Therapeutic effect of recombinant human erythropoietin on anaemia with erythropoietin deficiency in diabetic patients.

AIMS: The aim of this study was to investigate the therapeutic effect of recombinant human erythropoietin (rHuEpo) on anaemia with erythropoietin deficiency in diabetic patients. METHODS: Twenty diabetic patients with anaemia and Epo deficiency were enrolled. All patients were treated with rHuEpo (Epokine; 4000 U/day s.c., three times a week) for 8 weeks. RESULTS: The responder group (n = 14) had significant increments in haemoglobin compared with the non-responder group (n = 6) (P < 0.05). No significant differences were found between the responder and non-responder groups in terms of duration of diabetes mellitus, serum creatinine level, 24-h urine albumin excretion rates, frequency of diabetic microangiopathy, or HbA1c. There was no difference between the two groups in terms of serum iron and total iron-binding capacity (TIBC). Serum ferritin level was significantly higher in the responder group than in the non-responder group (240.3 +/- 108.4, 25.8 +/- 3.0 micro g/l, P < 0.05), as was transferrin saturation (32.7 +/- 7.9%, 21.2 +/- 5.3%, P < 0.05). CONCLUSIONS: rHuEpo could be useful in the treatment of anaemia with erythropoietin deficiency in diabetic patients, and the degree of iron storage and functional iron deficiency might be the main cause of hyporesponsiveness to rHuEpo.     

Effective treatment of the anaemia associated with multiple myeloma by recombinant human erythropoietin

We administered recombinant human erythropoietin to an anaemic patient with multiple myeloma (IgD, λ type) who had been dependent on blood transfusions. The recombinant human erythropoietin (60 units/kg) was given intravenously three times weekly and transfusion requirements, haemoglobin level, and reticulocyte responses were monitored. The patient had an increase in haemoglobin level and reticulocyte counts within 14 days, and no longer needed transfusions. No organ dysfunction or other toxic effects were observed. We consider that recombinant human erythropoietin may be a new method to treat anaemia associated with multiple myeloma.     

Correction of severe anaemia using immuno-regulated gene therapy is achieved by restoring the early erythroblast compartment

Patients with chronic renal failure usually require exogenous erythropoietin (epo) to alleviate anaemia resulting from inadequate epo production by the kidneys. We have recently shown that severe anaemia in genetically manipulated epo-deficient mice (EpoTAg) can be corrected by adoptively transferred epo-producing lymphocytes. The aim of this study was to investigate the precise effects of human epo administration by this route on erythropoietic development in epo-deficient mice. The erythroblast compartments of untreated and treated EpoTAg mice were analysed in comparison with wild-type mice. The early erythroblast population was reduced in the bone marrow of epo-deficient mice, whilst the number of erythroid colony-forming units (CFU-E) was not significantly compromised. This paucity in marrow early erythroblasts was restored to normal values in treated mutant mice. In addition, the early erythroblast population was expanded in the spleens of treated animals. These findings show that the early erythroblasts are important targets of epo and that epo corrects anaemia of epo-deficient mice by restoring marrow function and splenic erythropoiesis.     

Autoantibodies to erythropoietin receptor in patients with immune‐mediated diseases: relationship to anaemia with erythroid hypoplasia

The prevalence, clinical associations and pathogenic role of newly identified autoantibodies to the erythropoietin receptor (EPOR) in patients with anaemia were investigated. Sera from 203 patients with immune‐related or chronic kidney diseases were screened for anti‐EPOR antibodies by enzyme‐linked immunosorbent assay, and antibody specificity was evaluated by immunoprecipitating EPOR from AS‐E2 cells using purified immunoglobulin (Ig) fractions. In addition, the pathogenic role of anti‐EPOR antibodies was determined by examining their inhibitory effects on AS‐E2 cell proliferation. Clinical findings were compared between patients with and without anti‐EPOR antibodies, in all patients and those with systemic lupus erythematosus (SLE). Serum anti‐EPOR antibodies were detected in 52 patients. Purified IgG or IgM fractions from anti‐EPOR antibody‐positive sera immunoprecipitated EPOR and inhibited the EPO‐dependent proliferation of AS‐E2 cells in a dose‐dependent manner. Anti‐EPOR antibodies were associated with low haemoglobin concentrations and reticulocytopenia in all patients enrolled and those with SLE. Further, there was a negative correlation between the levels of anti‐EPOR antibodies and the number of bone marrow erythroblasts in patients who underwent bone marrow examinations. These findings suggest that EPOR autoantibodies are present in a subset of patients with anaemia and that impaired erythropoiesis can be mediated by anti‐EPOR antibodies, which functionally neutralize EPO activity.     

