Reticulocyte Response Research Articles

A Phase I, Single and Fractionated, Ascending‐Dose Study Evaluating the Safety, Pharmacokinetics, Pharmacodynamics, and Immunogenicity of an Erythropoietin Mimetic Antibody Fusion Protein (CNTO 528) in Healthy Male Subjects

The erythropoietin mimetic antibody fusion protein CNTO 528 was developed as a novel antibody fusion protein by constructing an active hematopoietic peptide onto an IgG1-based scaffold. This resulted in a molecule with a long circulating half-life and a prolonged effect of stimulating reticulocyte production and hemoglobin (Hgb) synthesis. To assess the safety, pharmacokinetics, and pharmacodynamics of CNTO 528, the authors gave 44 adult healthy male subjects single or fractionated doses of intravenous CNTO 528 or placebo. CNTO 528 was generally well tolerated. The maximum observed concentration (Cmax) and the area under the concentration versus time curve (AUC) increased in an approximately dose-dependent manner between the 0.09-mg/kg and 0.9-mg/kg doses. The maximum effect on the reticulocyte response occurred approximately 8 to 9 days after administration. A median increase in Hgb (> or =1 g/dL above baseline) was achieved 9 to 10 days after administration, with a maximum effect between 19 and 26 days. Two subjects in the 0.9-mg/kg dose group had elevated Hgb concentrations requiring phlebotomy. In this first-in-human study, CNTO 528 was well tolerated and effective in elevating and maintaining Hgb by at least 1 g/dL following a single intravenous administration, which suggests that an erythropoietin mimetic molecule, such as CNTO 528, may be an effective therapy for patients with anemia.

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.

The effect of severe hepatic impairment on the pharmacokinetics and haematological response of C.E.R.A.

ABSTRACTAim: To examine the effect of severe hepatic impairment (HI) on the pharmacokinetics (PK) and pharmacodynamics (PD) of the continuous erythropoietin receptor activator, C.E.R.A.Methods: A non-randomised, multicentre, single-dose, open-label study in patients with HI (n = 12) and healthy subjects (n = 12). After 2 weeks of screening, participants received a single intravenous dose of C.E.R.A. (200 µg), and were then followed for ≈8 weeks. The area under the concentration–time curve (AUC) from drug administration to last measurable concentration (AUClast), and maximum C.E.R.A. concentration (Cmax) were calculated to assess PK. The baseline-corrected area under the effect curve over 22 days (AUEcorr) for reticulocyte count was the primary PD parameter.Results: The PK profile was similar in patients and healthy subjects (AUClast: 6678 vs 6985 ng*h/mL; Cmax: 63 vs 75 ng/mL) C.E.R.A. produced a sustained erythropoietic response in bothgroups, with increases in reticulocyte counts peaking 7–9 days post-dose and returning to baseline by Day 22. Although mean AUEcorr was 64% lower in patients, this may have been an artefact of higher baseline reticulocyte counts. Lower reticulocyte responses in patients did not translate into lower responses for haemoglobin, haematocrit or erythrocytes, suggesting that HI had no clinically relevant effect on the PD of C.E.R.A. C.E.R.A. was well tolerated. Four AEs (none considered drug related) were reported in three patients (mild myocardial ischaemia; mild pyrexia and liver transplant; severe bacterial peritonitis [serious AE]); no AEs were reported in healthy subjects.Conclusions: Severe HI has no clinically relevant effect on PK parameters or haematological response after single-dose C.E.R.A.

Pharmacodynamic Analysis of Recombinant Human Erythropoietin Effect on Reticulocyte Production Rate and Age Distribution in Healthy Subjects

To evaluate the effect of recombinant human erythropoietin (rHuEPO) on the reticulocyte production rate and age distribution in healthy subjects. Extensive pharmacokinetic and pharmacodynamic data collected from 88 subjects who received a single subcutaneous dose of rHuEPO (dose range 20-160 kIU) were analysed. Four nonlinear mixed-effects models were evaluated to describe the time course of the percentage of reticulocytes and their age distribution in relation to rHuEPO pharmacokinetics. Model A accounted for stimulation of the production of progenitor cells in bone marrow, and model B implemented shortening of differentiation and maturation times of early progenitors in bone marrow. Model C was the combination of models A and B, and model D was the combination of model A with an increase in the maturation times of the circulating reticulocytes. Model evaluation was performed using goodness-of-fit plots, a nonparametric bootstrap and a posterior predictive check. Model D was selected as the best model, and evidenced accurate and precise estimation of model parameters and prediction of the time course of the percentage of reticulocytes. At baseline, the estimated circulating reticulocyte maturation time was 2.6 days, whereas the lifespan of the precursors in the bone marrow was about 5 days. The rHuEPO potency for the stimulatory effect (7.61 IU/L) was higher than that for the increase in reticulocyte maturation times (56.3 IU/L). There was a significant 1- to 2-day lag time in the reticulocyte response. The effect of rHuEPO on the reticulocyte age distribution consisted of a transient increase in the reticulocyte maturation time from baseline up to 6-7 days, occurring 1 day after administration. The dose-dependent amplitude of the changes in the age distribution lasted for 12-14 days. The model-predicted peak increase in the reticulocyte release rate ranged from 140% to 160% of the baseline value and was maximal on days 7-8 following rHuEPO administration. A semiphysiological model quantifying the effect of rHuEPO on the reticulocyte production rate and age distribution was developed. The validated model predicts that rHuEPO increases the reticulocyte production rate and modifies the reticulocyte age distribution in a dose-dependent manner.

