The Effect of Oral Iron Chelator Deferiprone on Iron Overload and Oxidative Stress in Patients with Myelodysplastic Syndromes: A Study by the Israeli MDS Working Group.

BACKGROUND: Most patients with lower risk myelodysplastic neoplasms or syndromes (MDSs) become RBC transfusion-dependent, resulting in iron overload, which is associated with an increased oxidative stress state. Iron-chelation therapy is applied to attenuate the toxic effects of this state. Deferiprone (DFP) is an oral iron chelator, which is not commonly used in this patient population, due to safety concerns, mainly agranulocytosis. The purpose of this study was to assess the effect of DFP, on oxidative stress parameters in iron-overloaded RBC transfusion-dependent patients with lower risk MDSs. METHODS: Adult lower risk MDS patients with a cumulative transfusion burden of >20 red blood cell units and evidence of iron overload (serum ferritin >1,000 ng/mL) were included in this study. DFP was administered (100 mg/kg/day) for 4 months. Blood samples for oxidative stress parameters and iron overload parameters were done at baseline and monthly: reactive oxygen species (ROS), phosphatidylserine, reduced glutathione, membrane lipid peroxidation, serum ferritin, and cellular labile iron pool. The primary efficacy variable was ROS. Tolerability and side effects were recorded as well. A paired t test was applied for statistical analyses. RESULTS: Eighteen patients were treated with DFP. ROS significantly decreased in all cell lineages: median decrease of 58.6% in RBC, 33.3% in PMN, and 39.8% in platelets (p < 0.01 for all). Other oxidative stress markers improved: phosphatidylserine decreased by 57.95%, lipid peroxidase decreased by 141.3%, and reduced gluthathione increased by 72.8% (p < 0.01 for all). The iron-overload marker and cellular labile iron pool decreased by 35% in RBCs, 44.3% in PMN, and 46.3% in platelets (p < 0.01 for all). No significant changes were observed in SF levels. There were no events of agranulocytosis. All AEs were grades 1-2. CONCLUSIONS: Herein, we showed preliminary evidence that DFP decreases iron-induced oxidative stress in MDS patients with a good tolerability profile (albeit a short follow-up period). No cases of severe neutropenia or agranulocytosis were reported. The future challenge is to prove that reduction in iron toxicity will eventually be translated into a clinically meaningful improvement.

Toxicity of iron overload and iron overload reduction in the setting of hematopoietic stem cell transplantation for hematologic malignancies.

Iron is an essential element for key cellular metabolic processes. However, transfusional iron overload (IOL) may result in significant cellular toxicity. IOL occurs in transfusion dependent hematologic malignancies (HM), may lead to pathological clinical outcomes, and IOL reduction may improve outcomes. In hematopoietic stem cell transplantation (SCT) for HM, IOL may have clinical importance; endpoints examined regarding an impact of IOL and IOL reduction include transplant-related mortality, organ function, infection, relapse risk, and survival. Here we review the clinical consequences of IOL and effects of IOL reduction before, during and following SCT for HM. IOL pathophysiology is discussed as well as available tests for IOL quantification including transfusion history, serum ferritin level, transferrin saturation, hepcidin, labile plasma iron and other parameters of iron-catalyzed oxygen free radicals, and organ IOL by imaging. Data-based recommendations for IOL measurement, monitoring and reduction before, during and following SCT for HM are made.

Uptake of non-transferrin iron by erythroid cells.

Most of the iron in the plasma is bound to transferrin (Tf) and is taken up by cells through their surface Tf receptors (TfRs). Under pathological conditions of iron-overload, the plasma iron which is in excess of the binding capacity of Tf is present as non-Tf-bound iron. We probed the uptake of non-Tf iron and its consequences on the oxidative status of peripheral RBC and reticulocytes as well as developing erythroid precursors grown in vitro. The cells were exposed to ferrous ammonium sulfate under Tf-supplemented and Tf-free conditions. Using flow cytometry techniques, we found that both the TfR-deficient mature RBC and their TfR-containing precursors at all stages of maturation can take up non-Tf iron that accumulates as redox-active labile iron and generates reactive oxygen species. Such a mechanism may account for ineffective erythropoiesis of developing precursors in the bone marrow and for the shortening of the lifespan of mature RBCs in the circulation.

Increased serum hepcidin levels during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndrome.

