František Trejtnar

Azole Antimycotics Differentially Affect Rifampicin-Induced Pregnane X Receptor-Mediated CYP3A4 Gene Expression

Azole antifungal drug ketoconazole has recently been demonstrated as an inhibitor of a ligand-induced pregnane X receptor (PXR)-mediated transcriptional regulation of the CYP3A4 gene through disruption of PXR interaction with steroid receptor coactivator (SRC)-1. In contrast, other clotrimazole-derived antifungal agents are known as potent inducers of CYP3A4 through PXR. In the present study, we examined effects of azole antimycotics clotrimazole, ketoconazole, econazole, oxiconazole, miconazole, fluconazole, and itraconazole on PXR-mediated expression of CYP3A4. We investigated individual effects of the tested azoles as well as their action on rifampicin-induced PXR-mediated transactivation and expression of CYP3A4 in LS174T cell line and primary human hepatocytes, their interactions with PXR ligand-binding domain, and azole-mediated recruitment of SRC-1 to PXR. In addition, applying the pharmacodynamic approach and dose-response analysis, we aimed to describe the nature of potential interactions of tested azole antimycotics coadministered with a prototypical PXR ligand rifampicin in transactivation of CYP3A4 gene. We describe additive and antagonistic interactions of partial and full agonists of PXR nuclear receptor in the therapeutic group of azole antimycotics in rifampicin-mediated transactivation of CYP3A4. We show that oxiconazole is a highly efficacious activator of CYP3A4 transactivation, which could be antagonized by rifampicin in a competitive manner. In addition, we show that activation of the CYP3A4 promoter is a complex process, which is not exclusively determined by azole-PXR interactions, and we suggest that the ability of some azoles to affect recruitment of SRC-1 to PXR modulates their net effects in transactivation of CYP3A4 both in the absence or presence of rifampicin.

Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase

The global burden of tuberculosis, its health and socio-economic impacts, the presence of drug-resistant forms and a potential threat of latent tuberculosis should serve as a strong impetus for the development of novel antituberculosis agents. We reported the in vitro activity of salicylanilide benzoates and pyrazine-2-carboxylates against Mycobacterium tuberculosis (minimum inhibitory concentrations as low as 0.5 μmol/L). Nineteen salicylanilide derivatives with mostly good antimycobacterial activity were evaluated for the inhibition of two essential mycobacterial enzymes, methionine aminopeptidase and isocitrate lyase, which are necessary for the maintenance of the latent tuberculosis infection. Salicylanilide derivatives act as moderate inhibitors of both mycobacterial and human methionine aminopeptidase and they also affect the function of mycobacterial isocitrate lyase. 4-Bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate was the most potent inhibitor of mycobacterial methionine aminopeptidase (41% inhibition at 10 μmol/L) and exhibited the highest selectivity. 5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide and 4-chloro-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate caused 59% inhibition of isocitrate lyase at 100 μmol/L concentration and (S)-4-bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 2-acetamido-3-phenylpropanoate produced 22% inhibition at 10 μmol/L; this rate is approximately comparable to 3-nitropropionic acid. Inhibition of those enzymes contributes at least in part to the antimicrobial activity of the compounds.

Iron reduction potentiates hydroxyl radical formation only in flavonols

Flavonoids, substantial components of the human diet, are generally considered to be beneficial. However, they may possess possible pro-oxidative effects, which could be based on their reducing potential. The aims of this study were to evaluate the ability of 26 flavonoids to reduce ferric ions at relevant pH conditions and to find a possible relationship with potentiation of hydroxyl radical production. A substantial ferric ions reduction was achieved under acidic conditions, particularly by flavonols and flavanols with the catecholic ring B. Apparently corresponding bell-shaped curves displaying the pro-oxidant effect of flavonols quercetin and kaempferol on iron-based Fenton reaction were documented. Several flavonoids were efficient antioxidants at very low concentrations but rather inefficient or pro-oxidative at higher concentrations. Flavonols, morin and rutin were progressively pro-oxidant, while 7-hydroxyflavone and hesperetin were the only flavonoids with dose-dependent inhibition of hydroxyl radical production. Conclusively, administration of flavonoids may lead to unpredictable consequences with few exceptions.

Antibacterial Activity of Salicylanilide 4-(Trifluoromethyl)-benzoates

The development of novel antimicrobial agents represents a timely research topic. Eighteen salicylanilide 4-(trifluoromethyl)benzoates were evaluated against Mycobacterium tuberculosis, M. avium and M. kansasii, eight bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and for the inhibition of mycobacterial isocitrate lyase. Some compounds were further screened against drug-resistant M. tuberculosis and for their cytotoxicity. Minimum inhibitory concentrations (MICs) for all mycobacterial strains were within 0.5–32 μmol/L, with 4-chloro-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 4-(trifluoromethyl)benzoate superiority. Gram-positive bacteria including MRSA were inhibited with MICs ≥ 0.49 μmol/L, while Gram-negative ones were much less susceptible. Salicylanilide 4-(trifluoromethyl)benzoates showed significant antibacterial properties, for many strains being comparable to standard drugs (isoniazid, benzylpenicillin) with no cross-resistance. All esters showed mild inhibition of mycobacterial isocitrate lyase and four compounds were comparable to 3-nitropropionic acid without a direct correlation between in vitro MICs and enzyme inhibition.

