The interactions of monomeric acridines and unsymmetrical bisacridines (UAs) with DNA duplexes: an insight provided by NMR and MD studies.

Members of a novel class of anticancer compounds, exhibiting high antitumor activity, i.e. the unsymmetrical bisacridines (UAs), consist of two heteroaromatic ring systems. One of the ring systems is an imidazoacridinone moiety, with the skeleton identical to the structural base of Symadex. The second one is a 1-nitroacridine moiety, hence it may be regarded as Nitracrine's structural basis. These monoacridine units are connected by an aminoalkyl linker, which vary in structure. In theory, these unsymmetrical dimers should act as double-stranded DNA (dsDNA) bis-intercalators, since the monomeric units constituting the UAs were previously reported to exhibit an intercalating mode of binding into dsDNA. On the contrary, our earlier, preliminary studies have suggested that specific and/or structurally well-defined binding of UAs into DNA duplexes might not be the case. In this contribution, we have revisited and carefully examined the dsDNA-binding properties of monoacridines C-1305, C-1311 (Symadex), C-283 (Ledakrin/Nitracrine) and C-1748, as well as bisacridines C-2028, C-2041, C-2045 and C-2053 using advanced NMR techniques, aided by molecular modelling calculations and the analysis of UV-VIS spectra, decomposed by chemometric techniques. These studies allowed us to explain, why the properties of UAs are not a simple sum of the features exhibited by the acridine monomers.

Acid-Base Equilibrium and Self-Association in Relation to High Antitumor Activity of Selected Unsymmetrical Bisacridines Established by Extensive Chemometric Analysis.

Unsymmetrical bisacridines (UAs) represent a novel class of anticancer agents previously synthesized by our group. Our recent studies have demonstrated their high antitumor potential against multiple cancer cell lines and human tumor xenografts in nude mice. At the cellular level, these compounds affected 3D cancer spheroid growth and their cellular uptake was selectively modulated by quantum dots. UAs were shown to undergo metabolic transformations in vitro and in tumor cells. However, the physicochemical properties of UAs, which could possibly affect their interactions with molecular targets, remain unknown. Therefore, we selected four highly active UAs for the assessment of physicochemical parameters under various pH conditions. We determined the compounds' pKa dissociation constants as well as their potential to self-associate. Both parameters were determined by detailed and complex chemometric analysis of UV-Vis spectra supported by nuclear magnetic resonance (NMR) spectroscopy. The obtained results indicate that general molecular properties of UAs in aqueous media, including their protonation state, self-association ratio, and solubility, are strongly pH-dependent, particularly in the physiological pH range of 6 to 8. In conclusion, we describe the detailed physicochemical characteristics of UAs, which might contribute to their selectivity towards tumour cells as opposed to their effect on normal cells.

Metabolic Profiles of New Unsymmetrical Bisacridine Antitumor Agents in Electrochemical and Enzymatic Noncellular Systems and in Tumor Cells.

New unsymmetrical bisacridines (UAs) demonstrated high activity not only against a set of tumor cell lines but also against human tumor xenografts in nude mice. Representative UA compounds, named C-2028, C-2045 and C-2053, were characterized in respect to their physicochemical properties and the following studies aimed to elucidate the role of metabolic transformations in UAs action. We demonstrated with phase I and phase II enzymes in vitro and in tumors cells that: (i) metabolic products generated by cytochrome P450 (P450), flavin monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) isoenzymes in noncellular systems retained the compound's dimeric structures, (ii) the main transformation pathway is the nitro group reduction with P450 isoenzymes and the metabolism to N-oxide derivative with FMO1, (iii), the selected UGT1 isoenzymes participated in the glucuronidation of one compound, C-2045, the hydroxy derivative. Metabolism in tumor cells, HCT-116 and HT-29, of normal and higher UGT1A10 expression, respectively, also resulted in the glucuronidation of only C-2045 and the specific distribution of all compounds between the cell medium and cell extract was demonstrated. Moreover, P4503A4 activity was inhibited by C-2045 and C-2053, whereas C-2028 affected UGT1A and UGT2B action. The above conclusions indicate the optimal strategy for the balance among antitumor therapeutic efficacy and drug resistance in the future antitumor therapy.