Response of the Erythroid Lineage to Irradiation.

Radiation causes acute marrow damage and anemia, however the relative sensitivity of erythroid progenitors and precursors to radiation damage is unknown. While acute anemia elicits a rapid recovery response, the characteristics of erythroid recovery following irradiation have not been well investigated. Mice display a linear response of radiation dose and red cell damage as measured by micronucleated reticulocytes (MN-RET) up to 1-2Gy. Higher radiation doses lead both to decreased MN-RET frequency and to delayed reticulocyte recovery (Dertinger, et al., Mutation Research, in press). To determine why marrow erythroid toxicity differs at low (< 1Gy) versus high (> 2 Gy) radiation doses, we developed a novel method to quantify erythroid precursors using multispectral imaging flow cytometry. C57Bl/6 mice were irradiated either with 0, 1 or 4 Gy and erythroid progenitors (BFU-E, CFU-E) and precursors (ProE, BasoE, PolyE, OrthoE) in the marrow were analyzed between 1 hr and 5 days later. 1 hr following 1Gy radiation, BFU-E and CFU-E were reduced to 63±5% and 13±1% of unirradiated controls, respectively. 24 hrs after 1Gy, there was no significant change in BFU-E, however, a striking recovery of CFU-E (93±21% of controls) was observed. This CFU-E surge was followed by a wave of recovering erythroid precursors in the marrow beginning at 48 hrs and peaking at 72 hrs post radiation, when BasoE, PolyE, and OrthoE reached > 150% of unirradiated controls. These precursors subsequently fell below 70% and 90% of controls at 4 and 5 days, respectively. We also characterized the kinetics of erythroid progenitors and precursors following high-dose radiation. 1 hr after 4Gy, BFU-E and CFU-E were reduced to 10% and 1% of controls, respectively. Each of these erythroid progenitors reached their nadir (1% of controls) at 48 hrs, and began to modestly recover at 72 hrs. By 5 days after 4Gy, BFU-E were only 16±6% of controls, while CFU-E had variably recovered to 127±62%. Erythroid precursors also reached their nadir at 48 hrs following 4Gy with levels of 1–2% of controls, but then recovered to 50–100% by day 5. We conclude that CFU-E are exquisitely sensitive targets of radiation. While BFU-E are more radioresistant, CFU-E recovered more robustly, consistent with the known cycling kinetics of these progenitors. Our results also indicate that the reticulocyte response following low versus high radiation doses is due to rapid CFU-E recovery and the subsequent generation of a wave of erythroid precursors. It is likely that the decrease in MN-RET seen after high-dose radiation is due to the rapid and nearly complete depletion of erythroid precursors leaving few cells able to repair and complete maturation. Finally, we speculate that the oscillation of CFU-E and erythroid precursor populations following 1Gy is due to the operation of feedback regulation. Compared to BFU-E and late-stage erythroid precursors, CFU-E and ProE exhibited a more complex temporal pattern of recovery, suggesting that they may be focal points of this regulation. Examination of marrow progenitor and precursor cell injury and death following acute irradiation will lead to a better understanding of the regulation of hematopoiesis and lay the foundation for interventional studies to mitigate the effects of acute irradiation.

