Erythropoietin-induced rheological changes of rat erythrocytes.

The effects of recombinant human erythropoietin (rhEPO) on red blood cell (RBC) rheological properties were investigated in rats. Rats received intramuscular injections of 150 U/kg/d rhEPO for 5 d, following which blood samples were obtained 1, 5 or 10 d later. RBC deformability was assessed by determining cell transit times through 5-microm micropores (CTA) and RBC shape recovery time constants via photometry, aggregation in plasma and dextran was measured by photometry and RBC electrophoretic mobility was determined in a cylindrical electrophoresis system. RBC aggregation was found to be significantly decreased on day 5 after rhEPO treatment (P < 0.05), yet was unchanged from control on days 1 and 10. Mean RBC micropore transit times remained unchanged, but the distributions of transit times were altered; compared with control, the 5th percentiles on both days 1 and 5 were decreased and the 95th percentile on day 1 was elevated. Electrophoretic mobility of RBCs in phosphate-buffered saline was significantly increased on day 5 after rhEPO treatment (P < 0.05), with mobility measurements in dextran 500 (MW = 500 kDa) solutions suggesting that the cells' surface properties related to the formation of a 'depletion layer' may be altered on day 1. These results indicate that the rheological behaviour of RBC as a consequence of rhEPO treatment are temporal and are affected by the presence of reticulocytes as well as by the average age of the circulating cells.

Effects of erythropoietin on platelet reactivity and thrombopoiesis in humans.

A recent study in dogs suggested that erythropoietin (EPO) not only promotes the synthesis of increased numbers of reticulated platelets but that these newly produced platelets are hyperreactive compared with controls. Because of the increasing use of EPO in the perioperative setting, we characterized the effects of EPO on platelet reactivity in healthy human volunteers. In a randomized, controlled trial, we studied the effects of EPO on platelet reactivity, thrombopoiesis, and endothelial activation in circumstances similar to those of autologous blood donation. Thirty healthy male volunteers received placebo or EPO (100 or 500 U/kg of body weight given intravenously) three times a week for 2 weeks and underwent phlebotomy on days 8 and 15. Thrombin receptor-activating peptide induced expression of P-selectin, and CD63 increased 2- to 3-fold during EPO treatment. The enhanced platelet reactivity was also reflected by a 50% increase in soluble P-selectin in plasma. Plasma E-selectin levels increased in a dose-dependent fashion by more than 100% during EPO treatment, indicating substantial activation of endothelial cells. A 10% to 20% increase in platelet counts was observed in both EPO groups on day 5. In the placebo group, platelets increased only several days after the first phlebotomy. The increase in platelet counts was not reflected by changes in the amounts of reticulated platelets or circulating progenitor cells. In summary, we found that EPO markedly enhances endothelial activation and platelet reactivity, which may adversely affect patients at cardiovascular risk. However, the increased platelet reactivity could be exploited in patients with platelet dysfunction. (Blood. 2000;95:2983-2989)

Pseudo-reticulocytosis as a result of malaria parasites.

Recently fully automated methods for enumerating reticulocytes have become available as an integral function in routine haematology analysers. In such methods, all intraerythrocytic nucleic acid is stained and can be regarded as representing reticulocytes. It has previously been shown that Howell-Jolly bodies may be counted as reticulocytes in automated flow cytometric methods. In the present paper, data from two patients are described indicating that severe malaria infection may lead to falsely increased reticulocyte counts, at least in the CELL-DYN(R) 4000 haematology analyser. In this instrument, the intraerythrocytic nuclear material of the parasites will be stained and counted as reticulocytes. This phenomenon appears to be independent of the type of Plasmodium infection. Clinical haematology laboratories should be aware of this potential source of pseudo-reticulocytosis.

Recombinant erythropoietin in the prevention of late anaemia in intrauterine transfused neonates with Rh-haemolytic disease.