Electrochemical simulation of metabolic reduction and conjugation reactions of unsymmetrical bisacridine antitumor agents, C-2028 and C-2053.

Electrochemistry (EC) coupled with analysis techniques such as liquid chromatography (LC) and mass spectrometry (MS) has been developed as a powerful tool for drug metabolism simulation. The application of EC in metabolic studies is particularly favourable due to the low matrix contribution compared to in vitro or in vivo biological models. In this paper, the EC(/LC)/MS system was applied to simulate phase I metabolism of the representative two unsymmetrical bisacridines (UAs), named C-2028 and C-2053, which contain nitroaromatic group susceptible to reductive transformations. UAs are a novel potent class of antitumor agents of extraordinary structures that may be useful in the treatment of difficult for therapy human solid tumors such as breast, colon, prostate, and pancreatic tumors. It is considered that the biological action of these compounds may be due to the redox properties of the nitroaromatic group. At first, the relevant conditions for the electrochemical conversion and product identification process, including the electrode potential range, electrolyte composition, and working electrode material, were optimized with the application of 1-nitroacridine as a model compound. Electrochemical simulation of C-2028 and C-2053 reductive metabolism resulted in the generation of six and five products, respectively. The formation of hydroxylamine m/z [M+H-14]+, amine m/z [M+H-30]+, and novel N-oxide m/z [M+H-18]+ species from UAs was demonstrated. Furthermore, both studied compounds were shown to be stable, retaining their dimeric forms, during electrochemical experiments. The electrochemical method also indicated the susceptibility of C-2028 to phase II metabolic reactions. The respective glutathione and dithiothreitol adducts of C-2028 were identified as ions at m/z 873 and m/z 720. In conclusion, the electrochemical reductive transformations of antitumor UAs allowed for the synthesis of new reactive intermediate forms permitting the study of their interactions with biologically crucial molecules.

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione: Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions.

Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. A significant route of phase II drug metabolism is conjugation with glutathione (GSH), which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes. The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA, C-2028. GSH-supplemented incubations of C-2028 with rat, but not with human, liver cytosol led to the formation of a single GSH-related metabolite. Interestingly, it was also revealed with rat liver microsomes. Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole. Therefore, the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate. In turn, incubations of C-2028 and GSH with human recombinant glutathione S-transferase (GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form, respectively. These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule. Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction. Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry. Mechanisms for their formation were proposed. The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important. This may affect the patient's drug clearance due to GST activity, loss of GSH, or the interactions with GSH-conjugated drugs. Moreover, GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Electrochemical and in silico approaches for liver metabolic oxidation of antitumor-active triazoloacridinone C-1305.

5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) is a promising antitumor compound developed in our laboratory. A better understanding of its metabolic transformations is still needed to explain the multidirectional mechanism of pharmacological action of triazoloacridinone derivatives at all. Thus, the aim of the current work was to predict oxidative pathways of C-1305 that would reflect its phase I metabolism. The multi-tool analysis of C-1305 metabolism included electrochemical conversion and in silico sites of metabolism predictions in relation to liver microsomal model. In the framework of the first approach, an electrochemical cell was coupled on-line to an electrospray ionization mass spectrometer. The effluent of the electrochemical cell was also injected onto a liquid chromatography column for the separation of different products formed prior to mass spectrometry analysis. In silico studies were performed using MetaSite software. Standard microsomal incubation was employed as a reference procedure. We found that C-1305 underwent electrochemical oxidation primarily on the dialkylaminoalkylamino moiety. An unknown N-dealkylated and hydroxylated C-1305 products have been identified. The electrochemical system was also able to simulate oxygenation reactions. Similar pattern of C-1305 metabolism has been predicted using in silico approach. Both proposed strategies showed high agreement in relation to the generated metabolic products of C-1305. Thus, we conclude that they can be considered as simple alternatives to enzymatic assays, affording time and cost efficiency.

Anticancer Imidazoacridinone C-1311 is Effective in Androgen-Dependent and Androgen-Independent Prostate Cancer Cells.