A phase I/II trial of recombinant human interleukin-6 in patients with aplastic anaemia

Summary. In a phase I/II study, 11 patients with marrow failure (10 with acquired aplastic anaemia and one with pancytopenic Fanconi anaemia) were treated with recombinant human interleukin-6 (rhIL-6) to assess the safety and tolerability of rhIL-6 and its effects on peripheral blood counts, bleeding complications and transfusion requirements. All patients with acquired aplastic anaemia were refractory to immunosuppressive treatment or had relapsed after immunosuppressive therapy and were not bone marrow transplantation candidates. Recombinant hIL-6 was to be given as a once-daily subcutaneous injection for 28 d at doses ranging from 0-5 to 5-0βg/kg. After an observation period of 2 weeks, five patients received a second treatment course of 28 d.
Only one patient had a sustained increase in platelet count from 18 000 to 72 000/ fA. Bleeding occurred in four patients and caused premature discontinuation of rhIL-6 therapy in three patients. A deterioration of pre-existing anaemia was observed in nine patients. No significant changes of leucocyte counts were observed during the first cycle. During the second cycle the peripheral blood monocyte counts decreased significantly. No significant changes in bone marrow cellularity were observed. Recombinant hIL-6 induced a dose-dependent increase in acute-phase reactants in all patients. Other adverse events included fever, headache, arthralgia, tachycardia and hypertension. In conclusion, rhIL-6 given alone at low doses does not increase platelet counts in the majority of patients with aplastic anaemia and can precipitate a sudden worsening of pre-existing anaemia and thrombocytopenia. This study was discontinued prematurely on account of the toxicity of rhIL-6 seen in patients with aplastic anaemia.     

The effect of intravenous iron on the reticulocyte response to recombinant human erythropoietin

We studied the effect of intravenous (i.v.) adminisration of 200 mg of iron sucrose following an i.v. bolus injection of recombinant human erythropoietin (r-HuEPO; 300 U/kg body weight) in seven subjects and compared it with seven subjects treated with r-HuEPO alone. Reticulocytes, serum erythropoietin (EPO) and ferritin levels were studied at baseline and daily for the following 8 d. Use of i.v. iron abolished the marked reduction in serum ferritin observed with r-HuEPO administration. Although the total number of reticulocytes was not affected by i.v. iron administration, the reticulocyte Hb content and retHb (a measure in g/l of the Hb contained in all reticulocytes) were increased in the i.v. iron/r-HuEPO group compared with the group who received r-HuEPO alone. Therefore i.v. iron significantly potentiates the haemopoietic response to r-HuEPO in normal subjects.     

Pharmacokinetics of intravenous recombinant human erythropoietin in patients with chronic renal failure

In order to improve our understanding of the dose-concentration and concentration-effect relationships, the pharmacokinetics of recombinant erythropoietin were studied after the initial dose (n = 6) and after repeated doses (n = 9) administered intravenously in patients with chronic renal failure. Several venous blood samples were collected before (to obtain the baseline concentration) and after an intravenous dose of erythropoietin. A radioimmunoassay was used to determine the erythropoietin concentration in the samples. The apparent volume of distribution at steady state was 4.2 +/- 0.91 (initial dose) and 3.7 +/- 0.61 (repeated dosing), which is close to the assumed plasma volume in these patients. The half-life was 5.3 +/- 1.3 h and 5.8 +/- 1.2 h in the two groups, respectively, and is therefore too short for any accumulation to be expected when dosing three times per week. Consequently, no difference in baseline values could be detected between the groups. The clearance of erythropoietin in the groups was estimated to be 11.4 +/- 7.0 ml min-1 and 7.8 +/- 3.8 ml min-1, respectively. Erythropoietin kinetics did not differ after repeated dosing compared to the single initial dose. Intravenous administration of erythropoietin will result in high peak concentrations followed by a rapid decline to basal values.     

Recombinant human erythropoietin for the treatment of the anaemia associated with autologous bone marrow transplantation

