HPLC analýza potenciálních léčiv odvozených od aroylhydrazonu II

1. ABSTRAKT Vysokoúčinná kvapalinová chromatografia (HPLC) je v oblasti liečiv jednou z najčastejšie používanou analytickou technikou. Železo je dôležitým biogénnym prvkom, no napriek tomu jeho zvýšené množstvo v organizme pôsobí toxicky. Výskum vysoko selektívnych a účinných biokompatibilných chelátorov železa bol pôvodne inšpirovaný potrebou mobilizovať železo v tkanivách s chronickým preťažením železa. Avšak v posledných rokoch je pozornosť venovaná využitia chelátorov železa tiež pri liečbe bežných onemocnení. Salicylaldehyd isonicotinoyl hydrazon (SIH), biokompatibilný chelátor železa odvodený od aroylhydrazonu, je v dnešnej dobe predmetom aktívneho výskumu ako potenciálne liečivo. Okrem schopnosti viazať železo, vykazuje aj ďalšie zaujímavé farmakologické účinky: antioxidačný, antiproliferatívny, kardioprotektívny, antimalarický a antimikróbny. Táto práca sa zaoberá vypracovaním optimálnych chromatografických podmienok HPLC separácie SIH a jeho potenciálnych metabolitov (isoniazid, acetylisoniazid, salicylaldehyd) a následne využitia tejto separácie k štúdiu možnosti izolácie uvedených látok z moči králika pomocou SPE ako úpravy vzorku. Najlepšia chromatografická analýza bola dosiahnutá použitím kolóny od firmy Phenomenex (2504,6 mm I. D.) s náplňou Prodigy 5u ODS3 100A (5 μm). Mobilná fáza bola v...1. ABSTRACT High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. Although iron is a vital element, excessive amounts in the body are highly toxic. The search for highly selective and effective iron chelating agents has been mainly inspired by the need to mobilize iron from tissues that are chronically overloaded with iron. However, recent investigations focused on the possibility to use iron chelators for the treatment of many other pathologies. Salicylaldehyde isonicotinoyl hydrazone (SIH), a biocompatible iron chelator derived from aroylhydrazone, is under extensive investigation as a promising drug candidate. Besides ability to bind iron, it shows interesting pharmacological effects: antioxidative, antiproliferative, cardioprotective, antimalarical and antimicrobic. The aim of this study was to develop optimal HPLC conditions for the separation of SIH and its potential metabolites (isoniazide, acetylisoniazide, salicylaldehyde) and to apply the method to the study focused on the isolation of analytes from rabbit urine using SPE. The best chromatographic analysis was achieved on a HPLC column (Phenomenex 250 4.6 mm I. D.) packed with Prodigy 5u ODS3 100A (5 μm) as a stationary phase. The mobile phase was composed of methanol :...Katedra farmaceutické chemie a kontroly léčivDepartment of Pharmaceutical Chemistry and Drug ControlFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

HPLC analysis of drug candidates from the group of aroylhydrazones II.

1. ABSTRACT High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. Although iron is a vital element, excessive amounts in the body are highly toxic. The search for highly selective and effective iron chelating agents has been mainly inspired by the need to mobilize iron from tissues that are chronically overloaded with iron. However, recent investigations focused on the possibility to use iron chelators for the treatment of many other pathologies. Salicylaldehyde isonicotinoyl hydrazone (SIH), a biocompatible iron chelator derived from aroylhydrazone, is under extensive investigation as a promising drug candidate. Besides ability to bind iron, it shows interesting pharmacological effects: antioxidative, antiproliferative, cardioprotective, antimalarical and antimicrobic. The aim of this study was to develop optimal HPLC conditions for the separation of SIH and its potential metabolites (isoniazide, acetylisoniazide, salicylaldehyde) and to apply the method to the study focused on the isolation of analytes from rabbit urine using SPE. The best chromatographic analysis was achieved on a HPLC column (Phenomenex 250 4.6 mm I. D.) packed with Prodigy 5u ODS3 100A (5 μm) as a stationary phase. The mobile phase was composed of methanol :..

Analytické hodnocení léčiv a potenciálních léčiv ze skupiny látek chelatujících železo

Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové Katedra Katedra farmaceutické chemie a kontroly léčiv Kandidát Mgr. Ján Stariat Školiteľ Prof. RNDr. Jiří Klimeš, CSc. Názov dizertačnej práce Analytické hodnotenie liečiv a potenciálnych liečiv zo skupiny látok chelatujúcich železo Vysokoúčinná kvapalinová chromatografia (HPLC) má v analýze liečiv dominantné postavenie, pretože patrí medzi robustné techniky, umožňujúce dosiahnutie účinnej separácie väčšiny analytov prítomných vo vzorku, a zároveň ich kvalitatívne aj kvantitatívne hodnotenie. Pokroky vo vývoji stacionárnych fáz a detekčných techník umožňujú výrazne skrátiť čas analýzy, a zároveň získať celé spektrum informácií o povahe analytov prítomných vo vzorkách aj vo veľmi nízkych koncentráciách. Nádorové ochorenia sa stále radia medzi významné príčiny smrti, z čoho vyplýva požiadavka na vývoj nových liečebných postupov. Stratégia založená na chelatácii železa vnútri nádorových buniek by mohla výraznou mierou prispieť k zlepšeniu prognózy u pacientov s tumormi rezistentnými voči štandardnej chemoterapii. Tento mechanizmus antiproliferativného pôsobenia je aktívne rozvíjaný u novej skupiny potenciálnych liečiv štruktúrne odvodených od thiosemikarbazonu, u ktorých bol cytostatický účinok pozorovaný in vitro aj in vivo Ďalší...Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Chemistry and Drug Control Candidate Mgr. Ján Stariat Supervisor Prof. RNDr. Jiří Klimeš, CSc. Title of Doctoral Thesis Analytical evaluation of drugs and drug candidates from the group of iron chelating agents High performance liquid chromatography (HPLC) ranks among the most important analytical techniques in the field of pharmaceutical analysis thanks to its robustness and the ability to separate and qualitatively and quantitatively analyse the compounds presented in various samples. The ongoing progress in the development of novel stationary phases and detection techniques allows rapid analysis and to get comprehensive characterization of the analytes presented in the complex samples even at very low concentrations. Cancer still remains a leading cause of death, thus the need for novel, efficient treatment strategies is of crucial importance. The mechanism based on iron chelation (Fe) inside the tumour cells represents one of the most promising strategy which could enhance the prognosis of patients suffering from cancer resistant to standard chemotherapy. Thiosemicarbazone iron chelators are currently under intensive development as novel anticancer drugs. Their strong antiproliferative...Department of Pharmaceutical Chemistry and Pharmaceutical AnalysisKatedra farmaceutické chemie a farmaceutické analýzyFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

HPLC analysis of drug candidates from the group of aroylhydrazones II.

1. ABSTRACT High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. Although iron is a vital element, excessive amounts in the body are highly toxic. The search for highly selective and effective iron chelating agents has been mainly inspired by the need to mobilize iron from tissues that are chronically overloaded with iron. However, recent investigations focused on the possibility to use iron chelators for the treatment of many other pathologies. Salicylaldehyde isonicotinoyl hydrazone (SIH), a biocompatible iron chelator derived from aroylhydrazone, is under extensive investigation as a promising drug candidate. Besides ability to bind iron, it shows interesting pharmacological effects: antioxidative, antiproliferative, cardioprotective, antimalarical and antimicrobic. The aim of this study was to develop optimal HPLC conditions for the separation of SIH and its potential metabolites (isoniazide, acetylisoniazide, salicylaldehyde) and to apply the method to the study focused on the isolation of analytes from rabbit urine using SPE. The best chromatographic analysis was achieved on a HPLC column (Phenomenex 250 4.6 mm I. D.) packed with Prodigy 5u ODS3 100A (5 μm) as a stationary phase. The mobile phase was composed of methanol :..

Analytical Evaluation of Drugs and Drug Candidates from the Group of Iron Chelating Agents

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Chemistry and Drug Control Candidate Mgr. Ján Stariat Supervisor Prof. RNDr. Jiří Klimeš, CSc. Title of Doctoral Thesis Analytical evaluation of drugs and drug candidates from the group of iron chelating agents High performance liquid chromatography (HPLC) ranks among the most important analytical techniques in the field of pharmaceutical analysis thanks to its robustness and the ability to separate and qualitatively and quantitatively analyse the compounds presented in various samples. The ongoing progress in the development of novel stationary phases and detection techniques allows rapid analysis and to get comprehensive characterization of the analytes presented in the complex samples even at very low concentrations. Cancer still remains a leading cause of death, thus the need for novel, efficient treatment strategies is of crucial importance. The mechanism based on iron chelation (Fe) inside the tumour cells represents one of the most promising strategy which could enhance the prognosis of patients suffering from cancer resistant to standard chemotherapy. Thiosemicarbazone iron chelators are currently under intensive development as novel anticancer drugs. Their strong antiproliferative..

HPLC methods for determination of two novel thiosemicarbazone anti-cancer drugs (N4mT and Dp44mT) in plasma and their application to in vitro plasma stability of these agents

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.7 page(s

In Vitro Characterization of the Pharmacological Properties of the Anti-Cancer Chelator, Bp4eT, and Its Phase I Metabolites.

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

Incubation of Bp4eT with MCF-7 cells caused increased caspase activity, whereas its metabolites did not.

<p>MCF-7 cells were incubated for: (A) 3 h; (B) 24 h; or (C) 72 h/37°C with 100 nM Bp4eT or its metabolites, Bp4eA and Bp4eS. The caspase activities were then assayed in cellular lysates. The activities were related to cell viabilities and the results were expressed as a percentage of control. The results are mean ± SD (<i>n</i> = 4 experiments). Statistical significance (ANOVA): * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 as compared to the control (untreated) group.</p

Therapeutic indices of Bp4eT and its metabolites against neoplastic cells.

<p>The therapeutic indices were calculated using the following ratio, IC₅₀ non-cancerous cells / IC₅₀ neoplastic cells. Results are means of <i>n</i> ≥ 4 experiments.</p><p>Therapeutic indices of Bp4eT and its metabolites against neoplastic cells.</p

Line drawings of the structures of Bp4eT and its metabolites and indication of <i>E/Z</i> isomerism.

<p>Bp4eT, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone; Bp4eA; <i>N</i>^<i>3</i>-ethyl-<i>N</i>^<i>1</i>-[phenyl(pyridine-2yl)methylene]formamidrazone; Bp4eS, 2-benzoylpyridine 4-ethylsemicarbazone.</p