OBJECTIVE: To evaluate the efficacy of recombinant human erythropoietin (rHuEPO) in prevention of late anaemia due to Rh-haemolytic disease in neonates subjected to one or more intrauterine transfusions (IUTs). STUDY DESIGN: Six neonates (GA 28-38 weeks, BW 980-3,360 g), subjected to one or more IUTs for Rh-haemolytic disease, were treated for 3 weeks with rHuEPO (200 U/kg/day, s.c.) after the second week of life to prevent late anaemia and consequently reduce the need for blood transfusions. All treated neonates were supplemented weekly with iron, vitamin E and folinic acid, intramuscularly. RESULTS: Of the 6 patients studied, 4 preterm neonates, after commencement of rHuEPO treatment, showed a decrease in Hct values with persistent reticulocytopenia, and consequent need for one or more transfusions with packed and filtered red cells (PFRC). These 4 neonates had received a greater blood volume with IUTs than the 2 other term neonates, who, after starting rHuEPO treatment, showed an increase in Hct values and in reticulocyte count, with no transfusion requirements after birth (247 +/- 47 vs. 84 +/- 76 ml). CONCLUSIONS: Our results seem to correlate the efficacy of erythropoietin treatment in prevention of late anaemia resulting from Rh-haemolytic disease to the severity of intrauterine anaemia and to gestational age. Erythropoietin, in fact, was less effective in cases of severe intrauterine anaemia requiring a high volume of PFRC; it was also less effective in the preterm babies, because of the simultaneous presence of anaemia of prematurity and other major diseases.

Toxicity of dolastatin 10 in mice, rats and dogs and its clinical relevance.

PURPOSE: Dolastatin 10 (DOL 10), an oligopeptide isolated from the sea hare Dolabella auricularia, has been shown to be a highly potent cytotoxic agent in a variety of human tumor cell lines. The purpose of this study was to conduct preclinical toxicity evaluations to determine the target organ(s) of toxicity and its reversibility, the dose-limiting toxicity and the maximum tolerated dose (MTD), and to use this information for arriving at a safe starting dose and dose schedule for phase I clinical trails. METHODS: DOL10 was administered as a single intravenous bolus dose to CD2F1 mice, Fischer-344 rats and beagle dogs. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues. RESULTS: The MTD (i. e. the highest dose that did not cause lethality but produced substantial toxicity) was approximately 1350 microg/m(2) body surface area (450 microg/kg) in mice, 450 microg/m(2) (75 microg/kg) in rats and /=1350 microg/m(2) in mice, >/=150 microg/m(2) in rats and >/=400 microg/m(2) in dogs. Decreased weight gain or actual weight loss was observed at doses >/=1350 microg/m(2) in mice, >/=600 microg/m(2) in rats and >/=450 microg/m(2) in dogs. In all three species, the primary target organ of toxicity was the bone marrow, as indicated by decreases in the numbers of erythroid cells, myeloid cells, and megakaryocytes in the femoral bone marrow and by decreased white blood cell (WBC) and reticulocyte counts in peripheral blood. Marked neutropenia (i.e. >50% decrease compared to control animal or baseline values) was the principal effect on WBCs and occurred within a week of dosing. A mild anemia was evident 1 week after administering the drug to rats and dogs. The hematologic effects were transient and reversed by study termination. Other lesions at the MTD levels were cellular depletion and necrosis in lymphoid organs (rats and dogs), marked depletion of extramedullary hematopoietic cellular elements in the spleen (rats), thymic atrophy (mice and dogs), and minimal cellular necrosis in the ileum (rats). More extensive and severe pathology was observed in animals sacrificed in a moribund condition or found dead. CONCLUSIONS: Myelotoxicity was dose-limiting in all three species with mice being the least sensitive. In a phase I clinical trial, granulocytopenia was dose-limiting. Moreover, the MTD of DOL10 for rats and dogs is comparable to the human MTD. Therefore, the results from the preclinical toxicology studies correctly predicted a safe starting dose, the dose-limiting toxicity, and the MTD in humans.

Granulocyte colony-stimulating factor-supported combined immunosuppressive therapy (antilymphocyte globulin, cyclosporine, and methylprednisolone) in patients with aplastic anemia: tolerability, efficacy, and changes in the progenitor cell compartment.

