Comparative study of dihydroartemisinin and artesunate safety in healthy Thai volunteers.

OBJECTIVE: As part of new drug development initiatives in Thailand, a new tablet formulation of dihydroartemisinin (DHA, an antimalarial drug) has been developed. Our previous bioequivalence study indicated that the new and reference DHA formulations were well tolerated; however, a significant decrease in hemoglobin was detected after a single 200-mg oral dose. To explore further, a clinical study with an emphasis on hematological parameters was conducted. METHODS: A single-center, randomized, single-blind, cross-over clinical study was conducted in 18 healthy volunteers with a dosage of 300 mg daily for 2 days. Artesunate was used as a comparator. Adverse events were monitored and laboratory parameters on study Days 0, 2, 5, and 7 post drug administrations were analyzed. RESULTS: Eighteen volunteers completed both rounds of the study. Both drugs were well tolerated. All adverse events were mild. Significant decrease in hemoglobin compared to baseline was detected for both drugs 7 days after administration (DHA: 0.48 g/dl, p = 0.007; artesunate 0.38 g/dl, p = 0.001). Transient bone marrow suppression was evidenced by reduction of reticulocytes with a lowest number on study Day 5 (artesunate 75% reduction in reticulocyte count; DHA 47%, p < 0.001 for both drugs compared to baseline). CONCLUSION: The present study confirmed our previous finding on significant decrease in hemoglobin. Artesunate appeared to have more negative effects on the numbers of reticulocytes and white blood cells than DHA. Systemic laboratory and toxicity profiles presented in this study may be used as a framework for future clinical studies of artemisinin and its derivatives.

Hematopoietic progenitor cells in the blood and bone marrow in various hematologic disorders.

Hematopoietic progenitor cells are present in the blood and the bone marrow. Changes in the numbers of hematopoietic progenitor cells reflect alteration of pluripotent stem cells. We discuss such changes in common hematologic diseases including aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and thalassemia. In aplastic anemia, the numbers of burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) are much decreased; the decrease still exists after recovery from therapy. In PNH, the numbers of progenitor cells are low, even in the presence of marrow hypercellularity. In thalassemia, the numbers of progenitor cells are much increased; more pronounced in splenectomized patients.

The unusual pathobiology of hemoglobin constant spring red blood cells.

Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other alpha-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV "normalizing" effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional alpha-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and alpha-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional alpha-thalassemic variants and found that in addition to oxidized beta-globin chains, oxidized alpha cs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized alpha cs- and beta-globin chains. The membrane alterations induced by alpha cs chains are more akin to those induced by beta A-globin chains than those induced by the alpha A-globin chains that accumulate in the beta-thalassemias. Thus, each globin chain, alpha cs, alpha A, beta A, appears to produce its own form of membrane perturbation.

An in vitro study on thalassemic erythroid precursors in liquid culture.

Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.

Thalassemic serum inhibits endothelial cell mitosis in vitro.

Human umbilical vein endothelial cells were cultured in vitro using Iscove's Modified Dulbecco's Medium (IMDM) supplemented with either pooled normal human serum, or pooled thalassemic serum, or autologous umbilical cord serum, or fetal bovine serum. The mitotic activity was determined under the inverted phase contrast microscope and the number of mitotic cells was counted. Our results showed that the mitotic cells decreased in endothelial cell culture with thalassemic serum as compared with normal human serum, autologous umbilical cord serum or fetal bovine serum. The percentage of mitotic cells decreased on day 3 in the presence of beta-thalassemia/HbE serum from both splenectomized and non-splenectomized patients as compared with normal or autologous umbilical cord serum. In the presence of alpha-thalassemic serum, a similar outcome was also observed. From this study we can conclude that the thalassemic sera might contain factors which affect the endothelial cell growth and proliferation by inhibiting mitosis in vitro.

Alterations and pathology of thalassemic red cells: comparison between alpha- and beta-thalassemia.

Two main types of thalassemia have been categorized according to defective production of the globin gene ie alpha-thalassemia and beta-thalassemia. We report different red cell abnormalities between these two types. The study included 139 thalassemic patients including 91 patients with hemoglobin (Hb) H disease (52 cases with the classical genotype and 39 cases with Hb Constant Spring) and 48 were beta-thalassemia/Hb E disease. The deformability index of thalassemic red cells measured by laser diffractometer was significantly lower than that of normal red cells. Increased susceptibility of the thalassemic red cells to monocyte phagocytosis was markedly noted. Few sialic acid molecules were scattered on red cell surface of thalassemic red cells. Reticulocytes with delayed maturation stage were also observed in thalassemia indicating enhanced release from the bone marrow. The alpha-thalassemic red cells had relatively better deformability, increased susceptibility to phagocytosis, reduced sialic acid content and greater degree irregular distribution of sialic acid on red cell surface as compared to beta-thalassemic red cells. The alpha-type with hemoglobin Constant Spring (Hb CS) had increased percentage of reticulocyte and young reticulocyte (high fluorescent intensity) as compared to beta-thalassemic red cells. The different abnormalities between alpha- and beta-thalassemic red cells may lead to different mechanism of red cell destruction and different severity of the disease.

Number and maturation of reticulocytes in various genotypes of thalassaemia as assessed by flow cytometry.

Ineffective erythropoiesis is a prominent defect leading to anaemic status in thalassaemic patients. Reticulocyte enumeration in the peripheral blood is a non-aggressive method of measuring bone marrow erythropoietic activity. We used an automated reticulocyte counter (Sysmex R-3000) to determine the number and maturation level of circulating reticulocytes among various types of thalassaemia: non-splenectomized beta-thalassaemia/haemoglobin E (beta E) and splenectomized cases (beta E-S), classical haemoglobin H disease (H), haemoglobin H disease with haemoglobin Constant Spring (H/CS), homozygous haemoglobin Constant Spring (CS/CS), homozygous haemoglobin E (EE), heterozygous thalassaemics and other rare combinations. Haemoglobin H disease has a higher absolute count than beta-thalassaemia (beta E), indicating relatively better compensatory erythropoiesis in haemoglobin H disease. Those with CS genes (H/CS and CS/CS) have poorer reticulocyte maturation than any other type of thalassaemia with rather high absolute numbers, especially in H/CS. This indicates a severer degree of ineffective erythropoiesis in beta-thalassaemia (beta E), which reflects an insufficient rise in reticulocyte number in comparison with alpha-thalassaemia (H). The presence of haemoglobin Constant Spring is associated with abnormally low reticulocyte maturation due to enhanced erythrocyte production or direct effect of Constant Spring globin itself, both still unexplained with the current information. The splenectomized beta E has increased reticulocyte number and cells with high DNA content, probably nucleated red cells, designated as the upper particle count parameter. However, there is the same degree of reticulocyte maturation in non-splenectomized and splenectomized beta E patients, suggesting a role for splenic pooling of reticulocytes.