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Solid-phase microextraction (SPME) is an alternative method to dialysis and ultrafiltration for the determination of plasma protein binding (PPB) of drugs. It is particularly advantageous for complicated analytes where standard methods are not applicable. Di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a lead compound of novel thiosemicarbazone anti-cancer drugs, which entered clinical trials in 2016. However, this agent exhibited non-specific binding on filtration membranes and had intrinsic chelation activity, which precluded standard PPB methods. In this study, using a simple and fast procedure, we prepared novel SPME fibers for extraction of DpC based on a metal-free, silicon string support, covered with C18 sorbent. Reproducibility of the preparation process was demonstrated by the percent relative standard deviation (RSD) of ≤ 9.2% of the amount of DpC extracted from PBS by several independently prepared fibers. The SPME procedure was optimized by evaluating extraction and desorption time profiles. Suitability of the optimized protocol was verified by examining reproducibility, linearity, and recovery of DpC extracted from PBS or plasma. All samples extracted by SPME were analyzed using an optimized and validated UHPLC-MS/MS method. The developed procedure was applied to the in vitro determination of PPB of DpC at two clinically relevant concentrations (500 and 1000 ng/mL). These studies showed that DpC is highly bound to plasma proteins (PPB ≥ 88%) and this did not differ significantly between both concentrations tested. This investigation provides novel data in the applicability of SPME for the determination of PPB of chelators, as well as useful information for the clinical development of DpC. Graphical abstract.

Di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a promising analogue of the dipyridyl thiosemicarbazone class currently under development as a potential anti-cancer drug. In fact, this class of agents shows markedly greater anti-tumor activity and selectivity than the clinically investigated thiosemicarbazone, Triapine®. However, further development of DpC requires detailed data concerning its metabolism. Therefore, we focused on the identification of principal phase I and II metabolites of DpC in vitro. DpC was incubated with human liver microsomes/S9 fractions and the samples were analyzed using ultra-performance liquid chromatography (UPLC(TM)) with electrospray ionization quadrupole-time-of-flight (Q-TOF) mass spectrometry. An Acquity UPLC BEH C(18) column was implemented with 2 mM ammonium acetate and acetonitrile in gradient mode as the mobile phase. The chemical structures of metabolites were proposed based on the accurate mass measurement of the protonated molecules as well as their main product ions. Ten phase I and two phase II metabolites were detected and structurally described. The metabolism of DpC occurred via oxidation of the thiocarbonyl group, hydroxylation and N-demethylation, as well as the combination of these reactions. Conjugates of DpC and the metabolite, M10, with glucuronic acid were also observed as phase II metabolites. Neither sulfate nor glutathione conjugates were detected. This study provides the first information about the chemical structure of the principal metabolites of DpC, which supports the development of this promising anti-cancer drug and provides vital data for further pharmacokinetic and in vivo metabolism studies.

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.

This study was focused on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method development for quantification of a novel potential anticancer agent, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), in aqueous media. Solid Bp4eT was found to consist predominantly of the Z isomer, while in aqueous media, both isomers coexist. Sufficient separation of both isomers was achieved on a Synergi 4u Polar RP column with a mobile phase composed of 2 mM ammonium formate, acetonitrile, and methanol (30:63:7; v/v/v). The photo diode array analysis of both isomers demonstrated different absorption spectra which hindered UV-based quantification. However, an equal and reproducible response was found for both isomers using an MS detector, which enables the determination of the total content of Bp4eT (i.e., both E- and Z- isomeric forms) by summation of the peak areas of both isomers. 2-Hydroxy-1-naphthylaldehyde 4-methyl-3-thiosemicarbazone (N4mT) was selected as the internal standard. Quantification was performed in selective reaction monitoring using the main fragments of [M+H](+) (240 m/z for Bp4eT and 229 m/z for N4mT). The method was validated over 20-600 ng/ml. This procedure was applied to a preformulation study to determine the proper vehicle for parenteral administration. It was found that Bp4eT was poorly soluble in aqueous media. However, the solubility can be effectively improved using pharmaceutical cosolvents. In fact, a 1:1 mixture of PEG 300/0.14 M saline markedly increased solubility and may be a useful drug formulation for intravenous administration. This investigation further accelerates development of novel anticancer thiosemicarbazones. The described methods will be useful for analogs currently under development and suffering the same analytical issue.