Neutropenic infections are the major cause of morbidity and mortality in the treatment of aplastic anemia (AA) with antilymphocyte globulin (ALG), cyclosporin A (CSA), and methylprednisolone (MP). Recent data suggest a beneficial effect of administering G-CSF as an adjunct to immunosuppression. We have treated 11 consecutive patients with AA using a combined immunosuppressive regimen including ALG, CSA, and MP plus G-CSF at a dose of 5 microg/kg/day until neutropoietic recovery. In addition to measuring routine hematological parameters we have performed serial determinations of reticulocyte counts and in vitro progenitor cell cultures before and after therapy in order to assess their predictive value for treatment response and to determine the impact of therapy on early hematopoiesis. One patient died on day 34 of neutropenic septicemia. At 1 year, 81% of patients showed response to treatment. The median time to ANC values >0.5 and >1.0 x 10(9)/l were 19 and 35 days, respectively. Reticulocyte counts started to recover after 6 weeks, and transfusion independence was observed on day 52 for red blood cell transfusions and on day 53 for platelet concentrates. All patients with detectable colony formation in peripheral blood achieved a complete hematological remission, as compared with only one of five patients without progenitor cell growth. Although normal ranges were rarely achieved, there was a small but definitive improvement in progenitor cell numbers as compared with baseline values in most patients. Our results confirm the good tolerability and high efficacy of this G-CSF-supported combined immunosuppressive therapy for AA. Detectable colony growth at diagnosis seems to predict a high chance for complete hematological response.

Distribution characteristics of entrapped recombinant human erythropoietin in liposomes and its intestinal absorption in rats.

Recombinant human erythropoietin (Epo) is frequently administered by intravenous (i.v.) injection for the clinical treatment of renal anemia. Oral (per os; p.o.) administration is desired as an alternative route to i.v. administration, and liposomes have been chosen as a drug carrier. We found previously that after a p.o. administration to rats of Epo entrapped in liposomes before gel filtration, the Epo was absorbed, but variability in the number of days of appearance and in the levels of pharmacological effects, i.e. , the peak of circulating reticulocyte counts (RTC), was observed. The purpose of the present study was to examine the distribution characteristics of Epo in liposomes and intestinal absorption of liposomal Epo in rats by using purified Epo entrapped in liposomes after gel filtration (Epo/liposomes). The distribution characteristics of Epo/liposomes were determined by measuring the Epo in liposomes by a radioimmunoassay, high-performance liquid chromatography and zeta potential measurements. We observed that the protein part of Epo was mostly entrapped in liposomes, and was not adsorbed by the liposomal membrane at middle and high Epo p.o. doses, but the zeta potential of the Epo/liposomes increased negatively with the increase in the Epo p.o. doses. These results suggest that the sialic acid part of Epo entrapped in liposomes may project out from liposomes, depending on the entrapped Epo concentration. Little Epo was adsorbed or penetrated into liposomes when it was added to empty liposomes. After the p. o. administration of Epo/liposomes, the peak of RTC appeared at a 2-day delay on day 6, without variation and without dose dependency in comparison with that after i.v. administration. These results suggest that one of the reasons for the variability may be because the non-entrapped Epo and/or Epo/liposomes itself affected the intestinal absorption of Epo/liposomes. In conclusion, Epo/liposomes without nonentrapped Epo may be clinically useful for the oral administration of Epo.

Safety and efficacy of erythropoietin in children with chronic renal failure.

