NoBlood

Annals of the New York Academy of Sciences 850:129-138

ELIEZER A. RACHMILEWITZ^a,c and MEMET AKER<sup>b


a</sup>Department of Hematology, Hadassah University Hospital, Jerusalem, Israel
^bDepartment of Pediatrics, Hadassah University Hospital, Jerusalem, Israel
^cCorresponding author: E. A. Rachmilewitz, MD, Head, Department of Hematology; Hadassah University Hospital, Ein Kerem; P.O. Box 12,000, Jerusalem, 91120 Israel; Tel: 972 2 677-6744; Fax 972-2-642-3067; E-mail erach@hadassah.org.il


ABSTRACT
The rationale for treatment with recombinant human erythropoietin (rHuEPO) in thalassemia came from studies in baboons, thalassemic mice and in erythroid cultures. The results demonstrated an increase in globin synthesis and consequently in fetal Hb (Hb F) resulting in improvement in erythropoietic parameters. In addition, endogenous serum Epo levels in various forms of thalassemia were inconsistent and not related to the severity of the anemia. Therefore, several preliminary studies with rHuEPO were performed, mainly on patients with ß thalassemia intermedia. The results indicate: a) a significant, dose-related (500 u/kg to 1000 u/kg x 3/week) increase in thalassemia erythropoiesis without changes in % of Hb F, MCV and MCH, mainly in splenectomized patients; b) the minimum effective dose is 500 u/kg x 3/week; c) there were no major side effects during the continuous treatment period of 9 months. In order to improve both quantitative and qualitative thalassemia erythropoiesis, several trials were undertaken combining rHuEPO with hydroxyurea (HU), which is known to increase % Hb F, MCV and MCH without a major effect on Hb levels. The designed trial included 3 to 6 months of HU alone (20 mg/kg x 4/week), or with rHuEPO alone (500 u/kg x 3/week or 375 u/kg x 2/week) or a combination of the two drugs. The results show an additive effect of the two drugs, in some of the patients. It is not known whether the addition of oral iron to rHuEPO is warranted for maximal erythropoietic response. The major limiting factor in designing large scale clinical trials is the relatively high cost of the drug. Nevertheless rHuEPO alone or in combination with other Hb F modulating drugs may have a positive effect in thalassemia with resulting improvement in the quality of life.

INTRODUCTION

Erythropoiesis is tightly regulated by the hormone erythropoietin, a glycoprotein hormone which induces the formation of red blood cells by stimulating proliferation, differentiation and maturation of erythroid progenitor cells. ¹ Recombinant human erythropoietin (rHuEPO) became available for clinical use in a variety of primary and secondary anemias after cloning of the gene more then ten years ago. ² Among the different types of anemia, a major component consists of congenital hemolytic anemia caused by mutations in the hemoglobin molecule, particularly sickle cell anemia and the thalassemia syndrome. The initial rationale of rHuEPO administration in hemoglobinopathies came following observations in anemic and non-anemic baboons ³ in thal. mice ⁴ and in thalassemia erythroid cultures, ⁵ where a significant increase in the percentage of fetal hemoglobin (HbF) concomitant with improvement in all erythroid parameters was observed. HbF was expected to ameliorate the severity and incidence of sickle cell crisis, ⁶ and to decrease the degree of hemolysis in thalassemia. ⁷ In the following review, we would like to summarize the available information on the role of rHuEPO in various forms of the thalassemia syndrome.

ENDOGENOUS SERUM EPO LEVELS IN THALASSEMIA
To date, there are no consistent results regarding endogenous EPO levels in thalassemia. The studies of Hammond et al., ⁸ Manor et al., ⁹ Beumi et al., ¹⁰ and Nisli et al., ¹¹ failed to demonstrate a direct inverse relationship between severity of the anemia in transfused or non-transfused patients with ß thalassemia intermedia (TI) and major (TM). In patients with sickle cell anemia, Sherwood et al. ¹² showed that serum EPO levels were lower than expected for the degree of their anemia. These findings are in contrast to the reports of Alessi et al. ¹³ and Dore et al. ¹⁴ who showed a significant increase in serum EPO levels in patients with TI and TM. The reasons for the variable results in the different studies are multifold. Among them are 1) different methodology of serum EPO determination, 2) the presence or absence of blood transfusions and the time of the assay vis Ã* vis the most recent transfusion, 3) the age of the patients, since very high serum EPO levels were found in young patients with TM and TI, comparable to the levels found in patients with aplastic anemia, ⁹ which were different from the levels found in older thalassemia patients with the same degree of anemia, 4) the presence or absence of the spleen ¹¹ and 5) last but not least, the circadian rhythm of EPO levels may also play a role. ¹⁵ Consequently, the decision whether to treat a patient with TI or TM with rHuEPO cannot be made on the basis of endogenous serum EPO levels as in other anemic disorders such as MDS or cancer-induced anemia.