Reduced erythroid cell and erythropoietin production in response to acute anemia in prion protein-deficient (Prnp−/−) mice

Cellular prion protein (PrPc) participates in the pathogenesis of prion diseases but its normal function remains unclear. PrPc is expressed on hematopoietic cells, including erythroid precursors. We investigated the role of PrPc in erythropoiesis in vivo with phenylhydrazine-induced acute anemia. Induction of equivalent anemia in wild-type (WT) and Prnp−/− mice resulted in a higher number of circulating reticulocytes, hematocrits and spleen weights in WT mice than in Prnp−/− mice on Days 5 and 7. Examination of bone marrow erythroid precursor cells (Ter119+) on Day 5 revealed no significant differences in the number of these cells between the two types of animals. However, a higher percentage of Ter119+ cells were going through apoptosis in Prnp−/− mice than in WT mice. Plasma erythropoietin (Epo) levels and Epo mRNA in kidneys peaked on Day 3 in response to anemia for both types of animals but rose less in Prnp−/− (5500 pg/ml ) than in WT (18,000 pg/ml) animals. Administration of recombinant human Epo to mice produced an equivalent reticulocyte response in both types of animals suggesting that the potential for erythroid generation is intact in Prnp−/− animals. These observations indicate that PrPc may modulate tissue hypoxia-sensing mechanisms or effect hypoxia target gene expression.

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: dose–response and time-course profiles measured by flow cytometry

A flow cytometric, anti-CD71-based method was used to measure peripheral blood reticulocyte and micronucleated reticulocyte frequencies in response to 137Cs total body irradiation (TBI). In three independent experiments, groups of five female C57BL/6N mice were irradiated at graded doses up to 3 Gy, and peripheral blood specimens were collected at 43 h post-irradiation. Whereas the frequency of reticulocytes declined over the range of doses studied, micronucleated reticulocyte incidence was observed to increase in a dose-dependent manner up to 1 Gy. At doses greater than approximately 1 Gy, micronucleated reticulocyte frequencies declined with increasing exposure. These responses were highly reproducible, with significant effects on reticulocyte and micronucleated reticulocyte frequencies observed for the lowest dose studied (0.125 Gy). A time-course experiment was performed to test whether radiation-induced cell cycle delay may explain saturation of the micronucleated reticulocyte endpoint at doses >1 Gy. For this experiment, groups of four female C57BL/6N mice were exposed to 1, 1.5, or 2 Gy TBI, and blood collection occurred at 12 h intervals from 43 to 115 h post-exposure. Reduced reticulocyte frequencies were observed for each dose studied, and the recovery of reticulocytes was increasingly delayed with higher radiation doses. Maximal micronucleated reticulocyte frequencies were observed at 43 or 55 h, with progressively lower values at later time points. At no time did micronucleated reticulocyte frequencies induced by 1.5 or 2 Gy significantly exceed that observed for 1 Gy at 43 h. These time-course data suggest that radiation-induced cell cycle delay cannot account for the micronucleated reticulocyte downturn phenomenon observed at doses greater than 1 Gy. An alternate hypothesis is discussed whereby apoptotic elimination of severely damaged bone marrow erythroid precursors plays a dominant role in saturating the radiation-induced micronucleated reticulocyte response observed for C57BL/6N mice.

An Assessment of Recombinant Human Erythropoietin Effect on Reticulocyte Production Rate and Lifespan Distribution in Healthy Subjects

An empirical pharmacodynamic model was developed to assess the effect of recombinant human erythropoietin (rHu-EPO) treatment on the reticulocyte production rate and lifespan distribution. Single doses of rHu-EPO at levels 20, 40, 60, 90, 120, and 160 kIU were administered to healthy volunteers (n = 8 per dose level). Erythropoietin plasma concentrations as well as hematologic responses were measured up to 42 days. The hematological data were used to determine explicit relationships between reticulocyte and red blood cell counts (RBC) and the reticulocytes' production rate and lifespan distribution. The parameter estimates obtained by simultaneous fitting of the model to the reticulocyte and RBC data revealed that rHu-EPO transiently increased the reticulocyte lifespan from the baseline value of 1.7 days to 3.4 days and the effect lasted for 8.3 days. The dose dependent increase in the reticulocyte production had the maximal value of 77.5 10(9) cells/l/day and was followed by a rebound that was less than 9% of the baseline value. Both reticulocyte and RBC responses were preceded by a dose-independent lag time of 1.7 days. The effect of rHu-EPO on the reticulocyte production rate and lifespan distribution was characterized. The results of the present study can be further utilized in building more mechanistic pharmacodynamic models of rHu-EPO stimulatory effects.

Optimising Haemoglobin Response to Exogenous Erythropoietin in Anaemic Patients Treated in Routine Haemato-Oncology Practice.

