Xenoreceptors of the nuclear receptor superfamily, such as pregnane X receptor (PXR), are liver-enriched ligand-activated transcription factors regarded as crucial sensors in xenobiotic exposure and detoxification. PXR controls transcription of many drug-handling genes and influx/efflux transporters, thus playing a crucial role in drug metabolism and excretion. Liver functions have been studied using primary human hepatocytes (PHHs), which, when conventionally cultured, undergo rapid de-differentiation, leaving them unsuitable for long-term studies. Recently, 3D PHHs called spheroids have emerged as an in vitro model that is similar to in vivo hepatocytes regarding phenotype and function and represents the first in vitro model to study the long-term regulation of drug-handling genes by PXR. In this study, we used mathematical modelling to analyze the long-term activation of PXR in 3D PHHs through expression kinetics of three key PXR-regulated drug-metabolizing enzymes, CYP3A4, CYP2C9, and CYP2B6 and the P-glycoprotein efflux transporter encoding gene, MDR1. PXR action in 3D PHHs was induced by the antibiotic rifampicin at two clinically relevant concentrations. The results confirmed that high rifampicin concentrations activated PXR nearly to its full capacity. The analysis indicated the highest PXR-induced transcription rate constant for CYP2B6. The rate constant dictating mRNA degradation associated with activated PXR was highest for CYP3A4. Moreover, we measured the metabolic activity of CYP3A4, CYP2C9, and CYP2B6 and quantified their metabolic rate constants. Metabolic activity rate constant of CYP3A4 was found to be the highest whereas that of CYP2B6 was found to be the lowest among the studied enzymes. Our results provide important insight into the regulation of PXR-target genes in 3D PHHs and show that mRNA expression and metabolic activity data can be combined with quantitative analysis to reveal the long-term action of PXR and its effects on drug-handling genes.

• MeSH
• biologické modely MeSH
• buněčné sféroidy * metabolismus   MeSH
• cytochrom P-450 CYP3A metabolismus   genetika   MeSH
• hepatocyty * metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• pregnanový X receptor * metabolismus   MeSH
• regulace genové exprese * MeSH
• rifampin farmakologie   MeSH
• steroidní receptory * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytochrom P-450 CYP3A MeSH
• pregnanový X receptor * MeSH
• rifampin MeSH
• steroidní receptory * MeSH

Rifampicin is a model ligand of the pregnane X receptor (PXR), the nuclear receptor involved in the regulation of cytochrome P450 3A4 (CYP3A4). Rifampicin forms several degradation products and metabolites of which 25-desacetylrifampicin is the most abundant in vivo. Here, we aimed to study both the stability and metabolism of rifampicin in media and 2D and 3D primary human hepatocytes (PHHs). Additionally, we analyzed interactions of rifampicin derivatives with PXR. We described that rifampicin gradually degrades by more than 50 % in the medium partly into quinone over 72 h. We observed 25-desacetylrifampicin in 2D PHHs but not in 3D PHHs. Contrary, rifampicin was converted into quinone in a one-direction process in media of 3D PHHs. The potency of rifampicin and its derivatives to activate human PXR was arranged as follows: 3-formylrifamycin SV > rifampicin quinone > rifampicin > rifampicin N-oxide > 25-desacetylrifampicin, respectively, but none activates mouse and rat PXR. The binding differences between rifampicin and 25-desacetylrifampicin were modeled in silico. Finally, we showed that overexpressed uptake organic anion transporting polypeptide 1B1 (OATP1B1) potentiated activation of PXR by rifampicin and rifampicin quinone, but overexpressed efflux multidrug resistance protein 1 (MDR1) decreased PXR activation by all derivatives.

• Klíčová slova
• Cytochrome P450, Hepatocyte spheroid, Metabolism, Molecular docking, Pregnane X receptor, Rifampicin,
• MeSH
• cytochrom P-450 CYP3A metabolismus   MeSH
• hepatocyty * metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• myši MeSH
• pregnanový X receptor * metabolismus   MeSH
• rifampin * farmakologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytochrom P-450 CYP3A MeSH
• pregnanový X receptor * MeSH
• rifampin * MeSH

Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy * metabolismus   MeSH
• genetická transkripce MeSH
• iniciace genetické transkripce * MeSH
• Mycobacterium smegmatis * metabolismus   genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií MeSH
• rifampin * farmakologie   MeSH
• sigma faktor * metabolismus   genetika   MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• bakteriální proteiny * MeSH
• DNA řízené RNA-polymerasy * MeSH
• rifampin * MeSH
• sigma faktor * MeSH
• transkripční faktory MeSH

• Klíčová slova
• BPaLM availability, Bedaquline testing, MDR-TB, MDR-TB testing in EU/EAA, Pretomanid testing,
• MeSH
• antituberkulotika * terapeutické užití   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti * MeSH
• multirezistentní tuberkulóza * farmakoterapie   diagnóza   mikrobiologie   MeSH
• Mycobacterium tuberculosis účinky léků   genetika   MeSH
• rifampin * terapeutické užití   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• komentáře MeSH
• Geografické názvy
• Evropa MeSH
• Názvy látek
• antituberkulotika * MeSH
• rifampin * MeSH

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.

