[Show abstract][Hide abstract] ABSTRACT: Cancer cells have a high iron requirement and many experimental studies, as well as clinical trials, have demonstrated that iron chelators are potential anti-cancer agents. The ligand, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), demonstrates both potent anti-neoplastic and anti-retroviral properties. In this study, Bp4eT and its recently identified amidrazone and semicarbazone metabolites were examined and compared with respect to their anti-proliferative activity towards cancer cells (HL-60 human promyelocytic leukemia, MCF-7 human breast adenocarcinoma, HCT116 human colon carcinoma and A549 human lung adenocarcinoma), non-cancerous cells (H9c2 neonatal rat-derived cardiomyoblasts and 3T3 mouse embryo fibroblasts) and their interaction with intracellular iron pools. Bp4eT was demonstrated to be a highly potent and selective anti-neoplastic agent that induces S phase cell cycle arrest, mitochondrial depolarization and apoptosis in MCF-7 cells. Both semicarbazone and amidrazone metabolites showed at least a 300-fold decrease in cytotoxic activity than Bp4eT towards both cancer and normal cell lines. The metabolites also lost the ability to: (1) promote the redox cycling of iron; (2) bind and mobilize iron from labile intracellular pools; and (3) prevent 59Fe uptake from 59Fe-labeled transferrin by MCF-7 cells. Hence, this study demonstrates that the highly active ligand, Bp4eT, is metabolized to non-toxic and pharmacologically inactive analogs, which most likely contribute to its favorable pharmacological profile. These findings are important for the further development of this drug candidate and contribute to the understanding of the structure-activity relationships of these agents.
[Show abstract][Hide abstract] ABSTRACT: Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator-boronyl salicylaldehyde isonicotinoyl hydrazone-BSIH along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC–UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7–115 and 4–115 μM, respectively, and applied to BSIH activation experiments in vitro. An LC–MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06–23 and 0.24–23 μM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC–UV and LC–MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and its chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggests that the concept of prochelation might be a promising strategy for further development of aroylhydrazone cytoprotective agents.
No preview · Article · Feb 2015 · Journal of Pharmaceutical and Biomedical Analysis
[Show abstract][Hide abstract] ABSTRACT: Salicylaldehyde isonicotinoyl hydrazone (SIH) is a lipophilic, tridentate iron chelator with marked anti-oxidant and modest cytotoxic activity against neoplastic cells. However, it has poor stability in an aqueous environment due to the rapid hydrolysis of its hydrazone bond. In this study, we synthesized a series of new SIH analogs (based on previously described aromatic ketones with improved hydrolytic stability). Their structure-activity relationships were assessed with respect to their stability in plasma, iron chelation efficacy, redox effects and cytotoxic activity against MCF-7 breast adenocarcinoma cells. Furthermore, studies assessed the cytotoxicity of these chelators and their ability to afford protection against hydrogen peroxide-induced oxidative injury in H9c2 cardiomyoblasts. The ligands with a reduced hydrazone bond, or the presence of bulky alkyl substituents near the hydrazone bond, showed severely limited biological activity. The introduction of a bromine substituent increased ligand-induced cytotoxicity to both cancer cells and H9c2 cardiomyoblasts. A similar effect was observed when the phenolic ring was exchanged with pyridine (i.e., changing the ligating site from O, N, O to N, N, O), which led to pro-oxidative effects. In contrast, compounds with long, flexible alkyl chains adjacent to the hydrazone bond exhibited specific cytotoxic effects against MCF-7 breast adenocarcinoma cells and low toxicity against H9c2 cardiomyoblasts. Hence, this study highlights important structure-activity relationships and provides insight into the further development of aroylhydrazone iron chelators with more potent and selective anti-neoplastic effects.
[Show abstract][Hide abstract] ABSTRACT: Creatine, phosphocreatine and adenine nucleotides are highly polar markers of myocardial metabolism that are poorly retained on reversed-phase silica sorbents. Zirconia represents an alternative material to silica with high promise to be used in hydrophilic interaction chromatography. This study describes the first systematic investigation of the ability of ZrO2 to separate creatine, phosphocreatine, adenosine 5'-monophosphate, adenosine 5'-diphosphate and adenosine 5'-triphosphate and compares the results with those obtained on TiO2 . All analytes showed a hydrophilic-interaction-chromatography-like retention pattern when mobile phases of different strengths were tested. Stronger retention and better column performance were achieved in an organic-rich mobile phases as compared to aqueous conditions, where poor retention and insufficient column performance were observed. The effect of mobile phase pH and ionic strength was evaluated as well. The analysis of myocardial tissue demonstrated that all compounds were separated in a relevant biological material and thus proved ZrO2 as a promising phase for the hydrophilic interaction chromatography of biological samples that deserves further investigation. This article is protected by copyright. All rights reserved.
