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.

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.

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.

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.

[Glucuronidation of antitumour therapeutics--detoxification, mechanism of resistance or prodrug formation?]. / Glukuronidacja leków przeciwnowotworowych--detoksyfikacja, mechanizm opornosci czy sposób na forme proleku?

The physiological role of phase I and II of xenobiotic biotransformations is their detoxification and better excretion outside the organism. UDP-glucuronosyltransferases (UGTs) being the enzymes of phase II metabolism catalyse the conjugation of glucuronic acid to the lipophilic substrate by its specific nucleophilic group. UGT isoenzymes of various substrate specificities and different expression profiles in selected tissues belong to the large UGT superfamily. Usually, glucuronidation is the detoxification process, but sometimes (morphine, tamoxifen) glucuronides express biological activity higher than or comparable to the native compound. The level of UGT gene expression is individual for patients, because of their genetic status as well as epigenetic conditions. Also, xenobiotics are able to modulate UGT level and gene expression by the interaction with nuclear receptors. Moreover, one can find a lower level of UGT in the tumour compared to normal tissue, which results in the protection against deactivation of the drug and in the promotion of its selective activity in tumor tissue. On the other hand, UGT activity is considered as the possible cause of resistance to chemotherapy. Metabolism by hepatic and intestinal UGT isoenzymes is responsible for the "first-pass effect", whereas acquired resistance consists in the induction of UGT gene expression by the chemotherapeutic or its metabolite. Moreover, UGT induction can be associated with the induction of membrane transporters, particularly proteins of the ABC family, responsible for drug excretion outside the cell. The above resistance effects can be fortified by the overexpression of selected UGT isoenzymes sometimes observed in specific types of tumours. It is also considered that many advanced tumours are characterized by a higher level of ß-glucuronidase. This enzyme has a chance to be the molecular target of directed antitumour therapy, as it catalyses ß-glucuronide hydrolysis, leading to active aglycones.

Novel resveratrol-based substrates for human hepatic, renal, and intestinal UDP-glucuronosyltransferases.

Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by ß-glucuronidase hydrolysis and LC-MS/MS analysis. NI-12a was glucuronidated at both the -COOH and -OH functions, NI-ST-05 formed a novel N-O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent Km values of 240 and 6.2 µM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation.

CYP3A4-dependent cellular response does not relate to CYP3A4-catalysed metabolites of C-1748 and C-1305 acridine antitumor agents in HepG2 cells.

High CYP3A4 expression sensitizes tumor cells to certain antitumor agents while for others it can lower their therapeutic efficacy. We have elucidated the influence of CYP3A4 overexpression on the cellular response induced by antitumor acridine derivatives, C-1305 and C-1748, in two hepatocellular carcinoma (HepG2) cell lines, Hep3A4 stably transfected with CYP3A4 isoenzyme, and HepC34 expressing empty vector. The compounds were selected considering their different chemical structures and different metabolic pathways seen earlier in human and rat liver microsomes C-1748 was transformed to several metabolites at a higher rate in Hep3A4 than in HepC34 cells. In contrast, C-1305 metabolism in Hep3A4 cells was unchanged compared to HepC34 cells, with each cell line producing a single metabolite of comparable concentration. C-1748 resulted in a progressive appearance of sub-G1 population to its high level in both cell lines. In turn, the sub-G1 fraction was dominated in CYP3A4-overexpressing cells following C-1305 exposure. Both compounds induced necrosis and to a lesser extent apoptosis, which were more pronounced in Hep3A4 than in wild-type cells. In conclusion, CYP3A4-overexpressing cells produce higher levels of C-1748 metabolites, but they do not affect the cellular responses to the drug. Conversely, cellular response was modulated following C-1305 treatment in CYP3A4-overexpressing cells, although metabolism of this drug was unaltered.

CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells.