• Klíčová slova
• Induction, Inhibition, P-glycoprotein, Rifabutin, Rifampicin,
• MeSH
• P-glykoprotein metabolismus   MeSH
• rhodamin 123 metabolismus   MeSH
• rifabutin * farmakologie   MeSH
• rifampin * farmakologie   MeSH
• simulace molekulového dockingu MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• P-glykoprotein MeSH
• rhodamin 123 MeSH
• rifabutin * MeSH
• rifampin * MeSH

Rifampicin is a clinically important antibiotic that binds to, and blocks the DNA/RNA channel of bacterial RNA polymerase (RNAP). Stalled, nonfunctional RNAPs can be removed from DNA by HelD proteins; this is important for maintenance of genome integrity. Recently, it was reported that HelD proteins from high G+C Actinobacteria, called HelR, are able to dissociate rifampicin-stalled RNAPs from DNA and provide rifampicin resistance. This is achieved by the ability of HelR proteins to dissociate rifampicin from RNAP. The HelR-mediated mechanism of rifampicin resistance is discussed here, and the roles of HelD/HelR in the transcriptional cycle are outlined. Moreover, the possibility that the structurally similar HelD proteins from low G+C Firmicutes may be also involved in rifampicin resistance is explored. Finally, the discovery of the involvement of HelR in rifampicin resistance provides a blueprint for analogous studies to reveal novel mechanisms of bacterial antibiotic resistance.

• Klíčová slova
• HelD/HelR, RNA polymerase, antibiotics, bacteria, resistance, rifampicin,
• MeSH
• antibakteriální látky farmakologie   MeSH
• Bacteria * genetika   metabolismus   MeSH
• bakteriální léková rezistence MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• DNA MeSH
• rifampin * farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• rifampin * MeSH

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

• Klíčová slova
• Klenow (exo-) DNA polymerase, ferrocene-labeled nucleotides, nucleoside triphosphates, single-nucleotide polymorphism (SNP), single-point mutation, solid-phase primer elongation,
• MeSH
• jednonukleotidový polymorfismus MeSH
• metaloceny MeSH
• Mycobacterium tuberculosis * genetika   MeSH
• oxidace-redukce MeSH
• rifampin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metaloceny MeSH
• rifampin MeSH

The alkaline milieu of chronic wounds severely impairs the therapeutic effect of antibiotics, such as rifampicin; as such, the development of new drugs, or the smart delivery of existing drugs, is required. Herein, two innovative polyelectrolyte nanoparticles (PENs), composed of an amphiphilic chitosan core and a polycationic shell, were synthesized at alkaline pH, and in vitro performances were assessed by 1H NMR, elemental analysis, FT-IR, XRD, DSC, DLS, SEM, TEM, UV/Vis spectrophotometry, and HPLC. According to the results, the nanostructures exhibited different morphologies but similar physicochemical properties and release profiles. It was also hypothesized that the simultaneous use of the nanosystem and an antioxidant could be therapeutically beneficial. Therefore, the simultaneous effects of ascorbic acid and PENs were evaluated on the release profile and degradation of rifampicin, in which the results confirmed their synergistic protective effect at pH 8.5, as opposed to pH 7.4. Overall, this study highlighted the benefits of nanoparticulate development in the presence of antioxidants, at alkaline pH, as an efficient approach for decreasing rifampicin degradation.