THE EFFECT OF rHuEPO ON HbF SYNTHESIS IN EXPERIMENTAL SYSTEMS

The preliminary results of rHuEPO in experimental animals such as baboons ³ and thalassemia mice ⁴ were very encouraging and suggested that -globin chain synthesis is markedly increased following administration of rHuEPO at a dose at least 5- to 10-fold higher than that used to correct the anemia of patients with chronic renal failure. ¹⁶ Encouraging results were also obtained in synchronous erythroid cultures from TI and TM patients where alteration of rHuEPO concentrations resulted in a marked increase in HbF synthesis. On the basis of these results as well as the preliminary trials in patients with sickle cell anemia, ¹⁷ the foundation has been laid for preliminary trials of rHuEPO therapy in thalassesmia.

THE EFFECT OF rHuEPO ON PATIENTS WITH THALASSEMIA AND CHRONIC RENAL FAILURE

One of the first successful indications for treatment with rHuEPO was to correct the anemia of patients with chronic renal failure either before or after instituting dialysis. ¹⁶ The effective dose is in a range of 25 to 100 u/kg given subcutaneously twice a week. By coincidence, a certain percentage of the patients with anemia of chronic renal failure who were treated with rHuEPO, had - or ß-thalassemia trait. One study ¹⁸ included 3 patients with ß-thalassemia trait and one with -thalassemia trait. The dose requirements of rHuEPO in order to achieve a target Hb level of > 10g/dl and the ongoing maintenance dose were higher when compared to matched dialysis patients without thalassemia trait (Fig.1). One patient with HbH disease did not respond to 250 u/kg/week, unlike the patient with HbH disease reported by Winearls et al. who did respond to a dose of 600 u/kg/week. ¹⁹





In another study, 4 patients with ß thalassemia minor required higher doses of rHuEPO (75 u/kg x 3/week) compared with non-thalassemic controls. ²⁰ Lai et al. ²¹ also observed a smaller and delayed increase in Hb levels in three patients with -thalassemia trait and one with ß-thalassemia trait using a higher rHuEPO dose of 175 u/kg/w compared to non-thalassemia dialysis patients who received an average of 120 u/kg/w. In two patients with end-stage renal failure and ß-thalassemia trait, who were transfusion dependent, administration of rHuEPO resulted in stable Hb levels obviating the need for further transfusions. ²² A very intriguing observation has been recently reported in a group of 12 anemic hemodialyzed patients, in whom the level of HbF increased from <3% to a peak value of 48% after one month of treatment with rHuEPO (45 u/kg m x 3/week). A correlation was found between % HbF and the reticulocyte count, indicating the influence of the changes of erythroid activity on HbF levels. ²³ These astonishing results will have to be verified by additional studies. If indeed rHuEPO administration in thalassemia will increase the amount of HbF production, one could expect that patients with thalassemia and chronic renal failure would respond better to rHuEPO when compared to controls without thalassemia. However, as mentioned above, the available reports to date do not demonstrate the advantage expected from the results of the study by Bellizi et al. ²³ The conclusion from the present studies indicate that the presence of - or ß-thalassemia trait does not prevent correction of the anemia by rHuEPO in patients with chronic renal failure undergoing dialysis. But in order to reach a satisfactory therapeutic target of Hb level in such patients, one needs more time and higher doses of rHuEPO when compared to patients with end-stage renal failure without thalassemia