Summary. Patients with solid tumours undergoing highdose chemotherapy with autologous bone marrow transplantation use an average of 10 units of packed red blood cells (PRBC) while awaiting haemopoietic reconstitution. They are also known to have inappropriately low endogenous erythropoietin levels for their degree of anaemia. This pilot study was designed to determine the effects of recombinant human erythropoietin (rHuEPO) on erythroid recovery and PRBC transfusion requirements. Ten patients received high-dose chemotherapy (days - 7 to -3), bone marrow reinfusion (day 0), and then rHuEPO (day 1 onward). RHuEPO (200 units/kg intravenous bolus daily), along with iron supplementation, was administered for 28 d or until a haematocrit (Hct) of 35% (independent of transfusions) was reached, whichever occurred first. PRBCs were routinely given for Hct 25% and platelets for counts < 20000/μl. Eight (80%) patients developed a brisk reticulocytosis (median peak reticulocyte count 0.32 × 10⁹/l) and a haematocrit 30% independent of red blood cell transfusions within 32 d of receiving marrow, as compared to 20/37 (54%) similarly treated controls. An unexpected finding was the more rapid engraftment in myeloid and platelet lineages in a subset of rHuEPO-treated patients. Quick return of red blood cells (17 v 33d) (P = 0.0001), platelets (14 v 19d) (P = 0.04), and neutrophils (13 v 25d) (P = 0.01) (with circulating myeloblasts and early myeloid forms) characterized recovery from an ifosfamide-based intensification with rHuEPO support. Similar trilineage enhancement of haemopoiesis did not occur with the possibly more myeloablative cyclophosphamide-based regimens. Despite the enhancement by rHuEPO on reticulocytosis, there was no significant decrease in PRBC transfusion requirements. RHuEPO proved to be a well-tolerated agent in enhancing reticulocytosis following high-dose chemotherapy. further study to elucidate the activity of erythropoietin on both erythroid and non-erythroid growth and maturation appears warranted.     

Serum erythropoietin concentration in anaemia of visceral leishmaniasis (kala-azar) before and during antimonial therapy

Serum erythropoietin (Epo) concentrations and variables of red cell and iron status were studied in 27 Sudanese patients who were treated with sodium stibogluconate for visceral leishmaniasis (kala-azar). Blood haemoglobin increased from 6.4 (±1.7 SD) to 9.5 (±1.4) g/dl during treatment. Serum ferritin decreased concomitantly. Serum iron levels were unchanged whereas the total iron binding capacity increased slightly. The pre-treatment serum Epo concentration in relation to the blood haemoglobin concentration was not as high as expected from the one in primary haematological diseases, indicating that there is a relative lack of Epo in anaemic kala-azar patients. Serum Epo further decreased during stibogluconate therapy. The normal dependence of the serum Epo level on the blood haemoglobin concentration was lost during mid-term antimonial treatment, but it recovered thereafter. Cell culture studies with the human hepatoma cells HepG2 showed that stibogluconate ( 30 μg/ml) inhibited Epo gene expression. Thus, effective treatment of kala-azar with stibogluconate results in improvement of anaemia, although the drug itself may impair Epo production.     

Growth differentiation factor 15 in anaemia of chronic disease,iron deficiency anaemia and mixed type anaemia

Recently, the iron and erythropoiesis‐controlled growth differentiation factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in β‐thalassaemia patients, thereby increasing iron absorption despite iron overload. To access the diagnostic and pathogenic impact of GDF15 in inflammatory anaemia the association of GDF15 expression with serum iron parameters and hepcidin was studied in patients suffering from iron deficiency anaemia (IDA), anaemia of chronic disease (ACD) and ACD subjects with true iron deficiency (ACD/IDA). GDF15 was significantly increased in both ACD and ACD/IDA, but not in IDA subjects as compared to controls. In contrast, hepcidin levels were significantly lower in IDA and ACD/IDA subjects than in ACD patients . IDA and ACD/IDA, but not ACD, showed an association between GDF15 and soluble transferrin receptor, an indicator of iron requirement for erythropoiesis. However, GDF15 did not correlate to hepcidin in either patient group. While GDF15 levels were linked to the needs for erythropoiesis and iron homeostasis in IDA, the immunity‐driven increase of GDF15 may not primarily affect iron homeostasis and hepcidin expression. This indicates that other ACD‐related factors may overcome the regulatory effects of GDF15 on hepcidin expression during inflammation.     

Iron metabolism in transgenic mice with hypoplastic anaemia due to incomplete deficiency of erythropoietin

Iron overload is a serious complication of many forms of anaemia, arising in part from mechanisms associated with compensatory increases in erythropoiesis. To investigate other mechanisms by which anaemia itself may perturb iron metabolism, without the confounding effects of compensatory erythropoiesis, we studied transgenic mice with a partially disabling insertion in the erythro-poietin gene, which manifested as incomplete erythropoietin deficiency.
Mice were studied aged 7–8 weeks. Haemoglobin concentrations were 6.6 ± 0.8 g/dl in mice homozygous for the modified erythropoietin gene, 12.9 ± 2.2 g/dl in heterozygous mice and 14.1 ± 1.0 g/dl in controls. Homozygous mice showed significant hepatic iron loading (2-fold increase in liver non-haem iron, compared with heterozygous mice and normal controls, with iron staining principally in the periportal hepatocytes). Absorption studies using ⁵⁹Fe showed increased uptake from the lumen of an in vivo isolated duodenal segment in homozygous mice, although at this point in time overall transfer of radioiron to the circulation and other tissues (mucosal transfer) was not different from controls. These observations demonstrate that anaemia can lead to hepatic iron loading even in the absence of increased erythropoiesis, and are consistent with the possibility that anaemic hypoxia can enhance mucosal iron uptake by the duodenal enterocyte.     

