Di(2-pyridyl)ketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di(2-pyridyl)ketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) are novel, highly potent and selective anti-tumor and anti-metastatic drugs. Despite their structural similarity, these agents differ in their efficacy and toxicity in-vivo. Considering this, a comparison of their pharmacokinetic and pharmaco/toxico-dynamic properties was conducted to reveal if these factors are involved in their differential activity. Both compounds were administered to Wistar rats intravenously (2 mg/kg) and their metabolism and disposition were studied using UHPLC-MS/MS. The cytotoxicity of both thiosemicarbazones and their metabolites was also examined using MCF-7, HL-60 and HCT116 tumor cells and 3T3 fibroblasts and H9c2 cardiac myoblasts. Their intracellular iron-binding ability was characterized by the Calcein-AM assay and their iron mobilization efficacy was evaluated. In contrast to DpC, Dp44mT undergoes rapid demethylation in-vivo, which may be related to its markedly faster elimination (T1/2 = 1.7 h for Dp44mT vs. 10.7 h for DpC) and lower exposure. Incubation of these compounds with cancer cells or cardiac myoblasts did not result in any significant metabolism in-vitro. The metabolism of Dp44mT in-vivo resulted in decreased anti-cancer activity and toxicity. In conclusion, marked differences in the pharmacology of Dp44mT and DpC were observed and highlight the favorable pharmacokinetics of DpC for cancer treatment.
The iron chelator, 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone (Bp4eT), was identified as a lead compound of the 2-benzoylpyridine thiosemicarbazone series, which were designed as potential anti-cancer agents. This ligand has been shown to possess potent anti-proliferative activity with a highly selective mechanism of action. However, further progress in the development of this compound requires data regarding its metabolism in mammals. The aim of this study was to identify the main in vitro and in vivo phase I metabolites of Bp4eT using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two metabolites were detected after incubation of this drug with rat and human liver microsomal fractions. Based on LC-MS(n) analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N (3)-ethyl-N (1)-[phenyl(pyridin-2-yl)methylene]formamidrazone, with both resulting from the oxidation of the thiocarbonyl group. The identity of these metabolites was further shown by LC-MS/MS analysis of these latter compounds which were prepared by oxidation of Bp4eT with hydrogen peroxide and their structures confirmed by nuclear magnetic resonance and infrared spectra. Both the semicarbazone and the amidrazone metabolites were detected in plasma, urine, and feces after i.v. administration of Bp4eT to rats. In addition, another metabolite that could correspond to hydroxylated amidrazone was found in vivo. Thus, oxidative pathways play a major role in the phase I metabolism of this promising anti-tumor agent. The outcomes of this study will be further utilized for: (1) the development and validation of the analytical method for the quantification of Bp4eT and its metabolites in biological materials; (2) to design pharmacokinetic experiments; and to (3) evaluate the potential contribution of the individual metabolites to the pharmacodynamics/toxico-dynamics of this novel anti-proliferative agent.
Novel thiosemicarbazone metal chelators are extensively studied anti-cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC-MS/MS method for the simultaneous quantification of 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1-E and M1-Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C18 column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid-phase extraction on 96-well plates. This method was validated over the concentration range of 0.18-2.80 μM for Bp4eT, 0.02-0.37 μM for both M1-E and M1-Z, and 0.10-1.60 μM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration-time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti-cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class.
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