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.

• MeSH
• analýza rozptylu MeSH
• financování organizované MeSH
• interpretace statistických dat MeSH
• isoniazid analogy a deriváty   analýza   metabolismus   MeSH
• králíci MeSH
• lidé MeSH
• naftaleny krev   MeSH
• prasata MeSH
• protinádorové látky krev   MeSH
• pyridoxal analogy a deriváty   analýza   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• stabilita léku MeSH
• thiosemikarbazony krev   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• zvířata MeSH

• Publikační typ
• abstrakt z konference MeSH

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.

• MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• potkani Wistar MeSH
• preklinické hodnocení léčiv MeSH
• protinádorové látky metabolismus   farmakokinetika   farmakologie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• thiosemikarbazony metabolismus   farmakokinetika   farmakologie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Di-2-pyridylketone isonicotinoyl hydrazone (PKIH) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) novel iron chelators which possess marked anti-cancer activity in vivo. However, further progress in the development of these drug candidates requires precise and convenient methods for their qualitative and quantitative analysis. The aim of this study was to develop and validate HPLC methods suitable for the purity and stability evaluation of Dp44mT and PKIH and subsequently to employ these methods in stress tests addressing their chemical stability. The chromatographic analyses of both chelators were accomplished via HPLC using a Discovery HSF5 column (25 cm x 4 mm; 5 microm). For separation of Dp44mT and its synthetic precursors, the mobile phase was composed of a mixture of 2 mM EDTA and acetonitrile in a ratio 60:40 (v/v). A desirable separation of PKIH from its synthetic precursors was achieved with a mixture of 0.01 M phosphate buffer (pH 3.0), methanol and acetonitrile in a ratio of 65:21:14 (v/v/v) with the addition of EDTA (2 mM). In order to confirm the utility of these HPLC methods for measuring these drugs and their stability, Dp44mT and PKIH were subjected to chemical stress tests. These experiments showed that Dp44mT was relatively stable against hydrolytic degradation, but quite sensitive to oxidation. On the other hand, PKIH was slightly sensitive to acid-catalyzed hydrolysis, but it was relatively stable under other tested conditions. Furthermore, these studies confirmed the utility of these methods not only for appropriate evaluation of purity but also stability. The analytical methods developed and validated in this study, as well as the basic data on the chemical stability, should further support the development of both these novel anti-cancer chelators as promising drug candidates.

• MeSH
• chelátory železa analýza   MeSH
• chelátory analýza   MeSH
• financování organizované MeSH
• koncentrace vodíkových iontů MeSH
• protinádorové látky analýza   MeSH
• reprodukovatelnost výsledků MeSH
• stabilita léku MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH

Recent studies have demonstrated that several chelators possess marked potential as potent anti-neoplastic drugs and as agents that can ameliorate some of the adverse effects associated with standard chemotherapy. Anti-cancer treatment employs combinations of several drugs that have different mechanisms of action. However, data regarding the potential interactions between iron chelators and established chemotherapeutics are lacking. Using estrogen receptor-positive MCF-7 breast cancer cells, we explored the combined anti-proliferative potential of four iron chelators, namely: desferrioxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), (E)-N'-[1-(2-hydroxy-5-nitrophenyl)ethyliden] isonicotinoyl hydrazone (NHAPI), and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), plus six selected anti-neoplastic drugs. These six agents are used for breast cancer treatment and include: paclitaxel, 5-fluorouracil, doxorubicin, methotrexate, tamoxifen and 4-hydroperoxycyclophosphamide (an active metabolite of cyclophosphamide). Our quantitative chelator-drug analyses were designed according to the Chou-Talalay method for drug combination assessment. All combinations of these agents yielded concentration-dependent, anti-proliferative effects. The hydrophilic siderophore, DFO, imposed antagonism when used in combination with all six anti-tumor agents and this antagonistic effect increased with increasing dose. Conversely, synergistic interactions were observed with combinations of the lipophilic chelators, NHAPI or Dp44mT, with doxorubicin and also the combinations of SIH, NHAPI or Dp44mT with tamoxifen. The combination of Dp44mT with anti-neoplastic agents was further enhanced following formation of its redox-active iron and especially copper complexes. The most potent combinations of Dp44mT and NHAPI with tamoxifen were confirmed as synergistic using another estrogen receptor-expressing breast cancer cell line, T47D, but not estrogen receptor-negative MDA-MB-231 cells. Furthermore, the synergy of NHAPI and tamoxifen was confirmed using MCF-7 cells by electrical impedance data, a mitochondrial inner membrane potential assay and cell cycle analyses. This is the first systematic investigation to quantitatively assess interactions between Fe chelators and standard chemotherapies using breast cancer cells. These studies are vital for their future clinical development.