Patients' response to treatment with human recombinant erythropoietin (Epo) for chemotherapy-associated anaemia varies between 50% and 70%. The cost-effectiveness of Epo may be maximized by targeting treatment at patients most likely to respond. In this study, modified ASH/ASCO guidelines were combined with a systematic approach to iron supplementation in order to maximize haemoglobin (Hb) response in iron deficient patients. Patients with haematological malignancy and chemotherapy-associated anaemia received Epo alone (n = 25) or Epo plus intravenous iron sucrose (iv iron) if functional iron deficiency was present (n=24). The cohort had a male/female ratio of 48:52% and a mean age 62.8 years (SD 12.8; range 24 to 81). Baseline measurements were repeated at 4-weekly intervals for a period of 32 weeks. Overall, 80% of cases experienced a Hb response greater than 1g/dl, with 65% of patients achieving a major Hb response (≥ 2g/dl increase above baseline). There was no difference in Hb response between the Epo alone vs. Epo plus iv iron groups (∅ 2.72 vs. 2.84 g/dl; p=0.831), indicating equivalent outcomes for functionally iron deficient patients treated with effective iv iron supplementation and iron replete patients. General Linear Modelling was used to explore the independent contributions of demographics, baseline iron parameters, and disease progression in predicting Hb response to Epo. In the final model three independent variables emerged as significant, which together accounted for 40% of variance in Hb response. Progression of the underlying malignancy was associated with a mean reduction in Hb response of 1.9g/dl (p<0.001). Lower baseline Hb was associated with an increased response - each 1g/dl decrement was associated with a mean increased response of 0.87g/dl (p=0.012). Early change in reticulocyte count (between baseline and early follow up (< 4 weeks) was found to be positively associated with Hb response - each additional 1% change was associated with a 0.016g/dl increase in Hb response (p=0.006). These findings are in contrast to previous reports that the response to Epo wasindependent of the outcome of the underlying malignancy anddiminished at lower Hb levels.Disease progression, baseline Hb and early increases in reticulocytes were all highly significantly correlated with the Hb response to Epo. In fact, in this cohort, all cases of non-response to Epo or loss of a response were associated with disease progression. To maximise response rates to Epo requires detection of functional iron deficiency and supplementation with iv iron. Furthermore, early reticulocyte response and disease progression should be closely followed and can help guide when to stop Epo therapy. This would allow rational use of resources and improve the health economics of Epo therapy by focusing on those most likely to benefit.

Autoimmune Hemolytic Anemia in HIV: A Case Series and Review of the Literature.

Background: Patients infected with HIV are more likely to have positive direct antiglobulin tests (Toy, Am J of Hem 1985) but there have only been a few reports of HIV-related AIHA (autoimmune hemolytic anemia) in the literature. Here we report three new cases of AIHA in HIV infected patients diagnosed from July 2003 to September 2005 at UCSD.Methods: Patients were identified by clinical presentation to the HIV service. All cases were considered highly likely by hematologists to have AIHA (lab data table 1).Results: Patient #1 was a 43 yo Caucasian male with CD4 < 50 prior ITP and untreated recently diagnosed visceral KS. On presentation, he reported dyspnea and had hepatosplenomegaly. His Hb was 5.2, retic 14%, LDH 459, Ibili 0.7, haptoglobin <7; + microspherocytes on smear. He was started on high-dose glucocorticoids (GC) before PRBC transfusion, and tolerated it well other than one fever to 100F. He received prednisone (pred) 60mg daily with taper. 3 weeks later at follow-up he self-tapered pred to 20mg and Hb was 11.1. Once off pred, his Hb remained stable at 11 when he died from PML approximately 4 months after remission of AIHA. Patient #2 is a 43 yo Latino male presenting with syncope, dizziness, dyspnea and palpations. His Hb was 5.4, retic 8%, LDH 366, Ibili 1.2, haptoglobin < 7, with smears showing extensive microspherocytes. He was treated with high-dose GC and received PRBC with heparin prophylaxis. His Hb recovered to 12. He was tapered off GC over 8 months and remains in remission for the past 2 months with Hb 14. Patient #3 is a 38 yo Latino male with dyspnea. His Hb was 5.7, retic 8%. He symptomatically improved with PRBC, and subsequently had a rapid response to GC. Hb peaked at 13.5 through tapering of pred by 10 mg weekly to 30 mg daily. The patient missed follow-up and continued the rapid taper, hemolysis recurred 1 month after pred cessation. Pred 60mg qd was restarted with resolution of hemolysis; this recurred even with slow taper (2mg/week) below 20mg daily. Pred again increased to 60 mg daily and danazol 600mg bid added with resolution of hemolysis. Pred taper to 20 mg was tolerated.Conclusions: AIHA in HIV patients has been described mainly in single case reports and abstracts (Koduri, Am Jo Hematology 2002). Only a few reports include long-term follow-up and fewer have included precipitants for AIHA. Within 2 years, we have three HIV patients with documented AIHA under different clinical manifestations and responses to treatment. One patient had a recent diagnosis of KS, the 2nd had received penicillin for syphilis and the 3rd had no recent medical complications. All 3 patients tolerated transfusion which resulted in symptomatic improvement and responded successfully to pred. Patient #3 who had a high CD4, was the only one with recurrence of hemolysis upon pred taper; perhaps a higher CD4 count may be associated with more persistent AIHA. In the literature, there are 14 other cases of idiopathic AIHA in adults with HIV (summary table to be presented at meeting). Of the 6 cases with reported CD4 counts, all were less than 200. 4/14 cases had inadequate reticulocyte response demonstrating existence of additional factors contributing to anemia. Most cases (10/14) responded to standard AIHA treatments including GC, IVIG and splenectomy. Overall 5/14 had died at the time of the reports; 2 of these deaths were related to PCP after remission of AIHA. 2/8 cases receiving PRBC died of DIC that occurred after transfusion.Table 1CaseHb gm/dlRetic %LDH IU/LHapto mg/dlIBili mg/dlCD4 #/ml15.213.9459<70.98225.48.1366<71.28635.78.1317<72.1424

Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo

AbstractMalarial anemia is a global public health problem and is characterized by a low reticulocyte response in the presence of life-threatening hemolysis. Although cytokines, in particular tumor necrosis factor-α (TNF-α), can suppress erythropoiesis, the grossly abnormal bone marrow morphology indicates that other factors may contribute to ineffective erythropoiesis. We hypothesized that the cytotoxic hemozoin (Hz) residues from digested hemoglobin (Hb) significantly contribute to abnormal erythropoiesis. Here, we show that not only isolated Hz, but also delipidated Hz, inhibits erythroid development in vitro in the absence of TNF-α. However, when added to cultures, TNF-α synergizes with Hz to inhibit erythropoiesis. Furthermore, we show that, in children with malarial anemia, the proportion of circulating monocytes containing Hz is associated with anemia (P < .001) and reticulocyte suppression (P = .009), and that this is independent of the level of circulating cytokines, including TNF-α. Plasma Hz is also associated with anemia (P < .001) and reticulocyte suppression (P = .02). Finally, histologic examination of the bone marrow of children who have died from malaria shows that pigmented erythroid and myeloid precursors are associated with the degree of abnormal erythroid development. Taken together, these observations provide compelling evidence for inhibition of erythropoiesis by Hz.

Urinary excretion of darbepoetin after intravenous vs subcutaneous administration to preterm neonates

Previous studies suggest that darbepoetin might stimulate erythropoiesis in preterm neonates more effectively if injected subcutaneously (s.c.) than if infused intravenously (i.v.). It has been postulated that this is because very high plasma concentrations after i.v. dosing result in urinary loss of the drug. However, this theory has not been tested systematically, and no direct comparisons have been made between s.c. and i.v. dosing of darbepoetin in preterm neonates. Preterm neonates were eligible for this pilot study if they were born at < or =32 weeks gestation with a weight of < or =1500 g, and had a hemoglobin < or =10.5 g/dl. The darbepoetin was given (4 microg/kg) i.v., over 4 h, if an i.v. was already in place and s.c. if no i.v. was in place. Urine was collected for drug quantification before dosing and for 48 h after. Blood was obtained for immature reticulocyte fraction (IRF), absolute reticulocyte count (ARC) and reticulocyte % before and 96 h after dosing. Ten preterm neonates were studied: five received i.v. and five received s.c. darbepoetin. No adverse effects of the administrations were detected. IRF, ARC and reticulocyte % increased in the i.v. and s.c. recipients, and no difference in magnitude of increase was apparent between the groups. Before the darbepoetin was administered, none of the patients had any erythropoietin (Epo) detected in their urine. After i.v. dosing, no darbepoetin was detected in any of the samples, on any of the subjects, over the subsequent 48 h. After s.c. dosing, three of the patients had minimal urinary Epo detected. The patient with the largest urinary loss of drug had only 0.13% of the administered dose detected in the urine. Thus, essentially no urinary loss of drug was observed following either i.v. or s.c. darbepoetin dosing. Darbepoetin loss into the urine was below detectable limits among seven patients, while three had minimally detectable urinary losses. i.v. and s.c. dosing resulted in approximately equivalent increases in reticulocyte response when measured 96 h after dosing. On this basis, we speculate that if a patient who is to receive darbepoetin has an i.v. in place, the drug can be given i.v., thus avoiding any discomfort associated with an s.c. injection.