No preview · Article · May 2014 · Journal of Separation Science
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2012 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Significance:
Anthracyclines (doxorubicin, daunorubicin, or epirubicin) rank among the most effective anticancer drugs, but their clinical usefulness is hampered by the risk of cardiotoxicity. The most feared are the chronic forms of cardiotoxicity, characterized by irreversible cardiac damage and congestive heart failure. Although the pathogenesis of anthracycline cardiotoxicity seems to be complex, the pivotal role has been traditionally attributed to the iron-mediated formation of reactive oxygen species (ROS). In clinics, the bisdioxopiperazine agent dexrazoxane (ICRF-187) reduces the risk of anthracycline cardiotoxicity without a significant effect on response to chemotherapy. The prevailing concept describes dexrazoxane as a prodrug undergoing bioactivation to an iron-chelating agent ADR-925, which may inhibit anthracycline-induced ROS formation and oxidative damage to cardiomyocytes.
A considerable body of evidence points to mitochondria as the key targets for anthracycline cardiotoxicity, and therefore it could be also crucial for effective cardioprotection. Numerous antioxidants and several iron chelators have been tested in vitro and in vivo with variable outcomes. None of these compounds have matched or even surpassed the effectiveness of dexrazoxane in chronic anthracycline cardiotoxicity settings, despite being stronger chelators and/or antioxidants.
The interpretation of many findings is complicated by the heterogeneity of experimental models and frequent employment of acute high-dose treatments with limited translatability to clinical practice.
Dexrazoxane may be the key to the enigma of anthracycline cardiotoxicity, and therefore it warrants further investigation, including the search for alternative/complementary modes of cardioprotective action beyond simple iron chelation.
[Show abstract][Hide abstract] ABSTRACT: Salicylaldehyde isonicotinoyl hydrazone (SIH) is a lipophilic, orally-active tridentate iron chelator providing both effective protection against various types of oxidative stress-induced cellular injury and anticancer action. However, the major limitation of SIH is represented by its labile hydrazone bond that makes it prone to plasma hydrolysis. Recently, nine new SIH analogues derived from aromatic ketones with improved hydrolytic stability were developed. Here we analyzed their antiproliferative potential in MCF-7 breast adenocarcinoma and HL-60 promyelocytic leukemia cell lines. Seven of the tested substances showed greater selectivity than the parent agent SIH towards the latter cancer cell lines compared to non-cancerous H9c2 cardiomyoblast-derived cells. The tested chelators induced a dose-dependent dissipation of the inner mitochondrial membrane potential, an induction of apoptosis as evidenced by Annexin V positivity or significant increases of activities of caspases 3, 7, 8 and 9 and cell cycle arrest. With the exception of nitro group-bearing NHAPI, the studies of iron complexes of the chelators confirmed the crucial role of iron in the mechanism of their antiproliferative action. Finally, all the assayed chelators inhibited the oxidation of ascorbate by iron ions indicating lack of redox activity of the chelator-iron complexes. In conclusion, this study identified several important design criteria for improvement of the antiproliferative selectivity of the aroylhydrazone iron chelators. Several of the novel compounds--in particular the ethylketone-derived HPPI, NHAPI and acetyl-substituted A2,4DHAPI--merit deeper investigation as promising potent and selective anticancer agents.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2012 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: The most separations in HILIC mode are performed on silica-based supports. Nevertheless, recently published results have indicated that the metal oxides stationary phases also possess the ability to interact with hydrophilic compounds under HILIC conditions. This paper primarily describes the retention behaviour of model hydrophilic analytes (4-aminobenzene sulfonic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, 3,4-diaminobenzoic acid, 3-aminophenol and 3-nitrophenol) on the polybutadine modified zirconia in HILIC. The results were simultaneously compared with a bare zirconia and a silica-based HILIC phase. The mobile phase strength, pH and the column temperature were systematically modified to assess their impact on the retention of model compounds. It was found that the retention of our model hydrophilic analytes on both zirconia phases was mainly governed by adsorption while on the silica-based HILIC phase partitioning was primarily involved. The ability of ligand-exchange interactions of zirconia surface with a carboxylic moiety influenced substantially the response of carboxylic acids on the elevated temperature as well as to the change of the mobile phase pH in contrast to the silica phase. However, no or negligible ligand-exchange interactions were observed for sulfanilic acid. The results of this study clearly demonstrated the ability of modified zirconia phase to retain polar acidic compounds under HILIC conditions, which might substantially enlarge the application area of the zirconia-based stationary phases.