A prospective randomized study of the use of recombinant human erythropoietin (rHuEPO) in children with chronic renal disease was conducted to assess dosing requirements and side effects. Forty-four children with chronic renal failure, aged 4 months to 21 years, were studied. Twenty-five patients were pre dialysis, 10 on peritoneal dialysis, and 9 on hemodialysis. Patients received either 150 U/kg per week or 450 U/kg per week divided thrice weekly of rHuEPO for 12 weeks or until target hemoglobin (Hb) was attained. Dose was then adjusted to maintain a normal Hb. Eighty-two percent of patients reached target Hb by 7.9+/-5.6 weeks (mean+/-SD); 95% of patients in the high-dose group and 66% in the low-dose group reached target Hb within 12 weeks. The overall median rHuEPO dose at target Hb was 150 U/kg per week. Hemodialysis patients tended to require more rHuEPO to maintain a normal Hb (median 250 U/kg per week). Transfusion requirements and panel-reactive antibody levels decreased during the 12 weeks. Iron deficiency and/or hypertension occurred in 30% of children. In conclusion, rHuEPO at 150 U/kg per week is safe and effective in treating anemia in children with chronic renal disease.

Modulation of erythropoiesis in rat bone marrow erythroblastic islands by cyclooxygenase inhibition.

We designed our study to explore how the inhibition of prostaglandins (PGs) could affect erythropoiesis in bone marrow erythroblastic islands (EIs). To this end, we used hypoxic-stimulated rats-hypobaric hypoxia (42.55 kPa/6 h)-pretreated or not with indomethacin (4 mg/kg/3 days). Blood sampling was done at 0 h, 24 h, and 72 h after hypoxia. The study included estimations of the plasma erythropoietin (EPO) level (by radioimmunoassay), peripheral blood, number of EI from classes I to V per femur, rate of immature cell's differentiation into erythroblasts, and rate of repeated participation of macrophages in new EI reconstruction. Plasma EPO rose significantly (p < 0.01) in all hypoxic rats: 40.5+/-10.15 mU/ml and 46.75+/-16.28 mU/ml and at 0 h versus 13.83+/-6.82 mU/ml in controls. An increased rate of cell differentiation into erythroblasts in EIs (p < 0.01), an enhanced reconstruction in involuted EIs, and a reduced number of maturing EIs (p < 0.01) were observed in all hypoxic animals. However, in indomethacin-pretreated rats, the stimulation of bone marrow erythropoiesis was better expressed. Our results favor the concept that PG inhibition does not attenuate the erythropoietic response to hypoxia and support the hypothesis about the important role of EI macrophages as a local regulator of bone marrow erythropoiesis.

The effect of recombinant human erythropoietin on platelet counts is strongly modulated by the adequacy of iron supply.

The effect of recombinant human erythropoietin (rHuEpo) on megakaryopoiesis remains controversial. Treatment with rHuEpo in renal failure patients has been associated with a slight elevation of platelet counts. In animal studies, high doses of rHuEpo produced an increase of platelet counts followed by a gradual return to normal after 7 to 15 days or even a substantial degree of thrombocytopenia. However, because iron deficiency is also known to be associated with thrombocytosis, (functional) iron deficiency during rHuEpo could be contributing to these observations. We investigated the impact of iron supply on changes in platelet counts induced by rHuEpo. Rats were either fed normal food (normal rats) or received 1% carbonyl iron for 2 weeks or 3 months, as well as during the experiment, to achieve iron supplementation or overload, respectively. Rats of all three categories then received daily intravenous injections of rHuEpo (10, 50, or 150 U) or normal saline (0 U) for 20 days. With 0 to 10 U rHuEpo, platelets remained stable. In normal rats receiving 50 to 150 U rHuEpo, platelets increased to 120% to 140% of baseline at 4 to 12 days to level off at 120% at 16 to 20 days. This response was less sustained in splenectomized animals. Iron-supplemented rats receiving 50 to 150 U rHuEpo also increased platelets initially, but the peak was at day 4, followed by a gradual return to baseline and even a moderate thrombocytopenia later on. Iron-overloaded rats receiving 50 to 150 U rHuEpo also had increased platelets at day 4, but the duration of platelet increase was shorter, and they experienced a more pronounced degree of thrombocytopenia in proportion to the dose of rHuEpo. Because the early elevation of platelets was of larger magnitude than hematocrit changes, it is unlikely that it could be accounted for by shrinkage of plasma volume. Because it was observed in all three iron conditions, there appears to be some direct positive effect of rHuEpo on platelet production. However, after this transient effect, expanded erythropoiesis appears to exert a negative impact upon platelet production. Secondary thrombocytopenia was not related to splenic pooling, and its very slow correction after cessation of rHuEpo therapy is not compatible with changes in platelet survival. Rather, it is consistent with stem cell competition between erythroid and megakaryocytic development. However, this secondary thrombocytopenia is masked by (functional) iron deficiency in rats not receiving an adequate iron supply from food or stores.