The androgen receptor (AR) plays a critical role in prostate cancer (PCa) development and metastasis. Thus, blocking AR activity and its downstream signaling constitutes a major strategy for PCa treatment. Here, we report on the potent anti-PCa activity of a small-molecule imidazoacridinone, C-1311. In AR-positive PCa cells, C-1311 was found to inhibit the transcriptional activity of AR, uncovering a novel mechanism that may be relevant for its anticancer effect. Mechanistically, C-1311 decreased the AR binding to the prostate-specific antigen (PSA) promoter, reduced the PSA protein level, and, as shown by transcriptome sequencing, downregulated numerous AR target genes. Importantly, AR-negative PCa cells were also sensitive to C-1311, suggesting a promising efficacy in the androgen-independent PCa sub-type. Irrespective of AR status, C-1311 induced DNA damage, arrested cell cycle progression, and induced apoptosis. RNA sequencing indicated significant differences in the transcriptional response to C-1311 between the PCa cells. Gene ontology analysis showed that in AR-dependent PCa cells, C-1311 mainly affected the DNA damage response pathways. In contrast, in AR-independent PCa cells, C-1311 targeted the cellular metabolism and inhibited the genes regulating glycolysis and gluconeogenesis. Together, these results indicate that C-1311 warrants further development for the treatment of PCa.

Design, synthesis and high antitumor potential of new unsymmetrical bisacridine derivatives towards human solid tumors, specifically pancreatic cancers and their unique ability to stabilize DNA G-quadruplexes.

New promising unsymmetrical bisacridine derivatives (UAs), have been developed. Three groups including 36 compounds were synthesized by the condensation of 4-nitro or 4-methylacridinone, imidazoacridinone and triazoloacridinone derivatives with 1-nitroacridine compounds linked with an aminoalkyl chain. Cytotoxicity screening revealed the high potency of these compounds against several tumor cell lines. Particularly, imidazoacridinone-1-nitroacridine dimers strongly inhibited pancreatic Panc-1, Mia-Pa-Ca-2, Capan-2 and prostate cancer DU-145 cell growth. The studied compounds showed very strong antitumor activity (T/C> 300%) against Walker 256 rat adenocarcinoma. The selected 26 UAs were tested against 12 human tumor xenografts in nude mice, including colon, breast, prostate and pancreatic cancers. The studies on the molecular mechanism of action demonstrated that these unsymmetrical dimers significantly responded to the presence of G-quadruplex not to dsDNA. Structure-activity relationships for UAs potency to G-quadruplex stabilization indicated that thermal stability of this drug-G-quadruplex complex depended not only on the structure of heterocyclic rings, but also on the properties of dialkylamino chains of the ring linkers. In conclusion, the presented studies identified the new group of effective antitumor agents against solid human tumors, particularly pancreatic Panc-1, BxPC-3 and Mia-Pa-Ca-2 and strongly indicated their distinctive interactions with DNA. In contrast to monomers, G-quadruplex not dsDNA is proposed to be the first molecular target for these compounds.

Enhanced Activity of P4503A4 and UGT1A10 Induced by Acridinone Derivatives C-1305 and C-1311 in MCF-7 and HCT116 Cancer Cells: Consequences for the Drugs' Cytotoxicity, Metabolism and Cellular Response.

Activity modulation of drug metabolism enzymes can change the biotransformation of chemotherapeutics and cellular responses induced by them. As a result, drug-drug interactions can be modified. Acridinone derivatives, represented here by C-1305 and C-1311, are potent anticancer drugs. Previous studies in non-cellular systems showed that they are mechanism-based inhibitors of cytochrome P4503A4 and undergo glucuronidation via UDP-glucuronosyltranspherase 1A10 isoenzyme (UGT1A10). Therefore, we investigated the potency of these compounds to modulate P4503A4 and UGT1A10 activity in breast MCF-7 and colon HCT116 cancer cells and their influence on cytotoxicity and cellular response in cells with different expression levels of studied isoenzymes. We show that C-1305 and C-1311 are inducers of not only P4503A4 but also UGT1A10 activity. MCF-7 and HCT116 cells with high P4503A4 activity are more sensitive to acridinone derivatives and undergo apoptosis/necrosis to a greater extent. UGT1A10 was demonstrated to be responsible for C-1305 and C-1311 glucuronidation in cancer cells and glucuronide products were excreted outside the cell very fast. Finally, we show that glucuronidation of C-1305 antitumor agent enhances its pro-apoptotic properties in HCT116 cells, while the cytotoxicity and cellular response induced by C-1311 did not change after drug glucuronidation in both cell lines.