Alpha erythropoietin in the management of anaemia of prematurity: a report of three cases in Nigeria

Anaemia of prematurity is a common problem encountered in most special care baby units warranting often repeated blood transfusions with its inherent dangers. We present three patients in Nigeria who were very low birth weight premature babies on whom recombinant erythropoietin was used as an efficient replacement for blood transfusions. All three patients showed a progressive drop in haemoglobin concentration from the first to the sixth to eight week of life with development of heamic murmurs and insignificant reticulocyte responses, and were planned for transfusion therapy. The parents of all the patients strongly refused transfusion therapy (one on religious grounds) warranting the use of recombinant erythropoietin to which there was a significant response in all three patients with elevation of haemoglobin concentration, PCV, and reticulocyte counts, and obviating the planned transfusions. No significant side effects were also noticed. We suggest further controlled trials to establish this mode of therapy.

Living high–training low: effect on erythropoiesis and aerobic performance in highly-trained swimmers

The "living high-training low" model (LHTL), i.e., training in normoxia but sleeping/living in hypoxia, is designed to improve the athletes performance. However, LHTL efficacy still remains controversial and also little is known about the duration of its potential benefit. This study tested whether LHTL enhances aerobic performance in athletes, and if any positive effect may last for up to 2 weeks after LHTL intervention. Eighteen swimmers trained for 13 days at 1,200 m while sleeping/living at 1,200 m in ambient air (control, n=9) or in hypoxic rooms (LHTL, n=9, 5 days at simulated altitude of 2,500 m followed by 8 days at simulated altitude of 3,000 m, 16 h day(-1)). Measures were done before 1-2 days (POST-1) and 2 weeks after intervention (POST-15). Aerobic performance was assessed from two swimming trials, exploring .VO(2max) and endurance performance (2,000-m time trial), respectively. Reticulocyte, serum EPO and soluble transferrin receptor responses were not altered by LHTL, whereas reticulocytes decreased in controls. In POST-1 (vs. before): red blood cell volume increased in LHTL only (+8.5%, P=0.03), .VO(2max) tended to increase more in LHTL (+8.1%, P=0.09) than in controls (+2.5%, P=0.21) without any difference between groups (P=0.42) and 2,000-m performance was unchanged with LHTL. In POST-15, both performance and hematological parameters were similar to initial levels. Our results indicate that LHTL may stimulate red cell production, without any concurrent amelioration of aerobic performance. The absence of any prolonged benefit after LHTL suggests that this LHTL model cannot be recommended for long-term purposes.

Zidovudine-induced pure red cell aplasia presenting after 4 years of therapy

Reversible pure red cell aplasia is a recognized complication of both zidovudine and lamivudine, typically occurring within the first 3 months of therapy [1,2]. We report the case of a 29-year-old man with HIV, severe haemophilia A and hepatitis C, who developed reversible pure red cell aplasia 4 years after commencing zidovudine. The patient presented to our centre in September 2004 with a 2-week history of exertional dyspnoea and palpitations. He had severe haemophilia A and chronic hepatitis C (genotype 3B), which had never been treated. HIV was diagnosed in 1984; highly active antiretroviral therapy (HAART) was commenced in February 1999 with zidovudine, lamivudine and indinavir. In 2002, his HAART regimen was changed to Trizivir (zidovudine, abacavir and lamivudine). As he had previously developed Stevens–Johnson syndrome on co-trimoxazole, monthly nebulized pentamidine was used as Pneumocystis carinii prophylaxis. His haemophilia A was managed with plasma-derived factor VIII on demand (Fandhi 3000 units), which he used two to three times a month. He was taking no other medications. On examination, he appeared pale but otherwise well. There was conjunctival pallor, but no jaundice. Respiratory and abdominal examination was normal. He had a hyperdynamic circulation with a grade 3/6 ejection systolic flow murmur confined to the left sternal edge. There were no symptoms or signs of bleeding. Initial laboratory investigations showed a severe macrocytic anaemia with a haemoglobin level of 3.5 g/dl and mean corpuscular volume of 115 fl. The reticulocyte count was low at 3 × 109/l. Platelet and white cell counts were normal. The blood film showed an oval macrocytosis. On review of previous blood counts, the patient had had a macrocytosis (range 117–122 fl) without anaemia since commencing HAART in 1999. At the time of presentation, his HIV-RNA level was less than 50 copies/ml and the CD4 cell count was 391 cells/μl. Serum ferritin, B12 and folate were normal, as were plasma methylmalonic acid and homocysteine (functional markers of B12 and folate deficiency, respectively). Renal and liver function tests were normal. The serum erythropoietin level was appropriately raised at 584 U/l. Bone marrow aspirate and trephine biopsy were performed with factor VIII cover. The aspirate showed a megaloblastic picture with maturation arrest of the erythroid series at the early erythroblast stage. The trephine biopsy was hypercellular and megaloblastic with disorganized haematopoeisis. Parvovirus IgG was positive but IgM was negative. Mycoplasma serology was negative. Bone marrow trephine stains for acid-fast bacilli, fungi, parvovirus, human herpes virus 8 and Epstein–Barr virus were negative. Polymerase chain reaction testing of bone marrow aspirate for parvovirus B19 and HHV-6 DNA was negative. Peripheral blood flow cytometry for paroxysmal nocturnal haemoglobinuria was negative. Four units of packed red cells were transfused, and the patient was monitored for 10 days. As he failed to mount a reticulocyte response and the virology findings proved negative, his HAART regimen was changed to lamivudine, tenofovir and efavirenz. Eight days after stopping zidovudine, he mounted a reticulocyte response of 120 × 109/l. The full blood count normalized 20 days after the cessation of zidovudine. Six months later, he remains well with a normal full blood count and normal mean corpuscular volume. Pure red cell aplasia is a well-documented adverse reaction of HAART regimens containing both zidovudine and lamivudine. In this instance, the discontinuation of zidovudine, but not of lamivudine, led to the prompt resolution of the anaemia. This is the first published case of zidovudine-induced pure red cell aplasia occurring so late in zidovudine therapy, and illustrates that this complication is not restricted to the first 3 months of treatment.