Comparison of biological activity and safety of recombinant canine erythropoietin with that of recombinant human erythropoietin in clinically normal dogs.

OBJECTIVE: To determine whether recombinant canine erythropoietin (rcEPO) stimulates erythropoiesis in dogs without causing the immunogenicity problem (ie, erythroid hypoplasia) associated with recombinant human erythropoietin (rhEPO). ANIMALS: 13 clinically normal dogs. PROCEDURE: Dogs were randomly assigned to 2 groups; 1 group (n = 6) received rhEPO, whereas the other group (7) received rcEPO. Both groups received SC injections of diluent for 4 weeks before initiating treatment with erythropoietin (100 U/kg of body weight, SC, 3 times/wk). Hematocrit and absolute reticulocyte count were monitored weekly, CBC were done monthly, and bone marrow aspirates for cytologic evaluation were obtained before and at 4, 8, 16, and 24 weeks during treatment. RESULTS: Weekly mean Hct and absolute reticulocyte count increased in both groups of dogs during the first 2 weeks of treatment. For dogs receiving rhEPO, precipitous decreases in reticulocyte number and more gradual decreases in Hct were associated with development of erythroid hypoplasia. Dogs receiving rhEPO developed erythroid hypoplasia by week 4 (n = 4), 8 (1), or 16 (1). With cessation of rhEPO treatment after diagnosis of erythroid hypoplasia, RBC production recovered 5 to 11 weeks (median, 7 weeks) later. In contrast, rcEPO treatment caused sustained increases in Hct and reticulocytosis. None of the dogs receiving rcEPO developed erythroid hypoplasia. CONCLUSIONS: rcEPO stimulated erythrocyte production in clinically normal dogs during a 24-week period without causing the erythroid hypoplasia encountered in rhEPO-treated dogs. CLINICAL RELEVANCE: Because rcEPO did not cause erythroid hypoplasia, rcEPO may represent an improved option, compared with rhEPO, for treatment of erythropoietin-dependent anemia in dogs.

Reticulocytes and solvents: an epidemiological study.

OBJECTIVES: The aim of the study was to look for a rise in reticulocyte levels in workers exposed to various solvents, in comparison to non exposed control subjects. METHODS: A cohort of exposed workers was selected on the criterion of exposure to solvents, among employees regularly attending the Centre of Occupational Medicine of Molsheim (France) during the second trimester of 1995. Controls were selected over the same period from the voluntary blood donors of the Transfusion Centre of Strasbourg (France). Complete blood counts and flow cytometric reticulocyte counts were determined in all blood samples. RESULTS: Analysis of the haematological parameters displaying significant differences revealed the existence of higher levels of circulating reticulocytes and lymphocytes in workers exposed to solvents than in control subjects. These variations did not appear to depend on the intensity or length of exposure. CONCLUSIONS: This study demonstrates the occurrence of an increase in circulating reticulocytes in relation to occupational exposure to solvents, without as yet providing sufficient information to allow elucidation of the underlying mechanism. An increase in total circulating lymphocytes was observed in the same group of exposed workers. The concomitant rise in absolute values of these two elements of the blood counts is to our knowledge described here for the first time in an epidemiological study.

[Recombinant human erythropoietin: method of in vivo evaluation using normocythemic mice]. / Eritropoyetina recombinante humana: Método de valoración in vivo con ratones normocitémicos.