• MeSH
• aldehydy farmakologie   MeSH
• chelátory železa farmakologie   MeSH
• cyklofosfamid analogy a deriváty   MeSH
• deferoxamin farmakologie   MeSH
• doxorubicin MeSH
• fluoruracil MeSH
• hydrazony farmakologie   MeSH
• lidé MeSH
• methotrexát MeSH
• MFC-7 buňky MeSH
• paclitaxel MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky farmakologie   MeSH
• protokoly protinádorové kombinované chemoterapie farmakologie   MeSH
• synergismus léků MeSH
• tamoxifen MeSH
• thiosemikarbazony farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

V tomto projektu budou vyvinuty a validovány moderní analytické a bioanalytické metody (LC/MS/MS) vhodné pro studium osudu dvou nových thiosemikarbazonových protinádorových léčiv (Dp44mT a Dpcyc) v organismu. Tyto metody budou posléze využity jako validní nástroje pro studium biotransformace (in vitro i in vivo) a farmakokinetiky těchto léčiv. Zvláštní pozornost bude věnována metabolismu i způsobu eliminace uvedených látek, ale také studiu možného vztahu mezi biotransformací a toxikologickým profilem (především kardiotoxicitou, jenž byla zaznamenána u Dp44mT). Na základě výsledků bude navržena konkrétní obměna struktury léčiva s cílem optimalizovat farmakokinetický, popřípadě toxikologický profil uvedených thiosemikarbazonů.; Novel, modern analytical and bioanalytical methods (LC/MS/MS) will be developed and validated in order to study the fate of two novel thiosemicarbazone anti-cancer drugs (Dp44mT and Dpcyc) in an organism. Thereafter, these methods will be utilized as valid tools to study biotransformation of these compounds both in vitro and in vivo and to evaluate their pharmacokinetics. Particular attention will be paid on elimination and metabolism of the thiosemicarbazones as well as the investigation of the possible involvement of particular Dp44mT metabolite in cardiotoxicity observed after administration of this compound. Based on the results of this project the rational chemical structure modification of the thiosemicarbazones will be suggested to optimize pharmacokinetic profile and potentially also toxicological profile.

• MeSH
• biotransformace účinky léků   MeSH
• cytostatické látky terapeutické užití   MeSH
• klinické laboratorní techniky metody   využití   MeSH
• medicína založená na důkazech MeSH
• nádory farmakoterapie   MeSH
• proliferace buněk účinky léků   MeSH
• thiosemikarbazony farmakokinetika   farmakologie   chemie   klasifikace   škodlivé účinky   terapeutické užití   MeSH
• vztahy mezi strukturou a aktivitou MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• farmacie a farmakologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Combining low-dose chemotherapies is a strategy for designing less toxic and more potent childhood cancer treatments. We examined the effects of combining the novel thiosemicarbazones, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), or its analog, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), with the standard chemotherapies, celecoxib (CX), etoposide (ETO), or temozolomide (TMZ). These combinations were analyzed for synergism to inhibit proliferation of three pediatric tumor cell-types, namely osteosarcoma (Saos-2), medulloblastoma (Daoy) and neuroblastoma (SH-SY5Y). In terms of mechanistic dissection, this study discovered novel thiosemicarbazone targets not previously identified and which are important for considering possible drug combinations. In this case, DpC and Dp44mT caused: (1) up-regulation of a major protein target of CX, namely cyclooxygenase-2 (COX-2); (2) down-regulation of the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), which is known to affect TMZ resistance; (3) down-regulation of mismatch repair (MMR) proteins, MSH2 and MSH6, in Daoy and SH-SY5Y cells; and (4) down-regulation in all three cell-types of the MMR repair protein, MLH1, and also topoisomerase 2α (Topo2α), the latter of which is an ETO target. While thiosemicarbazones up-regulate the metastasis suppressor, NDRG1, in adult cancers, it is demonstrated herein for the first time that they induce NDRG1 in all three pediatric tumor cell-types, validating its role as a potential target. In fact, siRNA studies indicated that NDRG1 was responsible for MGMT down-regulation that may prevent TMZ resistance. Examining the effects of combining thiosemicarbazones with CX, ETO, or TMZ, the most promising synergism was obtained using CX. Of interest, a positive relationship was observed between NDRG1 expression of the cell-type and the synergistic activity observed in the combination of thiosemicarbazones and CX. These studies identify novel thiosemicarbazone targets relevant to childhood cancer combination chemotherapy.