New Unsymmetrical Bisacridine Derivatives Noncovalently Attached to Quaternary Quantum Dots Improve Cancer Therapy by Enhancing Cytotoxicity toward Cancer Cells and Protecting Normal Cells.

The use of nanoparticles for the controlled drug delivery to cells has emerged as a good alternative to traditional systemic delivery. Quantum dots (QDs) offer potentially invaluable societal benefits such as drug targeting and in vivo biomedical imaging. In contrast, QDs may also pose risks to human health and the environment under certain conditions. Here, we demonstrated that a unique combination of nanocrystals core components (Ag-In-Zn-S) would eliminate the toxicity problem and increase their biomedical applications. The alloyed quaternary nanocrystals Ag-In-Zn-S (QDgreen, Ag1.0In1.2Zn5.6S9.4; QDred, Ag1.0In1.0Zn1.0S3.5) were used to transport new unsymmetrical bisacridine derivatives (UAs, C-2028 and C-2045) into lung H460 and colon HCT116 cancer cells for improving the cytotoxic and antitumor action of these compounds. UAs were coupled with QD through physical adsorption. The obtained results clearly indicate that the synthesized nanoconjugates exhibited higher cytotoxic activity than unbound compounds, especially toward lung H460 cancer cells. Importantly, unsymmetrical bisacridines noncovalently attached to QD strongly protect normal cells from the drug action. It is worth pointing out that QDgreen or QDred without UAs did not influence the growth of cancer and normal cells, which is consistent with in vivo results. In noncellular systems, at pH 5.5 and 4.0, which relates to the conditions of endosomes and lysosomes, the UAs were released from QD-UAs nanoconjugates. An increase of total lysosomes content was observed in H460 cells treated with QDs-UAs which can affect the release of the UAs from the conjugates. Moreover, confocal laser scanning microscopy analyses revealed that QD-UAs nanoconjugates enter H460 cells more efficiently than to HCT116 and normal cells, which may be the reason for their higher cytotoxicity against lung cancer. Summarizing, the noncovalent attachment of UAs to QDs increases the therapeutic efficiency of UAs by improving cytotoxicity toward lung H460 cancer cells and having protecting effects on normal cells.

Electrochemical simulation of metabolism for antitumor-active imidazoacridinone C-1311 and in silico prediction of drug metabolic reactions.

The metabolism of antitumor-active 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) has been investigated widely over the last decade but some aspects of molecular mechanisms of its metabolic transformation are still not explained. In the current work, we have reported a direct and rapid analytical tool for better prediction of C-1311 metabolism which is based on electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS). Simulation of the oxidative phase I metabolism of the compound was achieved in a simple electrochemical thin-layer cell consisting of three electrodes (ROXY™, Antec Leyden, the Netherlands). We demonstrated that the formation of the products of N-dealkylation reactions can be easily simulated using purely instrumental approach. Newly reported products of oxidative transformations like hydroxylated or oxygenated derivatives become accessible. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass ion data and tandem mass spectrometry experiments. In silico prediction of main sites of C-1311 metabolism was performed using MetaSite software. The compound was evaluated for cytochrome P450 1A2-, 3A4-, and 2D6-mediated reactions. The results obtained by EC were also compared and correlated with those of reported earlier for conventional in vitro enzymatic studies in the presence of liver microsomes and in the model peroxidase system. The in vitro experimental approach and the in silico metabolism findings showed a quite good agreement with the data from EC/ESI-MS analysis. Thus, we conclude here that the electrochemical technique provides the promising platform for the simple evaluation of drug metabolism and the reaction mechanism studies, giving first clues to the metabolic transformation of pharmaceuticals in the human body.

Phase I and phase II metabolism simulation of antitumor-active 2-hydroxyacridinone with electrochemistry coupled on-line with mass spectrometry.

