ABSTRACT
Hydroxyurea (HU) induces fetal hemoglobin (Hb F) production in patients with sickle cell anemia. The therapeutic dosage of HU used for Hb F induction often elicits myelosuppression, which becomes its major associated complication. We examined the effect of HU on hemoglobin modulation and the role of radical scavengers on these induced changes. In vitro exposure of human blood to various concentrations of HU at predetermined time intervals induced a progressive dose-dependent oxidation (MetHb formation) of both adult (Hb AA) and sickle (Hb SS) hemoglobins. The oxidative effect of HU on Hb SS was 3 times greater than its effect on Hb AA. Similar but less profound changes were observed in H2O2-treated samples. Hb F was, however, observed to be relatively resistant to HU-induced oxidative damage. A substantial protective effect of Hb by alpha-tocopherol, ascorbic acid, and D-mannitol was observed during pretreatment of Hb AA and Hb SS blood samples. Analyses of the hemoglobins and their globin chain components by high-performance liquid chromatography revealed a considerable protective effect by these free radical scavengers. These results indicate that the HU-induced damage of hemoglobin and their component globin chains can be reduced by radical scavengers.
Subject(s)
Free Radical Scavengers/pharmacology , Hemoglobin, Sickle/metabolism , Hydroxyurea/pharmacology , Methemoglobin/metabolism , Adolescent , Adult , Antioxidants/pharmacology , Dose-Response Relationship, Drug , Endopeptidases/metabolism , Fetal Hemoglobin/metabolism , Heme/metabolism , Hemoglobin A/metabolism , Humans , Hydrogen Peroxide/pharmacology , Hydroxyurea/administration & dosage , Hydroxyurea/adverse effects , Infant, Newborn , Mannitol/pharmacology , Oxidation-Reduction , SpectrophotometryABSTRACT
A new procedure using high-performance liquid chromatography (HPLC) with ultraviolet detection to assay hydroxyurea (HU) levels in plasma has been developed. The drug was isolated from plasma by a direct deproteinization process with sulfosalicylic acid. Following neutralization of the acidic supernatant, an aliquot was loaded onto an Aminex HPX-72S column (300x7.8 mm). Chromatography was performed at 55 degrees C using a mobile phase consisting of acetonitrile-0.025 M ammonium sulfate buffer (pH 8.5) including 0.1% triethylamine, 0.01 M sodium sulfate, and 5 mM sodium heptane sulfonate. The UV absorbance of effluent was monitored at 214 nm. A flow-rate of 0.8 ml/min was used for analyzing HU in both human and mouse plasma. Under these conditions, the drug eluted at 12.6 min. The assay possessed linearity up to 425 microg/ml, with a lower limit of quantitation of 3.32+/-0.0004 microg/ml (mean+/-S.D., n=10). Intra-day and inter-day coefficients of variation were less than 8.5% and 8.7% respectively. Absolute differences were less than 7.4%. The method has been employed in clinical studies and the sensitivity of the assay was shown to be adequate for characterizing the plasma pharmacokinetics of HU in mice. In conclusion, the procedure described herein could be ideally suited for therapeutic monitoring of hydroxyurea.
Subject(s)
Antisickling Agents/blood , Chromatography, High Pressure Liquid/methods , Hydroxyurea/blood , Animals , Antisickling Agents/pharmacokinetics , Humans , Hydroxyurea/pharmacokinetics , Mice , Mice, Inbred ICR , Reproducibility of Results , Sensitivity and Specificity , Ultraviolet RaysABSTRACT
Trimidox (3,4,5-trihdroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase with accompanied growth inhibition and differentiation of mammalian cells. Hydroxyurea (HU) is the only ribonucleotide reductase inhibitor in clinical use for the treatment and management of sickle cell anemia, since this compound increases fetal hemoglobin (Hb F) production: a potent inhibitor of sickle hemoglobin (Hb SS) polymerization. However, the main limitations of HU is its lack of potency, myelosuppression and short half life. These studies investigated the effects of trimidox on the induction of hemoglobin and F-cells production in K562 erythroleukemia cells. Our study reveals that trimidox exhibits concentration dependent inhibitory effect on K562 cells with increase in benzidine positive normoblasts and F-cells production as well as morphological changes typical of erythroid differentiation. These findings provide the first evidence that the growth inhibitory differentiation of cells induced by trimidox enhance hemoglobin and F-cells production.