No preview · Article · Sep 2011 · Journal of Chromatography A
[Show abstract][Hide abstract] ABSTRACT: Catecholamines are stress hormones and sympathetic neurotransmitters essential for control of cardiac function and metabolism. However, pathologically increased catecholamine levels may be cardiotoxic by mechanism that includes iron-catalyzed formation of reactive oxygen species. In this study, five iron chelators used in clinical practice were examined for their potential to protect cardiomyoblast-derived cell line H9c2 from the oxidative stress and toxicity induced by catecholamines epinephrine and isoprenaline and their oxidation products. Hydroxamate iron chelator desferrioxamine (DFO) significantly reduced oxidation of catecholamines to more toxic products and abolished redox activity of the catecholamine-iron complex at pH 7.4. However, due to its hydrophilicity and large molecule, DFO was able to protects cells only at very high and clinically unachievable concentrations. Two newer chelators, deferiprone (L1) and deferasirox (ICL670A), showed much better protective potential and were effective at one or two orders of magnitude lower concentrations as compared to DFO that were within their clinically relevant plasma levels. Ethylenediaminetetraacetic acid (EDTA), dexrazoxane (ICRF-187, clinically approved cardioprotective agent against anthracycline-induced cardiotoxicity) as well as selected beta adrenoreceptor antagonists and calcium channel blockers exerted no effect. Hence, results of the present study indicate that small, lipophilic and iron-specific chelators L1 and ICL670A can provide significant protection against the oxidative stress and cardiomyocyte damage exerted by catecholamines and/or their reactive oxidation intermediates. This potential new application of the clinically approved drugs L1 and ICL670A warrants further investigation, preferably using more complex in vivo animal models.
[Show abstract][Hide abstract] ABSTRACT: Acyclic nucleoside phosphonates possess unique antiviral and antineoplastic activities; however, their polar phosphonate moiety is associated with low ability to cross biological membranes. We explored the potential of transdermal and topical delivery of 2,6-diaminopurine derivative cPr-PMEDAP.
In vitro diffusion of cPr-PMEDAP was investigated using formulations at different pH and concentration and with permeation enhancer through porcine and human skin.
Ability of 0.1-5% cPr-PMEDAP to cross human skin barrier was very low with flux values ~40 ng/cm(2)/h, the majority of compound found in the stratum corneum. The highest permeation rates were found at pH 6; increased donor concentration had no influence. The permeation enhancer dodecyl 6-dimethylaminohexanoate (DDAK, 1%) increased flux of cPr-PMEDAP (up to 61 times) and its concentration in nucleated epidermis (up to ~0.5 mg of cPr-PMEDAP/g of the tissue). No deamination of cPr-PMEDAP into PMEG occurred during permeation studies, but N-dealkylation into PMEDAP mediated by skin microflora was observed.
Transdermal or topical application of cPr-PMEDAP enabled by the permeation enhancer DDAK may provide an attractive alternative route of administration of this potent antitumor and antiviral compound.
Full-text · Article · Jun 2011 · Pharmaceutical Research
[Show abstract][Hide abstract] ABSTRACT: The ability of different stationary phases developed for the analysis of polar compounds (ZIC-HILIC, ZIC-pHILIC and Zorbax SB-Aq) to separate isoniazid, its metabolites (acetylisonazid, pyridoxal isonicotinoyl hydrazone, pyridoxal isonicotinoyl hydrazone 5-phosphate), pyridoxine, pyridoxal and pyridoxal 5-phosphate under MS compatible conditions was systematically investigated using HPLC-UV. The mobile phase strength, pH and buffer concentration were modified to assess their impact on the retention of these compounds. The best available separation of the compounds was achieved using 1 mM ammonium formate (pH≈6) and ACN (20:80, v/v) on ZIC-HILIC and employing 5 mM ammonium formate (pH 3.0) and ACN (40:60, v/v) on ZIC-pHILIC. A gradient profile using 0.5 mM ammonium formate (pH≈6) and MeOH (0-12 min: 10% MeOH, 12-15 min: 10-50% MeOH, 15-35 min: 50% MeOH, 35.0-35.2 min: 50-10% MeOH, 35.2-45.0 min: 10% MeOH) provided the best separation of the compounds on Zorbax SB-Aq. Subsequent LC-MS analysis demonstrated that ZIC-HILIC is useful for the analysis of pyridoxine, pyridoxal and pyridoxal isonicotinoyl hydrazone. However, the chromatographic conditions developed for the analysis of the compounds on Zorbax SB-Aq are capable of achieving the best separation of all compounds in this study with the higher sensitivity for most of the analytes.