Effective treatment of disease-related anaemia in B-chronic lymphocytic leukaemia patients with recombinant human erythropoietin

Nine B-chronic lymphocytic leukaemia (B-CLL) patients suffering from anaemia, due to no obvious cause except their disease, were treated with recombinant human erythropoietin (r-HuEPO). The treatment protocol provided a closed label phase of 3 months duration, during which the patients received r-HuEPO or placebo in a ratio of 2:1, followed by an open label phase, also of 3 months duration, during which r-HuEPO was administered to all patients three times a week s.c. r-HuEPO was given at a dose of 150 U/kg of body weight with an escalation of 50 U/kg up to a maximum of 300 U/kg three times a week. Complete response was achieved in 5/9 (55%) patients and partial response in 3/9 (33%). The response obtained was independent of the pretreatment serum EPO levels, the duration of anaemia, the concomitant administration of chemotherapy, the presence of splenomegaly, or the degree of bone marrow infiltration by lymphocytes. It appears that r-HuEPO is very effective in reversing the disease-related anaemia of B-CLL patients.     

A randomized double-blind placebo-controlled study with subcutaneous recombinant human erythropoietin in patients with low-risk myelodysplastic syndromes

To evaluate the effect of recombinant human erythropoietin (rHuEpo) on the haemoglobin level and transfusion requirement in low-risk myelodysplastic syndromes (MDS), 87 patients were enrolled in a randomized double-blind placebo-controlled study. 44 patients were assigned to epoetin α (150 U/kg/d s.c. for 8 weeks) and 43 to placebo arms. MDS types were homogenous in both groups: refractory anaemia (RA) 47.7–48.8%, refractory anaemia with ringed sideroblasts (RAS) 20.5–25.6%, refractory anaemia with excess of blasts (RAEB) (blasts < 10%) 31.8–25.6%. 14/38 evaluable patients responded to epoetin α versus 4/37 to placebo (P = 0.007). 50% of RA responded to epoetin α versus 5.9% to placebo (P = 0.0072), RAS 37.5% v 18.2% (P = 0.6) and RAEB 16.7% v 11.1% (P = 1.00). 60% of non-pretransfused patients responded to epoetin α (Hb 8.35 ± 0.73 to 10.07 ± 1.87 g/dl), whereas a slight decrease was observed in the placebo group (8.4 ± 0.66 to 8.19 ± 0.92 g/dl) (P = 0.0004). Percentage of transfused patients was similar in both arms. Basal erythropoietin (Epo) serum levels > 200 mU/l predicted for a non-response. At week 4 sTfR levels were increased > 50% in responders (P = 0.013), whereas an increase < 18% predicted for non-response (P = 0.006). Leucocyte and platelet counts were not influenced by epoetin α treatment. Adverse events occurred in 31.8% of the rHuEpo-treated versus 42.9% of the placebo-treated patients (P = 0.2), and seven patients did not complete the course. In conclusion, rHuEpo was effective in the treatment of low-risk MDS. RA subtype, no transfusions prior to rHuEpo therapy, and low basal Epo levels were associated with higher probability of response. Soluble transferrin receptor level at the fourth week was an early predictor of response.     

Potential roles of erythropoietin in the management of anaemia and other complications diabetes

Erythropoietin (EPO) is a haematopoietic cytokine, mainly generated in the renal cortex, and its secretion and action is impaired in chronic kidney disease (CKD). Early renal damage in diabetes mellitus (DM) is usually not detected because diabetes-induced nephron hypertrophy maintains glomerular filtration rate (GFR) and an elevated plasma creatinine concentration is a relatively late manifestation of diabetic nephropathy. However, anaemia occurs more frequently in subjects with DM when compared with those with non-DM renal disease. While reduced production and a blunted response to EPO occurs in DM with early renal damage, other factors including chronic inflammation, autonomic neuropathy and iron deficiency are also important. Although recombinant human erythropoietin (rhEPO) has been an effective therapeutic agent in CKD anaemia, it appears to be more effective in patients with DM, even in earlier stages. Nevertheless, patients with DM are also more likely to be iron deficient, a barrier to effective rhEPO therapy. The effect of treatment on the reliability of haemoglobin A_1c as an index of glycaemic control must be remembered. It is proposed that anaemia and its causes must be important components of care in subjects with early diabetic renal damage.     

