ascorbic-acid and 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone

An electron paramagnetic resonance (EPR) method was used to determine the concentration of the antitumor agent Triapine in BEAS-2B cells when Triapine was bound to iron (Fe). Knowledge of the concentration of Fe-Triapine in tumor cells may be useful to adjust the administration of the drug or to adjust iron uptake in tumor cells. An EPR spectrum is obtained for Fe(3+)-Triapine, Fe(3+)(Tp)

Topics: Ascorbic Acid; Cells, Cultured; Electron Spin Resonance Spectroscopy; Humans; Iron; Pyridines; Thiosemicarbazones; Transferrin

Thiosemicarbazones are a group of compounds that have received comprehensive investigation as anticancer agents. The antitumor activity of the thiosemicarbazone, 3-amino-2-pyridinecarboxaldehyde thiosemicarbazone (3-AP; triapine), has been extensively assessed in more than 20 phase I and II clinical trials. These studies have demonstrated that 3-AP induces methemoglobin (metHb) formation and hypoxia in patients, limiting its usefulness. Considering this problem, we assessed the mechanism of metHb formation by 3-AP compared with that of more recently developed thiosemicarbazones, including di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). This was investigated using intact red blood cells (RBCs), RBC lysates, purified oxyhemoglobin, and a mouse model. The chelation of cellular labile iron with the formation of a redox-active thiosemicarbazone-iron complex was found to be crucial for oxyhemoglobin oxidation. This observation was substantiated using a thiosemicarbazone that cannot ligate iron and also by using the chelator, desferrioxamine, that forms a redox-inactive iron complex. Of significance, cellular copper chelation was not important for metHb generation in contrast to its role in preventing tumor cell proliferation. Administration of Dp44mT to mice catalyzed metHb and cardiac metmyoglobin formation. However, ascorbic acid administered together with the drug in vivo significantly decreased metHb levels, providing a potential therapeutic intervention. Moreover, we demonstrated that the structure of the thiosemicarbazone is of importance in terms of metHb generation, because the DpT analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), does not induce metHb generation in vivo. Hence, DpC represents a next-generation thiosemicarbazone that possesses markedly superior properties. This investigation is important for developing more effective thiosemicarbazone treatment regimens.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Cell Proliferation; Deferoxamine; Drug Interactions; Erythrocytes; Humans; Hypoxia; Iron; Iron Chelating Agents; Kinetics; Methemoglobin; Mice; Oxidation-Reduction; Oxyhemoglobins; Pyridines; Thiosemicarbazones

Tumors are sensitive to iron (Fe) chelation therapy with the clinically used chelator desferrioxamine (DFO). Recently, the potent inhibitor of ribonucleotide reductase, Triapine, has entered clinical trials as an anticancer agent. This compound is a potential Fe chelator, but despite this, no investigations have examined its effect on cellular Fe metabolism. This is essential for understanding its mechanism of action and clinical effects.. We compared the effect of Triapine with DFO, and also with the novel Fe chelator, 311, which shows marked antiproliferative activity. This latter ligand was relevant to compare, because it is tridentate like Triapine and shares structural similarity. We assessed the effects of chelators on proliferation, Fe uptake, Fe efflux, the expression of cell cycle control molecules, and iron-regulatory protein-RNA-binding activity. Redox activity was determined by ascorbate oxidation, benzoate hydroxylation, plasmid DNA degradation, and the precipitation of cellular DNA. These studies have been performed using several neuroepithelioma and neuroblastoma cell lines and a variety of normal cell types including fibroblasts, umbilical vein endothelial cells, skeletal muscle cells, monocyte-derived macrophages, and bone marrow stem cells.. Triapine was twice as effective as DFO at mobilizing (59)Fe from prelabeled cells but was much less efficient than 311. In terms of preventing (59)Fe uptake from Tf, Triapine and DFO had similar activity, having far less efficacy than 311. All three of the chelators showed greater activity against the proliferation of neoplastic than of normal cells, the effect of 311 and Triapine being similar and these two chelators being significantly (P < 0.0001) more active than DFO. Complexation of Triapine with Fe had no appreciable effect on its antiproliferative activity, whereas addition of Fe totally inhibited the effects of DFO and 311. Furthermore, the Triapine Fe complex was shown to be redox active.. The cytotoxic mechanism of action of Triapine was different from that of DFO and 311, with the combined action of Fe chelation and free radical generation being involved.

Topics: Animals; Ascorbic Acid; Blotting, Northern; Blotting, Western; Cell Division; Chelating Agents; Deferoxamine; DNA; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Free Radicals; Glutathione; Humans; Iron; Iron Chelating Agents; Isoniazid; Models, Chemical; Oxidation-Reduction; Oxygen; Plasmids; Protein Binding; Pyridines; Rats; Thiosemicarbazones; Time Factors; Tumor Cells, Cultured

The objective of this study was to develop an injectable formulation of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) suitable for intravenous infusion. The solubility of 3-AP in different solvents and pH conditions was determined. The developed formulation underwent stability assessment and compatibility testing with large volume parenteral (LVP) solutions. The aqueous solubility of 3-AP was found to be 0.1 mg/ml and could only be increased marginally by altering the pH or adding surfactants. To achieve the desired concentration (> 4 mg/ml), 3-AP was formulated at 5-10 mg/ml in a nonaqueous system consisting of 70% polyethylene glycol 300 and 30% ethanol. However, 3-AP readily precipitated from this formulation when diluted with LVP solutions. Dilution-induced drug precipitation was eliminated by acidifying the solution with citric acid. Ascorbic acid, 0.1%, was found to minimize oxidative degradation of 3-AP. Accelerated stability data indicated that the formulation is compatible with the packaging components and is chemically stable at 2-8 degrees C, and retained > 90% of 3-AP at 40 degrees C for 3 months. Simulated infusion studies showed that the citric acid formulation was compatible with LVP solutions. However, because of the potential of extraction of plasticizers from polyvinyl chloride (PVC) plastic containers, it is recommended that the formulation be diluted in glass containers prior to administration.

Topics: Antineoplastic Agents; Antioxidants; Ascorbic Acid; Chemical Precipitation; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Drug Stability; Ethanol; Glass; Hydrogen-Ion Concentration; Infusions, Intravenous; Plasticizers; Polyethylene Glycols; Pyridines; Rubber; Solubility; Solvents; Spectrophotometry, Ultraviolet; Thiosemicarbazones