Hepcidin is a major regulator of iron metabolism. We evaluated changes in serum hepcidin during 3 months of therapy with the iron-chelator deferasirox in patients with low-risk myelodysplastic syndrome and iron overload. Serum hepcidin was found to be high in these patients, correlated with their iron and oxidative status, and further increased by treatment with deferasirox. These findings support the concept that the hepcidin level represents a balance between the stimulating effect of iron overload and the inhibitory effects of erythropoietic activity and oxidative stress. These preliminary findings favour the rationale for iron chelation therapy in such patients.

Oxidative stress contributes to hemolysis in patients with hereditary spherocytosis and can be ameliorated by fermented papaya preparation.

In the present study, we questioned the role of oxidative stress in hereditary spherocytosis (HS), where red blood cells (RBC) have a shortened survival due to primary deficiency in membrane proteins. Using flow cytometry techniques, we showed that RBC derived from 17 HS patients of seven families generate more reactive oxygen species, membrane lipid peroxides, and less reduced glutathione than normal RBC. Following in vitro incubation of HS-RBC from seven patients with a fermentation bioproduct of Carica papaya (fermented papaya preparation (FPP)) with known antioxidative properties, oxidative stress markers were significantly reduced. Similar results were obtained following treatment with FPP for 3 months of 10 adult HS patients, as well as decreased tendency to undergo hemolysis. The hemoglobin levels increased by >1 g/dl, mean corpuscular hemoglobin concentration decreased by >1 g/dl, and the reticulocyte count decreased by 0.93%. Concomitantly, lactic dehydrogenase decreased by 17% and indirect bilirubin by 50%. A significant decrease in malonyldialdehyde was also detected. These data indicate that oxidative stress plays an important role in the pathophysiology of HS which can be ameliorated by an antioxidant such as FPP. Additional clinical trials with FPP and other antioxidants are warranted.

The role of antioxidants and iron chelators in the treatment of oxidative stress in thalassemia.

On the basis of all the presented data, one can conclude that oxidative stress plays a major role in the pathophysiology of thalassemia and other congenital and acquired hemolytic anemias. Free extracellular (labile plasma iron, LPI) and intracellular (labile iron pool, LIP) iron species that have been identified in thalassemic blood cells are responsible for generation of oxidative stress by catalyzing formation of oxygen radicals over the antioxidant capacity of the cell. Consequently, there is a rationale for iron chelation to eliminate the free-iron species, which in this respect, act like antioxidants. In addition, antioxidants such as vitamin E and polyphenols are also capable of ameliorating increased oxidative stress parameters and, given together with iron chelators, may provide a substantial improvement in the pathophysiology of hemolytic anemias and particularly in thalassemia.

Amelioration of oxidative stress in red blood cells from patients with beta-thalassemia major and intermedia and E-beta-thalassemia following administration of a fermented papaya preparation.

In beta-hemoglobinopathies, such as beta-thalassemia (thal) and sickle cell anemia, the primary defects are mutations in the beta-globin gene. However, many aspects of the pathophysiology are mediated by oxidative stress. Fermented papaya preparation (FPP), a natural health food product obtained by biofermentation of carica papaya, has been shown to limit oxidative stress both in vitro and in vivo. We studied the effect of FPP on two groups of beta-thal patients: beta-thal, major and intermedia, (in Israel) and E-beta-thal (in Singapore). The results indicated that in both groups FPP treatment increased the content of reduced glutathione (GSH) in red blood cells (RBC), and decreased their reactive oxygen species (ROS) generation, membrane lipid peroxidation, and externalization of phosphatidylserine (PS), indicating amelioration of their oxidative status, without a significant change in the hematological parameters. Since the turnover of the erythron is relatively slow, it is possible that longer duration of treatment, probably with the addition of an iron chelator, is required in order to achieve the latter goals.

Fermented papaya preparation as redox regulator in blood cells of beta-thalassemic mice and patients.

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. Fermented papaya preparation (FPP) was tested for its antioxidant effects: the scavenging effect was determined spectrofluorometrically in a cell-free system using 2'-7'-dichlorofluorescin-diacetate (DCF). Both spontaneous and H(2)O(2)-induced DCF oxidations were decreased by FPP in a dose-dependent fashion. Using flow cytometry, it was shown that in vitro treatment of blood cells from beta-thalassemic patients with FPP increased the glutathione content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their reactive oxygen species, membrane lipid peroxidation and externalization of phosphatidylserine. These effects result in (a) reduced thalassemic RBC sensitivity to hemolysis and phagocytosis by macrophages; (b) improved PMN ability to generate oxidative burst - an intracellular mechanism of bacteriolysis, and (c) reduced platelet tendency to undergo activation, as reflected by fewer platelets carrying external phosphatidylserine. Oral administration of FPP to beta-thalassemic mice (50 mg/mouse/day for 3 months) and to patients (3 g x 3 times/day for 3 months), reduced all the above mentioned parameters of oxidative stress (p < 0.001 in mice and p < 0.005 in patients). These results suggest that FPP, as a potent antioxidant, might alleviate symptoms associated with oxidative stress in severe forms of thalassemia.