• Klíčová slova
• alkaline pH, ascorbic acid, polyelectrolyte nanoparticles, rifampicin,
• MeSH
• diferenciální skenovací kalorimetrie MeSH
• difrakce rentgenového záření MeSH
• koncentrace vodíkových iontů MeSH
• lékové transportní systémy * MeSH
• nanočástice chemie   ultrastruktura   MeSH
• polyelektrolyty chemie   MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• rifampin farmakologie   MeSH
• síran dextranu chemie   MeSH
• spektrofotometrie ultrafialová MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• statická elektřina MeSH
• uvolňování léčiv MeSH
• velikost částic MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyelektrolyty MeSH
• rifampin MeSH
• síran dextranu MeSH

Tuberculosis is a dreaded disease, which causes innumerable death worldwide. The emergence of drug resistance strains makes the situation devastating. Therefore, for better management of public health, it is mandatory to search for new anti-mycobacterial agents. In this context, the current study investigated two edible marine algae, Ulva lactuca and Ulva intestinalis, for the probable source of new anti-mycobacterial agents. To test the anti-mycobacterial activity, alcoholic extracts of these two algae were spotted on the Mycobacterium smegmatis lawn. Upon incubation, clear zone was observed at the spots. It indicated that these two extracts have anti-mycobacterial activity. In addition, their anti-biofilm property was also tested. It was found that both the extracts inhibit the mycobacterial biofilm development as well as they can disperse the preformed mycobacterial biofilm. Since these two are capable of dispersing preformed mycobacterial biofilm, it is possible that in the presence of either of these two extracts, isoniazid and rifampicin can kill biofilm encapsulated mycobacterium in combinatorial therapy. Consistent with the hypothesis, rifampicin and isoniazid killed mycobacteria that were present in biofilm. Thus, these two extracts augment the activity of rifampicin and isoniazid upon biofilm dispersal. Moreover, treatment of different cell lines with these two extracts exhibited no or little cytotoxic effects. Thus, these two agents have the potential to be good therapeutic agents against mycobacterial diseases.

• MeSH
• antibakteriální látky izolace a purifikace   farmakologie   MeSH
• biofilmy účinky léků   MeSH
• buněčné linie MeSH
• isoniazid farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mořské řasy chemie   MeSH
• Mycobacterium smegmatis účinky léků   MeSH
• rifampin farmakologie   MeSH
• rostlinné extrakty izolace a purifikace   farmakologie   MeSH
• synergismus léků MeSH
• Ulva chemie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• isoniazid MeSH
• rifampin MeSH
• rostlinné extrakty MeSH

The design of improved biopolymeric based hydrogel materials with high load-capacity to serve as biocompatible drug carriers is a challenging task with vital implications in health sciences. In this work, chitosan crosslinked dialdehyde xanthan gum interpenetrated hydroxypropyl methylcellulose gels were developed for the controlled delivery of different antibiotic drugs including ampicillin, minocycline and rifampicin. The prepared hydrogel scaffolds were characterized by rheology method, FTIR, SEM, TGA and compression analysis. In addition, gelation kinetics, swelling, in vitro degradation and drug release rate were studied under simulated gastrointestinal fluid conditions of pH 2.0 and 7.4 at 37 °C. Results demonstrated the gel composition and structure affected drug release kinetics. The release study showed more than 50% cumulative release within 24 h for all investigated antibiotic drugs. In vitro cell cytocompatibility using mouse embryonic fibroblast cell lines depicted ≥80% cell viability, indicating the gels are non-toxic. Finally, the antibacterial activity of loaded gels was evaluated against Gram-negative and positive bacteria (Escherichia coli, Staphylococcus aureus and Klebsiella pneumonia), which correlated well with swelling and drug release results. Overall, the present study demonstrated that the produced hydrogel scaffolds serves as promising material for controlled antibiotic delivery towards microbial growth inhibition.

• Klíčová slova
• Antibacterial activity, Antibiotic drug delivery, Biopolymers, Hydrogel,
• MeSH
• ampicilin farmakologie   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální polysacharidy chemie   MeSH
• biokompatibilní materiály chemie   MeSH
• buněčné linie MeSH
• chitosan chemie   MeSH
• deriváty hypromelózy chemie   MeSH
• Escherichia coli účinky léků   MeSH
• fibroblasty MeSH
• hydrogely chemická syntéza   chemie   farmakokinetika   toxicita   MeSH
• koncentrace vodíkových iontů MeSH
• mikrobiální testy citlivosti MeSH
• mikroskopie elektronová rastrovací MeSH
• minocyklin farmakologie   MeSH
• myši MeSH
• nosiče léků chemie   MeSH
• reologie MeSH
• rifampin farmakologie   MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• Staphylococcus aureus účinky léků   MeSH
• termogravimetrie MeSH
• uvolňování léčiv MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ampicilin MeSH
• antibakteriální látky MeSH
• bakteriální polysacharidy MeSH
• biokompatibilní materiály MeSH
• chitosan MeSH
• deriváty hypromelózy MeSH
• hydrogely MeSH
• minocyklin MeSH
• nosiče léků MeSH
• rifampin MeSH
• xanthan gum MeSH Prohlížeč