No preview · Article · Jun 2011 · Journal of Separation Science
[Show abstract][Hide abstract] ABSTRACT: Elevated catecholamine levels are known to induce damage of the cardiac tissue. This catecholamine cardiotoxicity may stem from their ability to undergo oxidative conversion to aminochromes and concomitant production of reactive oxygen species (ROS), which damage cardiomyocytes via the iron-catalyzed Fenton-type reaction. This suggests the possibility of cardioprotection by iron chelation. Our in vitro experiments have demonstrated a spontaneous decrease in the concentration of the catecholamines epinephrine and isoprenaline during their 24-h preincubation in buffered solution as well as their gradual conversion to oxidation products. These changes were significantly augmented by addition of iron ions and reduced by the iron-chelating agent salicylaldehyde isonicotinoyl hydrazone (SIH). Oxidized catecholamines were shown to form complexes with iron that had significant redox activity, which could be suppressed by SIH. Experiments using the H9c2 cardiomyoblast cell line revealed higher cytotoxicity of oxidized catecholamines than of the parent compounds, apparently through the induction of caspase-independent cell death, whereas co-incubation of cells with SIH was able to significantly preserve cell viability. A significant increase in intracellular ROS formation was observed after the incubation of cells with catecholamine oxidation products; this could be significantly reduced by SIH. In contrast, parent catecholamines did not increase, but rather decreased, cellular ROS production. Hence, our results demonstrate an important role for redox-active iron in catecholamine autoxidation and subsequent toxicity. The iron chelator SIH has shown considerable potential to protect cardiac cells by both inhibition of deleterious catecholamine oxidation to reactive intermediates and prevention of ROS-mediated cardiotoxicity.
Full-text · Article · Feb 2011 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.
Full-text · Article · Jan 2011 · Journal of Chromatography A
[Show abstract][Hide abstract] ABSTRACT: A new, sensitive LC–MS method for evaluation of the purity of Transkarbam 12 (T12), a novel and highly effective accelerant
of transdermal penetration, has been developed and validated. T12 and its impurities (6-aminohexanoic acid, AH, ε-caprolactam,
CA, and dodecyl 6-(6-aminohexanamido)hexanoate, DAH) were characterized by MS and MS–MS analysis. Separation was achieved
on a 150mm×3mm, 5-μm particle, phenyl–hexyl column. The mobile phase was a gradient prepared from water, formic acid,
and acetonitrile. The method was validated within the concentration range 50–250ngmL−1; correlation coefficients were >0.998. The accuracy of the method was from 98.6–105.0% for AH, 102.6–104.8% for CA, and 97.9–100.9%
for DAH. Precision was in the range 3.19–4.42% for AH, 3.22–5.81% for CA, and 4.8–8.78% for DAH. The selectivity of the method
and sample stability were also confirmed. The practical applicability of the method was proven by analysis of T12 bulk substance.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to develop and validate HPLC methods for the determination in plasma of two novel thiosemicarbazone anti-tumour drugs developed in our laboratories (Dp44mT and N4mT). The appropriate separations were achieved using a HS F5 HPLC column with the mobile phase composed of a mixture of either acetate buffer/EDTA or EDTA and acetonitrile (62:38 and 50:50, v/v, respectively). The plasma samples were pretreated with SPE (phenyl and C18, respectively). Furthermore, these methods were successfully applied to in vitro plasma stability experiments. The investigation has clearly shown that both thiosemicarbazones are markedly more stable in plasma than their aroylhydrazone forerunners.
No preview · Article · Jan 2009 · Journal of Chromatography B
[Show abstract][Hide abstract] ABSTRACT: RP-TLC retention behavior has been studied for a series of potential antituberculotic drugs from a group of substituted anilides of pyrazine-2-carboxylic acid. Lipophilicity was determined by use of reversed-phase thin-layer chromatography with methanol-water mobile phases. RMW values were obtained by extrapolation of RM (determined by RP-TLC) to methanol-free conditions. These experimental lipophilicity values were correlated with lipophilicity (log P) calculated by use of three software products. Good correlation was obtained between RMW and log P. The results of this study could be used in further experiments focused on investigation of the biological activity of these newly prepared compounds and other potential antituberculotic drugs with closely similar chemical structures.
No preview · Article · Dec 2006 · JPC - Journal of Planar Chromatography - Modern TLC