• Publikační typ
• časopisecké články MeSH

Dysregulation of iron homeostasis is one of the important processes in the development of many oncological diseases, such as pancreatic cancer. Targeting it with specific agents, such as an iron chelator, are promising therapeutic methods. In this study, we tested the cytotoxicity of novel azulene hydrazide-hydrazone-based chelators against pancreatic cancer cell lines (MIA PaCa-2, PANC-1, AsPC-1). All prepared chelators (compounds 4-6) showed strong cytotoxicity against pancreatic cancer cell lines and high selectivity for cancer cell lines compared to the healthy line. Their cytotoxicity is lower than thiosemicarbazone-based chelators Dp44mT and DpC, but significantly higher than hydroxamic acid-based chelator DFO. The chelator tested showed mitochondrial and lysosomal co-localization and its mechanism of action was based on the induction of hypoxia-inducible factor-1-alpha (HIF-1α), N-myc downstream-regulated gene-1 (NDRG1) and transferrin receptor 1 (TfR1). This strongly implies that the cytotoxic effect of tested chelators could be associated with mitophagy induction. Lipinski's rule of five analyses was performed to determine whether the prepared compounds had properties ensuring their bioavailability. In addition, the drug-likeness and drug-score were calculated and discussed.

• MeSH
• azuleny MeSH
• chelátory železa farmakologie   MeSH
• hydraziny MeSH
• hydrazony farmakologie   MeSH
• kyseliny hydroxamové MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory slinivky břišní * farmakoterapie   MeSH
• receptory transferinu MeSH
• thiosemikarbazony * farmakologie   MeSH
• železo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status.

• MeSH
• antracykliny * farmakologie   MeSH
• checkpoint kinasa 1 metabolismus   MeSH
• dítě MeSH
• doxorubicin metabolismus   farmakologie   MeSH
• inhibitory topoisomerasy II MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• P-glykoprotein metabolismus   MeSH
• poškození DNA MeSH
• protinádorová antibiotika MeSH
• thiosemikarbazony * farmakologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Tyrosine kinase inhibitors (TKIs) are frequently used in combined therapy to enhance treatment efficacy and overcome drug resistance. The present study analyzed the effects of three inhibitors, sunitinib, gefitinib, and lapatinib, combined with iron-chelating agents, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) or di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). Simultaneous administration of the drugs consistently resulted in synergistic and/or additive activities against the cell lines derived from the most frequent types of pediatric solid tumors. The results of a detailed analysis of cell signaling in the neuroblastoma cell lines revealed that TKIs inhibited the phosphorylation of the corresponding receptor tyrosine kinases, and thiosemicarbazones downregulated the expression of epidermal growth factor receptor, platelet-derived growth factor receptor, and insulin-like growth factor-1 receptor, leading to a strong induction of apoptosis. Marked upregulation of the metastasis suppressor N-myc downstream regulated gene-1 (NDRG1), which is known to be activated and upregulated by thiosemicarbazones in adult cancers, was also detected in thiosemicarbazone-treated neuroblastoma cells. Importantly, these effects were more pronounced in the cells treated with drug combinations, especially with the combinations of lapatinib with thiosemicarbazones. Therefore, these results provide a rationale for novel strategies combining iron-chelating agents with TKIs in therapy of pediatric solid tumors.

• Publikační typ
• časopisecké články MeSH