Here, we report the metabolic profile and the results of associated metabolic studies of 2-hydroxy-acridinone (2-OH-AC), the reference compound for antitumor-active imidazo- and triazoloacridinones. Electrochemistry coupled with mass spectrometry was applied to simulate the general oxidative metabolism of 2-OH-AC for the first time. The reactivity of 2-OH-AC products to biomolecules was also examined. The usefulness of the electrochemistry for studying the reactive drug metabolite trapping (conjugation reactions) was evaluated by the comparison with conventional electrochemical (controlled-potential electrolysis) and enzymatic (microsomal incubation) approaches. 2-OH-AC oxidation products were generated in an electrochemical thin-layer cell. Their tentative structures were assigned based on tandem mass spectrometry in combination with accurate mass measurements. Moreover, the electrochemical conversion of 2-OH-AC in the presence of reduced glutathione and/or N-acetylcysteine unveiled the formation of reactive metabolite-nucleophilic trapping agent conjugates (m/z 517 and m/z 373, respectively) through the thiol group. This glutathione S-conjugate was also identified after electrolysis experiment as well as was detected in liver microsomes. Summing up, the present work illustrates that the electrochemical simulation of metabolic reactions successfully supports the results of classical electrochemical and enzymatic studies. Therefore, it can be a useful tool for synthesis of drug metabolites, including reactive metabolites.

Binary Mixtures of Selected Bisphenols in the Environment: Their Toxicity in Relationship to Individual Constituents.

Bisphenol A (BPA) is one of the most popular and commonly used plasticizer in the industry. Over the past decade, new chemicals that belong to the bisphenol group have increasingly been used in industrial applications as alternatives to BPA. Nevertheless, information on the combined effects of bisphenol (BP) analogues is insufficient. Therefore, our current study aimed to find the biological response modulations induced by the binary mixtures of BP compounds. We determined the toxicity levels in Microtox and XenoScreen YES/YAS assays for several BP analogs alone, and for their binary mixtures. The results obtained constituted the database for chemometric intelligent data analysis to evaluate the possible interactions occurring in the mixtures. Several chemometric/biophysical models have been used (concentration addition-CA, independent action-IA and polynomial regression calculations) to realize this aim. The best fitting was found for the IA model and even in this description strong evidence for synergistic behaviors (modes of action) of some bisphenol analogue mixtures was demonstrated. Bisphenols A, S, F and FL were proven to be of significant endocrine threat (with respect to XenoScreen YES/YAS assay); thus, their presence in mixtures (including presence in tissues of living organisms) should be most strictly monitored and reported.

Drug-drug interaction potential of antitumor acridine agent C-1748: The substrate of UDP-glucuronosyltransferases 2B7, 2B17 and the inhibitor of 1A9 and 2B7.

BACKGROUND: The compound 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes - UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity. METHODS: UGT-mediated transformations with rat liver (RLM), human liver (HLM), and human intestine (HIM) microsomes and with 10 recombinant human isoenzymes were investigated. Studies on the ability of C-1748 to inhibit UGT were performed with HLM, HT29 colorectal cancer cell homogenate and the selected recombinant UGT isoenzymes. The reactions were monitored using HPLC-UV/Vis method and the C-1748 metabolite structure was determined with ESI-TOF-MS/MS analysis. RESULTS: Pseudo-molecular ion (m/z 474.1554) and the experiment with ß-glucuronidase indicated that O-glucuronide of C-1748 was formed in the presence of microsomal fractions. This reaction was selectively catalyzed by UGT2B7 and 2B17. High inhibitory effect of C-1748 was shown towards isoenzyme UGT1A9 (IC50=39.7µM) and significant but low inhibitory potential was expressed in HT29 cell homogenate (IC50=84.5µM). The mixed-type inhibition mechanism (Ki=17.0µM;Ki'=81.0µM), induced by C-1748 was observed for recombinant UGT1A9 glucuronidation, whereas HT29 cell homogenate resulted in noncompetitive inhibition (Ki=94.6µM). CONCLUSIONS: The observed UGT-mediated metabolism of C-1748 and its ability to inhibit UGT activity should be considered as the potency for drug resistance and drug-drug interactions in the prospective multidrug therapy.