Effects of rapamycin on accumulation of alpha-, beta- and gamma-globin mRNAs in erythroid precursor cells from beta-thalassaemia patients.

We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.

Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice.

OBJECTIVE: We previously demonstrated that cellular copper is involved in the regulation of proliferation and differentiation of hematopoietic progenitor cells. Modulation of cellular copper was achieved by supplementing the culture with a copper chelator that reduces cell copper content, or copper salts, which elevate the level of cellular copper. In the present study, we evaluated the effect of short-term (3-week) treatment with the copper chelator tetraethylenepentamine (TEPA) on short- and long-term (up to 11 weeks) ex vivo expansion of hematopoietic progenitors, as well as on their SCID engraftment potential. MATERIALS AND METHODS: Cord blood-derived purified CD34+ cells were grown in liquid medium supplemented with the cytokines stem cell factor, thrombopoietin, Flt3 ligand, and IL-6, and the chelator TEPA for the first 3 weeks and then for up to 11 weeks with cytokines alone. Control cultures were supplemented with cytokines alone for the entire culture duration. Cultured cells were characterized by immunophenotyping and cloning (CFUc). Transplantability was assayed by injection of repurified CD34+ cells into NOD/SCID mice. RESULTS: In the short term, TEPA supported increased percentages of early progenitors over control cultures incubated with cytokines alone (CD34(+)CD38-, p=0.001 and CD34(+)Lin-, p=0.016). In the long term, TEPA pretreated cultures showed prolonged expansion of CD34+ cells (p=0.01) and CFUc (p=0.002) compared with that of untreated cultures. The SCID engraftment potential of CD34+ cells repurified from the TEPA-treated cultures was higher compared with that of the control, i.e., only cytokine-treated cultures (p=0.03). CONCLUSION: TEPA enabled preferential proliferation of early progenitor cells with the phenotype CD34(+)CD38- and CD34(+)CD38- Lin- during the first weeks of culture, resulting in the observed increased long-term ex vivo expansion and engraftment capabilities.

The effect of tetraethylenepentamine, a synthetic copper chelating polyamine, on expression of CD34 and CD38 antigens on normal and leukemic hematopoietic cells.

We have previously found that the synthetic polyamine tetraethylenepentamine (TEPA) significantly delayed differentiation and prolonged expansion of cord-blood derived HPC in cytokine-supplemented cultures. Most HPC have the CD34+CD38+ phenotype, but the minority CD34+38- cells are primitive subset of HPC that have the potential for long-term repopulation in vivo. We investigated the effect of TEPA on the CD34/CD38 surface antigen expression of human myeloid leukemia cell lines as well as normal cord blood derived hematopoietic cells. Confirming previous results, our data showed that both the leukemic and normal cells increased their CD38 expression when grown in serum-containing medium or when treated with retinoic acid. In the present study, we found that TEPA inhibited CD38 under these conditions in both normal and leukemic cells. As for CD34, TEPA increased the proportion of CD34 cells in short- and long-term normal cultures but not in the leukemic cell lines. These results suggest that ex vivo expansion of HPC depends on the presence of CD34+CD38- cells and that TEPA prolongs HPC expansion by inhibiting the CD38- to CD38+ transition.

Cellular copper content modulates differentiation and self-renewal in cultures of cord blood-derived CD34+ cells.

Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood-derived CD34+ cells were cultured in liquid medium supplemented with Kit- ligand, FLt3, interleukin 6 (IL-6), thrombopoietin and IL-3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/10(7) cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/10(7) cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/10(7) cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony-forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34+ cells in low Cu was extended (12-16 weeks), and in high Cu was shortened (2-4 weeks), compared with control cultures (6-8 weeks). These effects required modulation of intracellular Cu only during the first 1-3 weeks of the culture; the long-term effects persisted thereafter, suggesting that the decision process for either self-renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.