AIM: To examine whether CYP3A4 overexpression influences the metabolism of anticancer agent imidazoacridinone C-1311 in CHO cells and the responses of the cells to C-1311. METHODS: Wild type CHO cells (CHO-WT), CHO cells overexpressing cytochrome P450 reductase (CPR) [CHO-HR] and CHO cells coexpressing CPR and CYP3A4 (CHO-HR-3A4) were used. Metabolic transformation of C-1311 and CYP3A4 activity were measured using RP-HPLC. Flow cytometry analyses were used to examine cell cycle, caspase-3 activity and cell apoptosis. The expression of pH 6.0-dependent ß-galactosidase (SA-ß-gal) was studied to evaluate accelerated senescence. ROS generation was analyzed with CM-H2 DCFDA staining. RESULTS: CYP3A4 overexpression did not change the metabolism of C-1311 in CHO cells: the levels of all metabolites of C-1311 increased with the exposure time to a similar extent, and the differences in the peak level of the main metabolite M3 were statistically insignificant among the three CHO cell lines. In CHO-HR-3A4 cells, C-1311 effectively inhibited CYP3A4 activity without affecting CYP3A4 protein level. In the presence of C-1311, CHO-WT cells underwent rather stable G2/M arrest, while the two types of transfected cells only transiently accumulated at this phase. C-1311-induced apoptosis and necrosis in the two types of transfected cells occurred with a significantly faster speed and to a greater extent than in CHO-WT cells. Additionally, C-1311 induced ROS generation in the two types of transfected cells, but not in CHO-WT cells. Moreover, CHO-HR-3A4 cells that did not die underwent accelerated senescence. CONCLUSION: CYP3A4 overexpression in CHO cells enhances apoptosis induced by C-1311, whereas the metabolism of C-1311 is minimal and does not depend on CYP3A4 expression.

Metabolic transformation of antitumor acridinone C-1305 but not C-1311 via selective cellular expression of UGT1A10 increases cytotoxic response: implications for clinical use.

The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.

Progress in targeting tumor cells by using drug-magnetic nanoparticles conjugate.

To limit cytotoxicity of anticancer drugs against healthy cells, an appropriate carrier should be synthesized to deliver the drug to the tumor tissue only. A good solution is to anchor a magnetic nanoparticle to the molecule of the drug and to use a properly directed external magnetic field. The synthesis of the conjugate of doxorubicin with magnetic nanoparticles (iron oxide) modified by us resulted in a substantial depression of the aggregation process of the nanoparticles and therefore allowed the correct examination of cytotoxicity of the modified drug. It has been shown, by performing the electrochemical microbalance measurements, that the use of magnetic field guaranteed the efficient delivery of the drug to the desired place. The change in the synthesis procedure led to an increase in the number of DOX molecules attached to one magnetic nanoparticle. The release of the drug took place at pH 5.8 (and below it), which pH characterizes the cancer cells. It has also been found that while the iron oxide magnetic nanoparticles were not cytotoxic toward human urinary bladder carcinoma cells UM-UC-3, the tumor cell sensitivity of the DOX-Np complex was slightly higher in comparison to the identical concentration of doxorubicin alone.

Modulation of CYP3A4 activity and induction of apoptosis, necrosis and senescence by the anti-tumour imidazoacridinone C-1311 in human hepatoma cells.

There is increasing evidence that the expression level of drug metabolic enzymes affects the final cellular response following drug treatment. Moreover, anti-tumour agents may modulate enzymatic activity and/or cellular expression of metabolic enzymes in tumour cells. We have investigated the influence of CYP3A4 overexpression on the cellular response induced by the anti-tumour agent C-1311 in hepatoma cells. C-1311-mediated CYP3A4 activity modulation and the effect of CYP3A4 overexpression on C-1311 metabolism have also been examined. With the HepG2 cell line and its CYP3A4-overexpressing variant, Hep3A4, experiments involving DAPI staining, cell cycle analysis, phosphatidylserine externalisation and senescence-associated (SA)-ß-galactosidase expression, were used to monitor the effects of C-1311 exposure. C-1311 cellular metabolism and CYP3A4 activity were investigated by high-performance liquid chromatography. C-1311 metabolism was very low in both hepatoma cell lines and slightly influenced by CYP3A4 expression. Interestingly, in HepG2 cells, C-1311 was an effective modulator of CYP3A4 enzymatic activity, being the inhibitor of this isoenzyme in Hep3A4 cells. Cell cycle analysis showed that HepG2 cells underwent a rather stable G(2) /M arrest following C-1311 exposure, whereas CYP3A4-overexpressing cells accumulated only slightly in this compartment. C-1311-treated cells died by apoptosis and necrosis, whereas surviving cells underwent senescence; however, these effects occurred faster and more intensely in Hep3A4 cells. Although CYP3A4 did not influence C-1311 metabolism, changes in CYP3A4 levels affected the C-1311-induced response in hepatoma cells. Therefore, inter-patient differences in CYP3A4 levels should be considered when assessing the potential therapeutic effects of C-1311.