PURPOSE: The implementation of an in vivo assay to determine the biological activity of human recombinant erythropoietin (Hu-r EPO) is essential. The purpose of this study was to perform and optimize the conditions of an easy in vivo bioassay suitable for routine testing of quality control of Hu-r EPO preparations. MATERIAL AND METHODS: Normocythemic 8 weeks female mice treated with different Hu-r EPO doses were employed. The reticulocyte response was measured by flow cytometry and by visual count in a Neubauer cell count chamber, after selective red blood cell haemolysis. A unique subcutaneous injection with blood extraction 96 hours later was the schedule employed. The reticulocyte count measured by both methods was plotted against the log dose of Hu-r EPO. RESULTS: The dose-response curve obtained was linear between 5 and 160 UI/mouse and the doses chosen for future assays were 10, 30 and 90 UI/mouse. The use of at least 6 animals per dose and not less than 3 assays to obtain reliable limits according to international regulations is convenient. Thirty assays were performed in four different samples and were analyzed by parallel lines (3 + 3) relating the response with the log dose. The coefficient of correlation between both methods was 0.989, so they are equivalent. CONCLUSIONS: This method is suitable because fewer animals and bioassays are necessary to obtain fiducial limits according to international requirements. It is in agreement with the tendency to reduce the number of animals used for bioassay because ethical and economic reasons.

Subchronic and chronic toxicity of ingested 1,3-dichloropropene in dogs.

The potential toxicologic effects to dogs of 1,3-dichloropropene (1, 3-D), a soil fumigant used for the control of nematodes, were investigated. The 13-week subchronic toxicity study consisted of male and female beagle dogs (4/sex/dose group) given approximately 0, 5, 15, or 41 mg 1,3-D/kg body wt/day (approximately equivalent amounts of cis and trans isomers) via their diets. The 1-year chronic toxicity study consisted of male and female beagle dogs (4/sex/dose group) provided diets delivering approximately 0, 0.5, 2. 5, or 15 mg/kg body wt/day. The test material was stabilized in the feed by microencapsulation in a starch/sucrose matrix (80/20). In both the 13-week and the 1-year studies, the primary effect of 1,3-D in male and female dogs ingesting a dosage of >/=15 mg/kg/day was hypochromic, microcytic anemia. The anemia was regenerative, with increased erythropoietic activity characterized by polychromasia of erythrocytes and increased numbers of reticulocytes in peripheral blood. In the 13-week study, the anemia in dogs given 41 mg/kg/day progressively worsened over time, while the anemia in dogs given 15 mg/kg/day remained relatively constant between 42 and 90 days of dosing. Partial reversal of the anemia of high-dose animals occurred during a 5-week recovery period following the 13-week dosing regimen. In the 13-week study, terminal fasted body weights of males given 15 or 41 mg/kg/day were decreased 3 and 28%, respectively, and body weights of females given 5, 15, or 41 mg/kg/day were decreased 4.5, 12, and 24%, respectively, relative to controls. Males given 5 mg/kg/day for 13 weeks had no change in body weights relative to controls. In the 1-year study, the hypochromic microcytic anemia in dogs given 15 mg/kg/day remained relatively constant in severity between 3 and 12 months of treatment. Histopathologic alterations associated with anemia in the 1-year study consisted of increased hematopoiesis of the bone marrow and increased extramedullary hematopoiesis of the spleen. Body weights of males given 15 mg/kg/day were 5-12% lower than controls during the first 13 weeks of the study and 13-19% lower than controls during the remaining 9 months. Body weights of females given 15 mg/kg/day were 5-14% lower than controls over the majority of the dosing period. Males and females given 0.5 or 2.5 mg/kg/day for 1 year had no change in body weights relative to controls. A no-observed-effect level of 2.5 mg/kg/day was established for male and female dogs from the 1-year study.

Influence of erythropoietin treatment on hemoglobin A1c levels in patients with chronic renal failure on hemodialysis.