Modulation of UDP-glucuronidation by acridinone antitumor agents C-1305 and C-1311 in HepG2 and HT29 cell lines, despite slight impact in noncellular systems.

BACKGROUND: Among the studied antitumor acridinone derivatives developed in our laboratory, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) exhibited cytotoxic and antitumor properties against several cancer types and were selected to be evaluated in preclinical and early-phase clinical trials. In the present work, we investigated the impact of C-1305 and C-1311 on UDP-glucuronosyltransferase (UGT) activity. METHODS: Enzyme activity modulation was studied using HPLC by analyzing standard UGT substrate metabolism in the presence and absence of antitumor drugs. The investigations were performed in two model systems: (i) under noncellular conditions, including human liver microsomes (HLM) and recombinant UGT1A1, 1A9 and 1A10 isoenzymes and (ii) in tumor cells. RESULTS: There was observed a slight impact of studied drugs on enzyme activity. Only UGT1A1 action was altered by both compounds. The modulatory effects of UGT activity in cellular systems depended on the tumor cell type. In the case of HepG2, C-1305 and C-1311 strongly induced UGT activity, particularly for C-1311, at concentrations significantly lower than the EC50. This effect contradicted irinotecan mediated UGT inhibition. HT29 colon tumor cells were less sensitive than HepG2 to enzyme modulation in the presence of the studied compounds, particularly C-1305, where enzymatic inhibition similar to that of irinotecan was observed. CONCLUSIONS: The results demonstrated that UGT activity modulation should be expected in the case of antitumor therapy with C-1305 or/and C-1311. Analysis of the results indicated that these modulations would occur via cellular regulatory pathways not by direct drug-enzyme interactions.

Stable nanoconjugates of transferrin with alloyed quaternary nanocrystals Ag-In-Zn-S as a biological entity for tumor recognition.

One way to limit the negative effects of anti-tumor drugs on healthy cells is targeted therapy employing functionalized drug carriers. Here we present a biocompatible and stable nanoconjugate of transferrin anchored to Ag-In-Zn-S quantum dots modified with 11-mercaptoundecanoic acid (Tf-QD) as a drug carrier versus typical anticancer drug, doxorubicin. Detailed investigations of Tf-QD nanoconjugates without and with doxorubicin by fluorescence studies and cytotoxic measurements showed that the biological activity of both the transferrin and doxorubicin was fully retained in the nanoconjugate. In particular, the intercalation capabilities of free doxorubicin versus ctDNA remained essentially intact upon its binding to the nanoconjugate. In order to evaluate these capabilities, we studied the binding constant of doxorubicin attached to Tf-QDs with ctDNA as well as the binding site size on the ctDNA molecule. The binding constant slightly decreased compared to that of free doxorubicin while the binding site size, describing the number of consecutive DNA lattice residues involved in the binding, increased. It was also demonstrated that the QDs alone and in the form of a nanoconjugate with Tf were not cytotoxic towards human non-small cell lung carcinoma (H460 cell line) and the tumor cell sensitivity of the DOX-Tf-QD nanoconjugate was comparable to that of doxorubicin alone.

The overexpression of CPR and P450 3A4 in pancreatic cancer cells changes the metabolic profile and increases the cytotoxicity and pro-apoptotic activity of acridine antitumor agent, C-1748.

Drug resistance is one of the major causes of pancreatic cancer treatment failure. Thus, it is still imperative to develop new active compounds and novel approach to improve drug efficacy. Here we present 9-amino-1-nitroacridine antitumor agent, C-1748, developed in our laboratory, as a candidate for pancreatic cancer treatment. We examined (i) the cellular response of pancreatic cancer cell lines: Panc-1, MiaPaCa-2, BxPC-3 and AsPC-1, differing in expression levels of commonly mutated genes for this cancer type, to C-1748 treatment and (ii) the role of P450 3A4 isoenzyme and cytochrome P450 reductase (CPR) in the modulation of this response. C-1748 exhibited the highest cytotoxic activity against MiaPaCa-2, while AsPC-1 cells were the most resistant (IC50: 0.015, 0.075µM, respectively). A considerable amount of apoptosis was detected in Panc-1 and MiaPaCa-2 cells but only limited apoptosis was observed in AsPC-1 and BxPC-3 cells as indicated by morphological changes and biochemical markers. Furthermore, only AsPC-1 cells underwent senescence. Since AsPC-1 cells were the most resistant to C-1748 as evidenced by the lowest P450 3A4 and CPR protein levels, this cell line was subjected to transient transfection either with P450 3A4 or CPR gene. The overexpression of P450 3A4 or CPR changed the pro-apoptotic activity of C-1748 and sensitized AsPC-1 cells to this drug compared to wild-type cells. However, metabolism was changed significantly only for CPR overexpressing cells. In conclusion, the antitumor effectiveness of C-1748 would be improved by multi-drug therapy with chemotherapeutics, that are able to induce P450 3A4 and/or CPR gene expression.