Serum erythropoietin and its relation with soluble transferrin receptor in patients with different types of anaemia in a locally defined reference population

Serum erythropoietin (Epo) and soluble transferrin receptor (sTR) were measured in a locally defined reference population (n=100): healthy volunteers (n=50); iron‐ deficiency anaemia (n=41) and haemolytic anaemia (n=9) (β‐thalassaemia, n= 4; autoimmune, n=5). Our data demonstrated an inverse relationship between erythroid activity and Epo levels. The regression line between Ln Epo and haemoglobin (Hb) was highly significant: P < 0.0001, r²=0.8275, Ln Epo=8.5346–0.04275 Hb, confidence limit 95%. The mean observed/predicted (O/P) ratio of Ln (Epo) was 1.01 ± 0.11. We demonstrated that the serum Epo concentration in this particular population correlated consistently with clinical measures of erythropoietic activity. sTR, a new index of erythropoiesis, varied from 16.1 to 148 nmol/l, mean 62.0 nmol/l in the anaemic patients’ group. The relationship between Ln Epo and Ln sTR was highly significant: P < 0.0001. We conclude that locally defined regression analyses are crucial for correct data interpretation and can indicate whether or not Epo production is appropiate or inappropiate. Serial determinations of sTR could help in the assessment of response to therapeutic doses of Epo.     

Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodysplastic syndromes: proposal for a predictive model

Previous studies have shown that approximately 40% of patients with myelodysplastic syndrome (MDS) and anaemia respond to treatment with human recombinant granulocyte-CSF (G-CSF) plus erythropoietin (epo). The present study was designed to investigate pre-treatment variables for their ability to predict erythroid responses to this treatment. 98 patients with MDS (30 RA, 31 RARS, 32 RAEB, five RAEB-t) were treated with a combination of G-CSF (0.3–3.0 μg/kg/d, s.c.) and epo (60–300 U/kg/d, s.c.) for at least 10 weeks. Minimum criteria for erythroid response was a 100% reduction of red blood cell (RBC) transfusion need or an increase in haemoglobin level of  1.5 g/dl. 35 patients (36%) showed responses to treatment. Medium duration of response was 11–24 months. In multivariate analysis, serum erythropoietin levels and initial RBC-transfusion need retained high statistical significance ( P  < 0.01). Using pre-treatment serum epo levels as a ternary variable (< 100, 100–500 or > 500 U/l) and RBC transfusion need as a binary variable (< 2 or  2 units per month), the analysis provided a predictive score for erythroid response. This score divided patients into three groups: one group with a high probability of erythroid responses (74%), one intermediate group (23%) and one group with poor responses to treatment (7%). This predictive scoring system could be used in decisions regarding use of these cytokines for treating the anaemia of MDS, both for defining patients who should not be given the treatment and for selecting patients for inclusion in prospective trials.     

Efficacy of a single,weekly dose of recombinant erythropoietin in myelodysplastic syndromes

Thirteen patients with low-to-intermediate risk myelodysplastic syndrome (MDS) received recombinant erythropoietin (r-EPO) at the single, weekly dose of 40.000 U for at least 8 weeks. Five patients (38.4%) achieved a major erythroid response (increased haemoglobin levels > 2 g/dl and/or transfusion independence), which is currently maintained after 3-11 months, without modification of r-EPO dose. This study suggests that 40.000 U r-EPO given once a week may be at least as effective as the more frequent (daily or three times a week) administrations of the drug usually employed in MDS patients.     

Levels of erythropoietin in patients with the anemias of chronic diseases and liver failure

Two mechanisms are felt to be responsible for the production of anemia in patients with chronic diseases. The first is failure to produce adequate amounts of erythropoietin (EP), and the second is failure to deliver iron to the bone marrow in amounts sufficient to support normal erythropoiesis. In order to evaluate these hypotheses we studied urine and serum EP levels and levels of 2,3-diphosphoglycerate in normal subjects, in patients with the anemia of chronic diseases, in patients with chronic liver disease, and in patients with a variety of other anemias. Based on the results, we propose first that insufficient production of EP is one of the major mechanisms responsible for anemia in patients with chronic diseases. Second, insufficient production of EP is, in part, responsible for anemia seen in patients with chronic liver disease. Third, serum and urine EP levels decrease with aging, and this correlates with the fall of hemoglobin levels seen in older normal subjects.