A synthetic peptide-based erythropoiesis stimulating agent (ESA) (Hematide) demonstrates dose dependent activity and is well tolerated in a phase 1 single dose, dose escalating study in normal healthy volunteers (NHV)

8180 Background: Hematide is an innovative, synthetic, pegylated, peptidic ESA that binds to and activates the erythropoietin (EPO) receptor. Although its amino acid sequence is unrelated to that of EPO, Hematide demonstrates in vitro and in vivo activities comparable to EPO and an extended serum half life in animal studies. This may allow less frequent dosing in patients with chronic anemia relative to EPO. Methods: A randomized double-blind placebo controlled dose escalation study was conducted to assess safety, PK and PD of Hematide administered as a single IV infusion to male NHV. Up to 7 cohorts of 7 subjects each (5 on Hematide and 2 on placebo) were planned with doses escalated unless or until stopping rules were met. Results: Three dose cohorts have been completed involving 21 NHV. As of this date, the study remains blinded and we assume increases in reticulocytes and hemoglobin (Hb) are due to Hematide. Injections were well tolerated with report of only a few Grade 1 adverse events and no pattern across cohorts. Reticulocyte increases were dose proportional; average reticulocyte peak concentrations (+/- SE) were 148 (+/- 19); 186 +/- (20); 230 (+/- 22) 109/L among the NHV with a reticulocyte response in Cohort 1 (n=4), Cohort 2 (n=5), and Cohort 3 (n=5) respectively, compared to 59 (+/- 5) 109/L among the 7 (pooled) non-responding subjects. In Cohort 3, an increase in Hb was seen in 5 subjects. The mean difference in changes from baseline Hb between these 5 responding and the 2 non-responding subjects was 1.0 g/dL at day 9 (p = 0.014) and remained detectable as of Day 35. Unblinded results, including PK data, will be presented. Conclusions: A single IV injection of Hematide administered to NHV was well tolerated and demonstrated potent dose dependent erythropoietic activity. These data will be used to plan Phase 2 studies of Hematide in patients with anemia related to cancer and renal failure. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Affymax Affymax Affymax, Amgen

Anemia and Diabetes in the Absence of Nephropathy

Patients with diabetes commonly have a greater degree of anemia for their level of renal impairment than those presenting with other causes of renal failure. To clarify the contribution and differing roles of diabetes and nephropathy in the development of anemia in diabetic patients, we examined the hematologic and hematinic parameters of diabetic patients without nephropathy. The study group was comprised of 62 patients with type 2 diabetes who had been followed for a median of 7 years. For the study, these patients had additional samples taken during their annual routine blood testing for the measurement of extra parameters, including serum ferritin, serum erythropoietin (Epo) levels, and the percentage of reticulocytes. These measurements were combined with the routine parameters Hb, hematocrit, HbA(1c), and glomerular filtration rate. In all, 8 of the 45 male patients (17.8%) and 2 of the 17 female patients (11.8%) were classified as anemic (Hb <13g/dl and <11.5g/dl, respectively). Although only a small number of the patients had anemia as defined by normal values, a retrospective analysis of individual patients over time revealed a sustained though small decrease in Hb from initial presentation. A statistically significant difference in Epo levels (P = 0.016 by Kruskal-Wallis test) was observed from the group with the lowest (Hb < or =11.5) to that with the highest (Hb > or =14.5) Hb values, with a median Epo value of 37 (interquartile range 24-42) vs. 13 (9-15) IU/l, respectively. In contrast, there was no evidence of an increased reticulocyte response to higher levels of Epo (r = 0.134 [Pearsons], P = 0.36). Reticulocyte counts ranged from 44 (38-57) to 76.5 (56-83) in the lowest and highest Hb groups, respectively. Although only a small number of subjects in the group were overtly anemic, all subjects had an ongoing, small but significant decrease in Hb since presentation. This study of diabetic patients without nephropathy shows an expected increase in Epo production in response to lowering levels of Hb but without the expected reticulocyte response.