Imidazoacridinone antitumor agent C-1311 as a selective mechanism-based inactivator of human cytochrome P450 1A2 and 3A4 isoenzymes.

BACKGROUND: 5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311), a promising antitumor agent that is also active against autoimmune diseases, was determined to be a selective inhibitor of the cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1311 might modulate the effectiveness of other drugs used in multidrug therapy. The present work aimed to identify the mechanism of the observed C-1311-mediated inactivation of CYP1A2 and CYP3A4. METHODS: The inactivation experiments were performed in vitro using the human recombinant CYP1A2 and CYP3A4 (Bactosomes). CYP isoenzyme activities were determined using the CYP-specific reactions, 7-ethoxycoumarin O-deethylation (CYP1A2) and testosterone 6ß-hydroxylation (CYP3A4). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by RP-HPLC with UV-Vis detection. RESULTS: The inhibition of CYPs by C-1311 was time-, concentration- and NADPH-dependent, which suggested a mechanism-based mode of action. Using a 10-fold dilution method and potassium ferricyanide we demonstrated the irreversible nature of the inhibition. In addition, the inhibition was attenuated by the presence of alternate substrates (alternative active site ligands) but not by a nucleophilic trapping agent (glutathione) or a reactive oxygen scavenger (catalase), which further supported a mechanism-based action. Substrate depletion partition ratios of 299 and 985 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively. CONCLUSIONS: Our results indicated that C-1311 is a potent mechanism-based inactivator of CYP1A2 and CYP3A4. This finding provided new insights into the mechanism of C-1311 antitumor action, particularly in relation to potential pharmacokinetic drug-drug interactions between C-1311 and/or its derivatives and the substrates of CYP isoforms.

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305.

1. 5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors. 2. It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy. 3. The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4. 4. Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, KI = 10.8 ± 2.14 µM) and testosterone 6ß-hydroxylation (CYP3A4, KI = 9.1 ± 2.82 µM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively. 5. In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug-drug interactions in potent multidrug therapy.

Improved cytotoxicity and preserved level of cell death induced in colon cancer cells by doxorubicin after its conjugation with iron-oxide magnetic nanoparticles.

A promising strategy for overcoming the problem of limited efficacy in antitumor drug delivery and in drug release is the use of a nanoparticle-conjugated drug. Doxorubicin (Dox) anticancer chemotherapeutics has been widely studied in this respect, because of severe cardiotoxic side effects. Here, we investigated the cytotoxic effects, the uptake process, the changes in cell cycle progression and the cell death processes in the presence of iron-oxide magnetic nanoparticles (Nps) and doxorubicin conjugates (Dox-Nps) in human colon HT29 cells. The amount of Dox participated in biological action of Dox-Nps was determined by cyclic voltammetry and thermogravimetric measurements. The cytotoxicity of Dox-Nps was shown to be two/three times higher than free Dox, whereas Nps alone did not inhibit cell proliferation. Dox-Nps penetrated cancer cells with higher efficacy than free Dox, what could be a consequence of Dox-Nps aggregation with proteins in culture medium and/or with cell surface. The treatment of HT29 cells with Dox-Nps and Dox at IC50 concentration resulted in G2/M arrest followed by late apoptosis and necrosis. Summing up, the application of iron-oxide magnetic nanoparticles improved Dox-Nps cell penetration compared to free Dox and achieved the cellular response to Dox-Nps conjugates similar to that of Dox alone.