Synthesis and in vitro evaluation of new derivatives of 2-substituted-6-fluorobenzo[d]thiazoles as cholinesterase inhibitors

A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthesised and characterised by IR, (1)H, (13)C and (19)F NMR spectroscopy and HRMS. Purity was checked by elemental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure-activity relationships reflecting the dependence of cholinesterase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinesterase was determined using molecular docking of the most active compound.

Highly sensitive fast determination of entecavir in rat urine by means of hydrophilic interaction chromatography–ultra-high-performance liquid chromatography–tandem mass spectrometry

Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3 ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1 ml, 30 mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100 pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).

Synthesis and biological activity of new salicylanilide N,N-disubstituted carbamates and thiocarbamates

The development of novel antimicrobial drugs represents a cutting edge research topic. In this study, 20 salicylanilide N,N-disubstituted carbamates and thiocarbamates were designed, synthesised and characterised by IR, (1)H NMR and (13)C NMR. The compounds were evaluated in vitro as potential antimicrobial agents against Mycobacterium tuberculosis and nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii) as well as against eight bacterial and fungal strains. Additionally, we investigated the inhibitory effect of these compounds on mycobacterial isocitrate lyase and cellular toxicity. The minimum inhibitory concentrations (MICs) against mycobacteria were from 4 μM for thiocarbamates and from 16 μM for carbamates. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited with MICs from 0.49 μM by thiocarbamates, whilst Gram-negative bacteria and most of the fungi did not display any significant susceptibility. All (thio)carbamates mildly inhibited isocitrate lyase (up to 22%) at a concentration of 10 μM. The (thio)carbamoylation of the parent salicylanilides led to considerably decreased cytotoxicity and thus improved the selectivity indices (up to 175). These values indicate that some derivatives are attractive candidates for future research.

Salicylanilide diethyl phosphates: synthesis, antimicrobial activity and cytotoxicity.

A series of 27 salicylanilide diethyl phosphates was prepared as a part of our on-going search for new antimicrobial active drugs. All compounds exhibited in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium strains, with minimum inhibitory concentration (MIC) values of 0.5-62.5μmol/L. Selected salicylanilide diethyl phosphates also inhibit multidrug-resistant tuberculous strains at the concentration of 1μmol/L. Salicylanilide diethyl phosphates also exhibited mostly the activity against Gram-positive bacteria (MICs ≥1.95μmol/L), whereas their antifungal activity is significantly lower. The IC50 values for Hep G2 cells were within the range of 1.56-33.82μmol/L, but there is no direct correlation with MICs for mycobacteria.

Sex differences in stereospecificity of oracin reductases in rat in vitro and in vivo.

In vitro and in vivo experiments to investigate possible stereospecific aspects of oracin reduction in relation to rat gender have been conducted. Incubation of oracin with rat microsomes, cytosol, and hepatocytes in the presence of various coenzymes and under aerobic or anaerobic conditions provided evidence for sex differences in the formation of 11-dihydrooracin (DHO) enantiomers. The greatest sex differences were seen in hepatocytes where females showed higher stereospecificity of the reductases than males. While female biotransformation enzymes preferentially generated approximately 82% of (+)-DHO, male enzymes gave only rise to 63% of (+)-DHO. Males displayed higher stereospecificity than females in the microsomal fraction. However, in the cytosolic fraction females exhibited higher stereospecificity than males. Similarly, in in vivo studies, the ratio of (+)- and (-)-DHO in faeces and urine gave no indication of the significant differences between the male and female rat. Enzyme stereospecificity has been defined as preferential formation of the (+)- or (-)-stereoisomer of 11-DHO by the respective enzyme. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as the chiral stationary phase with excellent resolution and stability.

Study of the biotransformation of a potential benzo[c]fluorene antineoplastic using high-performance liquid chromatography with high-speed-scanning ultraviolet detection

As the sum of benfluron metabolites found was only a part of the total amount applied, a search for undiscovered metabolites was undertaken in the extracts from isolated rat hepatocytes and in the bile and perfusate in the experiments with an isolated perfused rat liver. To identify the metabolites, high-performance liquid chromatography with UV spectral analysis was used, as benfluron derivatives exhibit characteristic absorption spectra. Administration of known metabolites to experimental animals and selective induction of certain metabolic pathways led to the finding of new metabolites and of the respective conjugates. Fast atom bombardment-mass spectrometry analysis was used to identify the newly found metabolites and conjugates.