Biomedical subjects

Frantisek Staud

Publications and source records attributed to Frantisek Staud.

10 recordsLinked to original sources

PubMed2006

Molecular determinants in the transport of a bile acid-derived diagnostic agent in tumoral and nontumoral cell lines of human liver.

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Biological Transport↗

PubMed2006

Expression and transport activity of breast cancer resistance protein (Bcrp/Abcg2) in dually perfused rat placenta and HRP-1 cell line.

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 microM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 microM) and abolished at high cimetidine concentrations (1000 microM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Synthesis and antimicrobial evaluation of new 2-substituted 5,7-di-tert-butylbenzoxazoles.

Various synthetic pathways of the 30 novel 2-substituted 5,7-di-tert-butylbenzoxazoles as new potential antimicrobial drugs are discussed. The 28 intermediates are described as well. The compounds were characterized by 1H and 13C NMR spectra, MS spectra, IR/UV spectra and by means of CHN analysis. The purity of the final compounds was checked by HPLC and their lipophilicity (log K) was also determined by means of RP-HPLC. In the present study, the correlation between RP-HPLC retention parameter log K (the logarithm of capacity factor K) and various calculated log P data is shown. The target compounds were tested for their in vitro antimycobacterial activity. Several compounds showed antituberculotic activity comparable with or higher than the standard isoniazide. In vitro cytotoxicity testing of the most active benzoxazoles and isoniazide as a reference drug was performed using MTT assay and compared with isoniazide as a reference drug. Structure-activity relationships among the chemical structures, the physical properties and the biological activities of the evaluated compounds are discussed in the article.

Anti-Bacterial Agents↗

PubMed2006

P-glycoprotein in the placenta: expression, localization, regulation and function.

Detailed understanding of the mechanisms employed in transfer of drugs across the placenta is essential for optimization of pharmacotherapy during pregnancy. Disclosure of drug efflux transporters as an "active component" of the placental barrier has brought new important insights into the field of transplacental pharmacokinetics. P-glycoprotein (P-gp, MDR1) is the first discovered and so far the best characterized of drug efflux transporters, whose role in the regulation of drug disposition to the fetus has been extensively studied. Expression of P-gp in the placental trophoblast layer was confirmed at the mRNA and protein levels in all phases of pregnancy, and several in vitro and in vivo studies demonstrated functional activity of the transporter in materno-fetal drug transport. P-gp is able to actively pump drugs and other xenobiotics from trophoblast cells back to the maternal circulation, providing thus protection to the fetus. This review summarizes the current knowledge on the expression, localization and function of P-gp in the placenta. In addition, we include the latest data concerning transcriptional regulation of placental P-gp expression and polymorphisms of the MDR1 gene. Clinical significance of placental P-gp and its future perspectives for pharmacotherapy during pregnancy are also discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents.

PURPOSE: Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood-brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined. METHODS: To study substrate affinity of tested AEDs to BCRP, transport experiments were performed in epithelial BCRP-expressing MDCKII-BCRP and MDCKII-parent cell lines cultured on microporous membrane. For detection of inhibitory potency of AEDs to BCRP, accumulation assays were carried out in MEF3.8-BCRP cells with known BCRP substrates, BODIPY FL prazosin and mitoxantrone. RESULTS: No obvious interactions of tested AEDs with BCRP transporter were observed. Therefore these drugs in relevant therapeutic concentrations are neither substrates nor inhibitors of BCRP. CONCLUSIONS: Based on our in vitro data we can conclude that resistance to treatment with the tested AEDs probably is not caused by the overexpression of BCRP in the BBB of epileptogenic brain tumors.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

Breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP) was identified 7 years ago as the most recent member of ABC drug efflux membrane transporters. It is a 655 amino acid peptide with an ability to extrude a wide variety of chemical compounds from the cells. Today, it is considered as one of three major transporters causing drug resistance in mammalian cells. It is also distributed in epithelia involved in drug disposition with major role in the placenta, liver and intestine. In addition, BCRP is responsible for the "side population" phenotype of stem cells and seems to play a significant role in protection against hypoxia. BCRP inhibitors are currently searched for to overcome drug resistance and to improve the pharmacokinetics, mainly intestinal absorption, of substrate drugs. Mutant BCRP has also been used as a selectable marker in stem cell gene therapy applications.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2004

Inhibition of experimental hepatic metastasis by targeted delivery of catalase in mice.