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.

Role of human UDP-glucuronosyltransferases in the biotransformation of the triazoloacridinone and imidazoacridinone antitumor agents C-1305 and C-1311: highly selective substrates for UGT1A10.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 µl · min⁻¹ · mg protein⁻¹, for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 µl · min⁻¹ · mg protein⁻¹. In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.

Influence of temperature and interactions with ligands on dissociation of dsDNA and ligand-dsDNA complexes of various types of binding. An electrochemical study.

Several medicinally important compounds that bind to dsDNA strands via intercalation (C-1311, C-1305, EtBr), major groove binding (Hoechst 33258) and covalent binding (cis-Pt) were examined. The obtained results suggest that both the transfer of conformation B to C and the denaturation process, for the ligand-dsDNA complexes, except for covalently bound cis-Pt, took place at higher temperatures compared to the unbound helix. Furthermore, much lower currents of electrooxidation of guanine at 100 °C, compared to the currents obtained at this temperature for dsDNA in the absence of ligands, suggest that the binding of ligands affects the way the dsDNA denaturates at increased temperatures and leads to formation of different forms of DNA single strands. The voltammetric results were compared with the data of two spectroscopic techniques: UV-Vis and CD.

The imidazoacridinone antitumor drug, C-1311, is metabolized by flavin monooxygenases but not by cytochrome P450s.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P(FMO), which is identified as an N-oxide derivative, was identical to P3(R) of liver microsomes. P3(R) was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3(R) = P(FMO). Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.

Flavin monooxygenases, FMO1 and FMO3, not cytochrome P450 isoenzymes, contribute to metabolism of anti-tumour triazoloacridinone, C-1305, in liver microsomes and HepG2 cells.

5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.

[UDP-glucuronyltransferases, proteins of endoplasmic reticulum--structure and mechanism of action]. / UDP-glukuronylotransferazy, bialka siateczki sródplazmatycznej--struktura i mechanizm dzialania.

UDP-glucuronyltranferase isoenzymes, UGTs, are responsible in mammals for conjugation of glucuronic acid generated by UDPGA with aglicon. UDPGA is bound to the appropriate group of I phase metabolite or, occasionally, to the native compound. As a result, the aglicon polarity increases, excreatable product in human urine is formed and, in turn, toxic effects are reduced. UGT protein structure consists of 2 domens. N-terminal catalyses of aglicon binding, whereas, C-terminal controls the addition of uridine-5'-diphosphoglucuronic acid. UGTs are anchored in endoplasmic reticulum, ER, by transmembrane fragment of C-terminal domain. The membrane location results in the latency of enzyme activity and demands specific transporters for cofactor as well as for conjugation products. There are NST and AT(ER), respectively. UDP-glucuronyltransferases exist usually as homo- and heterodimers, some of them are tetramers. 19 human UGT proteins are described by 3 gene subfamilies, UGT1A, UGT2A and UGT2B, which are expressed preferentially in the liver, but also in stomach, lung and intestine epithelium.

[UDP-glucuronyltransferases in detoxification and activation metabolism of endogenous compounds and xenobiotics]. / UDP-glukuronylotranferazy w metabolizmie detoksykacyjnym i aktywacyjnym zwiazków endogennych oraz ksenobiotyków.

Glucuronidation is a crucial pathway of metabolism and excretion of endogenous compounds and xenobiotics. UDP-glucuronyltransferases, UGT, catalyse transformations of bilirubine, steroids and thyroid hormones, bile acids as well as exogenous compounds, including drugs, carcinogens, environmental pollutants and nutrient components. From therapeutic point of view, the participation of UGTs in drug metabolism is of particular significance. Polymorphism of UGT1A and UGT2B genes resulted in various susceptibility of substrates to conjugation with glucuronic acid. Deactivation of xenobiotics and the following excretion of hydrophilic conjugates is a common task of glucuronidation, which should lead to detoxification. However, a lot of glucuronides were known, which expressed the comparable or even higher reactivity than that of the native compound. There are, among others, acyl glucuronides of carboxylic acids, morphine 6-O-glucuronide or retinoid glucuronides. They are able to bind cellular macromolecules with low or high strength and, as a consequence, their toxicity is saved or even increased, respectively.

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.