THE ROLE OF rHuEPO FOR PATIENTS WITH ß THALASSEMIA INTERMEDIA

The theoretical rationale for treatment of patients with thalassemia with rHuEPO was based on several observations: 1) the preliminary observations in experimental animals and in erythroid cultures that resulted in increase in the synthesis of HbF, ²⁴ 2) the discrepancy between endogenous serum EPO levels and the severity of the anemia in patients with thalassemia minor and intermedia, 3) a significant improvement in the quality of life in patients with ß-thalassemia intermedia who underwent splenectomy, which resulted in a temporary increase in Hb levels of 1-3g/dl. ²⁵ In the first trial, a relatively large dose of rHuEPO (l000 u/kg/week) was given twice a week to three patients with ß-thalassemia intermedia. ²⁶ The selected dose of rHuEPO was based on the doses used in baboons. The results showed an increase in total Hb levels by 2g/dl without any changes in % of HbF and in RBC indices. In a second preliminary trial, ²⁷ 2 out of 3 patients responded to a dose of subcutaneous injections of rHuEPO, (200-1000 u/kg x 3/week), by a 2-3 g/dl increase in Hb without changes in other erythroid parameters. An additional six patients treated for 10 weeks with similar amounts of rHuEPO failed to respond. ⁷
The longest trial of rHuEPO in ß-thalassemia intermedia was carried out in ten patients for 5-12 months. The results showed an increase in total Hb concentrations of 2-3 g/dl which was dependent on the dose of rHuEPO from 500 to 950 u/kg x 3/week (Fig.2). Seven splenectomized patients responded much better than three non-splenectomized patients. As in the preliminary reports, there were no changes in the percent of HbF, in globin-chain synthetic ratios, and in RBC indices throughout the period of study. Additional studies on the effects of rHuEPO in thalassemia were carried out by Nizli et al. in Turkey following administration of 500-1000 u/kg x 3/week for three months to ten patients with TI. In three patients, total Hb increased by at least 2 g/dl, which lasted as long as rHuEPO was given. ²⁹ In a second study, a sustained increase in Hb levels in 3 out of 16 patients with transfusion-dependent TM was observed, negating the need for further transfusions. Two patients did not require transfusions for more than 2 years. The positive response was confined to splenectomized patients, but apparently splenectomy was not the only factor that characterized the patients who responded to rHuEPO. ²⁹ As in the previous studies, there were no changes in any other erythroid parameters as well as in the levels of HbF. In one patient with thalassemia and severe iron overload, where iron chelation was not adequate, administration of rHuEPO was able to maintain higher Hb levels that permitted chronic phlebotomy and reduced the requirement for iron chelation therapy. ⁷ A similar therapeutic approach has been successfully applied in patients with chronic renal failure. ³⁰

THE IMPORTANCE OF ORAL IRON SUPPLEMENTATION WITH rHuEPO

In patients with anemia and chronic renal failure, several studies of iron metabolism and kinetics have indicated that oral or even intravenous (I.V.) iron supplementation is required in order to obtain an optimal hemopoietic response to rHuEPO. ³⁰ The basic difference between patients with anemia of chronic renal failure and patients with thalassemia is the fact that the latter have excess iron stored as ferritin or hemosiderin in many organs. In addition, their RBC also contain considerable amounts of iron that is not in the form of hemoglobin. The key question is whether upon administration of rHuEPO, the iron in their stores can be mobilized to meet the requirements created by the increased erythropoietic drive. From the present available data, the answer to the former question is not clear. Hypochromic microcytic RBC were formed following administration of rHuEPO without oral iron supplementation to five patients with sickle cell anemia, associated with a decrease in serum ferritin in three of them. This finding indicates that under erythropoietic stress, iron deficient erythropoiesis can occur in iron repleted subjects. ³¹ The availability of iron is rate limiting in response to rHuEPO in normal individuals. ^32,33 On the other hand, iron supplementation together with rHuEPO may increase the existing iron overload. ³⁴ The available data on oral iron in thalassemia patients who received rHuEPO is not sufficient to draw any conclusions. In most of the reported cases, oral iron supplementation was given in order to make sure of its availability for rHuEPO-mediated erythropoiesis, ^26,27 although in some patients the positive response to rHuEPO occurred without the addition of oral iron. ⁷ The only way to answer this question is by designing a well-controlled study of iron kinetics associated with an evaluation of the possible erythropoietic response in thalassemia patients who receive rHuEPO with or without oral iron supplementation.


Another approach is to try the effect of the oral iron chelator L1, which, unlike desferrioxamine (DF), is capable of removing free iron from iron-loaded thalassemic RBC. ³⁵ Since it is a weaker binder to iron than DF, it might release the chelated iron to meet the increased demands following administration of rHuEPO. A preliminary result of the positive role of L1 in promoting erythropoiesis in patients with rheumatoid arthritis and anemia has been previously reported. ³⁶

As pointed out by Olivieri, ⁷ rHuEPO in thalassemia patients may increase the risk of bone marrow expansion with the potential outcome of cortical destruction. Indeed, in one patient, mild bone pain was observed following a marked increase in Hb levels after receiving rHuEPO. ³⁷ However, in all the other reported cases, there were no major side effects that could be attributed to rHuEPO other than some intermittent skeletal discomfort which was controlled by bed rest and mild analgesics. An accelerated linear growth was found in the seven out of ten responding patients as expressed by changes in height standard deviation score for chronological age. ²⁸

The conclusions that can be drawn from the available data suggest that rHuEPO is capable of inducing erythropoietic response in patients with TI who are not regularly transfused. The magnitude of the response is dose related and was not uniform in all patients. Splenectomized patients seem to do better. It was rather disappointing that the erythropoietic drive resulted mainly in the production of thalassemic RBC since there were no changes in -chain synthesis, increased levels of HbF, and improvement of RBC indices. One of the major drawbacks of this treatment is the need for relatively high doses of rHuEPO in order to obtain a meaningful response. Since rHuEPO is an expensive drug, at this point its cost effectiveness will not allow it to be used worldwide, particularly in less developed countries where the number of thalassemia patients is relatively high and regular blood transfusion programs are still unavailable.