We studied the influence of erythropoietin (EPO) treatment on hemoglobin A1c (HbA1c) levels under conditions which eliminate the effect of changes in the blood glucose concentration. HbA1c levels, blood glucose, hematocrit (Hct) and reticulocyte counts were serially measured every two weeks after starting or stopping EPO administration in 15 non-diabetic hemodialysis patients. EPO treatment significantly influenced HbA1c levels, and the more erythropoiesis fluctuated by changing the dose of EPO, the more HbA1c levels changed, though there were no significant changes in blood glucose levels during the study period. The changes in HbA1c during the 2-week period correlated inversely with both the changes in Hct during the same 2 weeks and the reticulocyte counts at that time. We concluded that the change in Hct should be kept in mind when the HbA1c level is evaluated in EPO-treated patients and a formula should be proposed to correct HbA1c levels based on the change in Hct or the reticulocyte count.

Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin after single and multiple subcutaneous doses to healthy subjects.

OBJECTIVES: To understand the pharmacokinetic and pharmacodynamic properties of recombinant human erythropoietin (epoetin alfa) and to continue to optimize dosing regimens by determining whether administration of single high doses of epoetin alfa is as effective as repeated administration. METHODS: Epoetin alfa was administered as single subcutaneous doses of 300, 450, 600, 900, 1200, 1350, 1800, and 2400 IU/kg and in multiple subcutaneous dose regimens: 150 IU/kg 3 times a week for 4 weeks and 600 IU/kg once per week for 4 weeks in 2 open-label, randomized placebo-controlled studies in healthy volunteers. RESULTS: The absorption rate of epoetin alfa after subcutaneous administration was independent of dose, whereas clearance was dose-dependent in that it decreased with increasing dose. There was a linear relationship between response measured as percentage of reticulocytes area under the curve (AUC) and erythropoietin AUC for single doses up to 1800 IU/kg. Beyond the 1800 IU/kg dose, there was a saturation of response. The mean percentage of reticulocytes after single-dose regimens began to increase by days 3 to 4, reached their maximum at days 8 to 11, and returned to baseline values by day 22. In contrast, the mean percentage of reticulocytes after both multiple-dose regimens were maintained above baseline values through day 22 as both regimens stimulated modest but sustained increases in percentage of reticulocytes (1% to 2%). The mean percentage of reticulocytes AUC for 600 IU/kg epoetin alfa given once a week for 4 weeks was apparently greater than the mean percentage of reticulocytes AUC for 150 IU/kg 3 times a week for 4 weeks. Although daily oral iron supplementation was given, mean serum ferritin levels declined by approximately 75% through day 22 in subjects treated with multiple doses of epoetin alfa. CONCLUSIONS: These findings show that the pharmacologic response to epoetin alfa is a function of dose and dosing regimen. Repeated administration of epoetin alfa was more effective in stimulating a reticulocyte response than single-dose administration of the same total amount of epoetin alfa.

Expression and bioactivity of recombinant canine erythropoietin.

OBJECTIVE: To produce recombinant canine erythropoietin (rcEPO) and compare its biological activity with that of recombinant human EPO (rhEPO). ANIMALS: C57BL/6J mice. PROCEDURE: The gene encoding cEPO was isolated from a genomic library and subcloned into an eucaryotic expression vector. Production of rcEPO was achieved by stable transfection of the expression construct into Chinese hamster ovary cells. Biological activity was evaluated in vitro by analyzing the mitogenic activity of rcEPO on murine erythroid progenitor cells. In vivo bioactivity was assessed in mice by measuring the ability of rcEPO to increase blood reticulocyte counts. RESULTS: Size and glycosylation of rcEPO expressed in Chinese hamster ovary cells were similar to values for commercial rhEPO. Canine and human EPO stimulated proliferation of murine erythroid progenitor cells in vitro and murine reticulocytosis in vivo in a dose-dependent manner. CONCLUSIONS: Comparable biological activity was observed for rcEPO and rhEPO in the 2 murine-based assay systems studied. By avoiding interspecies variation in protein structure and the resulting potential for immunogenicity, rcEPO should represent a better option than rhEPO for treatment of dogs with erythropoietin-dependent anemia. CLINICAL RELEVANCE: Therapeutic use of rhEPO in companion animals is limited by its immunogenicity and the resulting potential to induce pure red cell aplasia. Development and availability of species-specific EPO preparations should avoid this problem.