William Bosworth Castle: pioneer of haematological clinical investigation.

William Bosworth Castle (1897-1990) spent his entire haematological career at the Harvard Medical Unit and Thorndike Memorial Laboratory at Boston City Hospital during the golden age of haematology. Castle's experiments in solving the puzzle of pernicious anaemia by identifying the intrinsic factor are models of clinical investigation and marked a new era in haematology. Castle's insatiable curiosity about the mechanisms of disease, his ability to design and conduct simple experiments to test hypotheses, and his ability to attract clinical investigators to the Thorndike Laboratory, and inspire them and make them feel like intellectual equals, fuelled the dramatic output of seminal work on nutritional anaemias, haemolysis, splenic function, haemoglobin physiology and coagulation. Indeed it was Castle's breadth of scientific achievements in haematology and his leadership of the Thorndike Memorial Laboratory that contributed to the notion that modern haematology was born in Boston but nurtured at the Thorndike Memorial Laboratory.

Pharmacokinetic and pharmacodynamic modeling of recombinant human erythropoietin after intravenous and subcutaneous dose administration in cynomolgus monkeys.

The pharmacokinetics (PK) and pharmacodynamics (PD) of recombinant human erythropoietin (rHuEpo) were investigated in monkeys. A two-compartment model with dual input and nonlinear disposition could adequately characterize the PK of rHuEpo upon three intravenous and six s.c. administrations. The kinetic model suggests rapid zero-order absorption of part of the s.c. dose (35%) followed by a slow first-order entry through the lymphatics. The s.c. treatments caused a delayed dose-dependent rise in reticulocyte numbers peaking between 100 and 200 h and returning to baseline by 300 to 400 h. This was followed by steady rises in red blood cell (RBC) and hemoglobin counts. A physiological catenary model based on a life span concept with rHuEpo stimulating the production of two cell populations (progenitor cells and erythroblasts) was applied. The model could adequately describe the reticulocyte responses upon the various s.c. treatments, giving estimates of maturation times for cells in the various stages of differentiation including the early progenitor cells (70.4 h), erythroblasts (15.0 h), and reticulocytes (141.6 h) that are close to the literature reported values. An Smax of 3.13 was estimated indicating a moderate maximum stimulation of erythropoiesis, whereas the SC50 was 842 IU/l. The model was used to effectively predict the increases in RBC and hemoglobin counts as well. In conclusion, the physiological PK/PD model developed could adequately describe the time course of rHuEpo effects, yielding realistic estimates of cell life span parameters.

Immature reticulocytes as an early predictor of engraftment in autologous and allogeneic bone marrow transplantation.

The purpose of this study was to evaluate reticulocyte parameters by means of flow cytometric reticulocyte counting in a group of patients who had undergone autologous and allogeneic bone marrow transplantation (BMT). The pattern of reticulocyte response and the predictive value of absolute neutrophil count (ANC), platelet count, number of CD34+ cell infused and graft source for reticulocyte response were studied. We compared absolute reticulocyte count (RetAbs), mean fluorescence index (MFI) and mean reticulocyte volume/mean corpuscular volume (MRV/MCV) ratio with conventional criteria (ANC and platelet count) in 22 allogeneic and 20 autologous BMT recipients. An abrupt increase in MRV/MCV ratio or a rise in MFI value were the earliest signs of erythropoietic recovery following allogeneic transplantation (63.6 and 22.8% of cases, respectively). In 13.6% of the cases, both parameters were observed simultaneously. All but three autologous transplant recipients showed changes in reticulocyte parameters earlier than ANC recovery. Granulocyte recovery and peripheral blood progenitor cells (PBPC) graft were predictive variables for RetAbs response in allogeneic transplant recipients. In the autologous group, predictive variables for RetAbs response were a high number of CD34+ infused cells and platelet recovery. An increase in the immature reticulocyte population is the earliest sign of haematopoietic recovery following BMT.