COMBINED ADMINISTRATION OF rHuEPO WITH MODULATORS OF HbF SYNTHESIS

As previously mentioned, the effect of rHuEPO was confined to an increase in the quantity of newly formed RBC. Therefore an attempt has been made to combine rHuEPO with drugs which are capable of improving the quality of thalassemic RBC by stimulating HbF synthesis. Most of the data has been obtained with hydroxyurea (HU), an antitumor agent that was initially tried in experimental animals. ³⁸ The use of HU was found to significantly decrease painful crises and the frequency of transfusions in patients with sickle cell anemia. ^6,17 On the other hand, the preliminary data on the use of HU in thalassemia suggests that although an increase in the percentage of HbF or in ß-globin chain synthesis, ³⁹ together with a consequent improvement in RBC indices has been found, it was not sufficient to increase significantly the level of total Hb and to modify the clinical course of the disease. ⁷ Therefore, the idea of combining an agent like rHuEPO which is capable of inducing a quantitative erythropoietic drive, together with an agent like HU, which is capable of inducing qualitative changes in the newly formed RBC, is most appealing. ⁴⁰ One can also assume that HU might spare non-cycling erythroid precursors which may undergo accelerated differentiation and maturation by adding rHuEPO. ⁴¹ This approach was first tried in sickle cell anemia and the results were not uniform. While Goldberg et al. ¹⁷ did not find any synergistic effect using the two drugs in 5 patients, Rodgers et al. ⁶ did find a synergistic effect of the two drugs in 4 patients with sickle cell anemia. The differences could be related to the dose of HU and rHuEPO, and to iron supplementation.
In thalassemia patients another theoretical advantage for the use of the two drugs in combination is that although rHuEPO may cause further bone marrow expansion, it may be neutralized to some extent by the effect of HU. In the first trial, rHuEPO was given alone s.c. for 12 weeks (500 u/kg x 3/w) together with oral iron and folic acid. In a subsequent period of 12 weeks HU was given (20 mg/kg) for 4 days every week while rHuEPO was given in the remaining 3 days of the week and was followed by an additional 12 weeks where HU (20 mg/kg x 4/w) was given alone. When each drug was given by itself, there was an increase in total Hb compared with pre-treatment levels. When the two drugs were given together, an additive positive effect on total Hb levels was found only in 2 out of 7 patients (Fig. 3). In all patients, the percent of Hb F and RBC indices improved when HU was given with or without rHuEPO. There were no significant HU-induced changes in other laboratory parameters or any major side effects which could be related to either drug throughout the trial period. In another short trial, rHuEPO (400 u/kg x 3/w and 800 u/kg x 2/w) was given together with HU (l500 mg/day x 4 days/week) for 12 weeks. In 8 out of 10 patients, total Hb increased with an average increment of 1.5g/dl. In some patients the percent HbF increased from 5% to 20% above baseline values in addition to improvement in RBC indices. ⁴³

SUMMARY AND CONCLUSIONS

From all the available data to date, one can conclude that treatment with rHuEPO increases the erythropoiesis of thalassemia RBC in patients with TI, in a dose-related pattern with an average increase of 1-3 g/dl over baseline levels. The minimum effective dose is at least 5 to 10 times higher than the dose used to correct the anemia in patients with chronic renal failure, starting in a range of 400 to 500 u/kg x 3/week and higher. It is not known why the response is not uniform in all patients. It seems that splenectomized patients respond better, most likely since in the absence of the spleen, the newly formed thalassemic RBC survive longer than in patients with an intact enlarged spleen. The response to rHuEPO does not correlate with the levels of endogenous serum EPO levels. At this point it is not known whether the addition of oral iron is mandatory for optimal response to rHuEPO. In order to improve the quality of the newly formed RBC, by increasing the synthesis of HbF and consequently the RBC indices, several agents have been tried. Among them are HU, short chain fatty acids ⁴⁴ and hemin. ⁴⁵ The preliminary data on combinations of rHuEPO with HU showed positive effects in several erythroid parameters but an additive effect on hemoglobin levels was not obtained in all treated patients. It is imperative to design well controlled clinical trials in order to ascertain what patient characteristics are predictive of a positive response to rHuEPO, the minimal effective dose, the time schedule and whether the addition of agents capable of inducing -globin chain synthesis besides HU will produce a synergistic effect that will improve both the quality and the quantity of erythropoiesis in patients with various forms of the thalassemia syndrome.

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