitude of increase in neutrophils and was associated with a mean increase in reticulocytes of 32,363/microL and a significant increase in mean hemoglobin of 1.04 +/- 0.34 g/dL over an 18-day interval. There was a significant increase in iron binding capacity and decreases in iron saturation and ferritin levels. In patients who were not recently transfused, there was an associated fall in endogenous erythropoietin levels. The increase in RBC production was most marked in patients who were severely anemic, transfusion-dependent, and who had elevated pretreatment EPO levels. There was no correlation between the increase in BFU-E and endogenous EPO levels or the time since last dose of zidovudine. The addition of recombinant EPO therapy three times weekly to patients did not result in further significant increases in BFU-E but did significantly increase hemoglobin. Our data suggest that recombinant G-CSF may be one of the hematopoietic factors that influences production of BFU-E and RBCs in humans.