Bovine liver catalase derivatives possessing diverse tissue distribution properties were synthesized, and their effects on hepatic metastasis of colon carcinoma cells were examined in mice. An intraportal injection of 1 x 10(5) colon 26 cells resulted in the formation of more than 50 metastatic colonies on the surface of the liver at 14 days after injection. An intravenous injection of catalase (CAT; 35000 units/kg of body weight) significantly (P < 0.001) reduced the number of the colonies in the liver. Galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT were also tested in the same system. Of these derivatives, Gal-CAT showed the greatest inhibitory effect on hepatic metastasis, and the number of colonies was significantly (P < 0.001) smaller than following treatment with catalase. High activities of matrix metalloproteinases (MMPs), especially MMP-9, were detected in the liver of mice bearing metastatic tumor tissues, which was significantly (P < 0.05) reduced by Gal-CAT. These results, combined with our previous finding that Gal-CAT can be efficiently delivered to hepatocytes, indicate that the targeted delivery of catalase to the liver by galactosylation is a promising approach to suppress hepatic metastasis. Decreased MMP activity by catalase delivery seems to be involved in its anti-metastatic effect.

Animals↗

PubMed2003

Examination of the functional activity of P-glycoprotein in the rat placental barrier using rhodamine 123.

Rhodamine 123 (Rho123), a model substrate of P-glycoprotein (P-gp), was used to evaluate the functional activity of P-gp efflux transporter in the rat placental barrier. The dually perfused rat-term placenta method was used. In our experiments, the materno-fetal transplacental passage of Rho123 did not meet the criteria of the first-order pharmacokinetics, suggesting an involvement of transporter-mediated process. Inhibitors of P-gp, such as [3'-keto-Bmt1]-[Val2]-cyclosporine (PSC833), cyclosporine (CsA), quinidine, and chlorpromazine, increased significantly the materno-fetal transplacental passage of Rho123 in the experiments under steady-state conditions. On the other hand, PSC833, CsA, and quinidine decreased the feto-maternal passage of Rho123. Similarly, in the experiments carried out under nonsteady-state conditions, CsA accelerated the passage of Rho123 in the materno-fetal direction and decreased its passage in the opposite direction. Feto-maternal transplacental clearances of Rho123 were found to be considerably higher than those in the materno-fetal course. Potent P-gp inhibitors, such as PSC833 or CsA, partially canceled the asymmetry. Negligible metabolism of Rho123 into its major demethylated metabolite rhodamine 110 was observed in the rat placenta. Expression of P-gp genes was detected using immunohistochemical, Western blotting, and reverse transcription-polymerase chain reaction methods preferentially in the second rat syncytiotrophoblast layer. In conclusion, these data suggest that P-gp limits the entry of Rho123 into fetuses and at the same time it accelerates the feto-maternal elimination of the model compound. Therefore, it seems plausible that pharmacokinetics of xenobiotics in the rat placental barrier could be controlled by P-gp in both directions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

P-glycoprotein expression and distribution in the rat placenta during pregnancy.

P-glycoprotein (P-gp) is a drug efflux transporter that limits the entry of various potentially toxic drugs and xenobiotics into the fetus and is thus considered a placental protective mechanism. In this study, P-gp expression was investigated in the rat chorioallantoic placenta over the course of pregnancy. Three methods have been employed: real-time RT-PCR, western blotting and immunohistochemistry. The expression of mdr1a and mdr1b genes was demonstrated as early as on the 11th gestation day (gd) and increased with advancing gestation. Western blotting analysis revealed the presence of P-gp in the rat placenta starting from gd 13 onwards. P-gp was localized in the developing labyrinth zone of the placenta on gd 13; from gd 15 up to the term P-gp was seen as a dot like continuous line in the syncytiotrophoblast layers. Our data confirm the presence of P-gp in the rat chorioallantoic placenta starting soon after its development, which may signify the involvement of P-gp in transplacental pharmacokinetics during the whole period of placental maturing.

ATP Binding Cassette Transporter, Subfamily B↗

PubMedDate not supplied

Expression and functional activity of breast cancer resistance protein (BCRP, ABCG2) transporter in the human choriocarcinoma cell line BeWo.

1. Breast cancer resistance protein (BCRP, ABCG2) is a drug efflux transporter that is believed to affect the drug disposition of several drugs and xenobiotics. In the present study, we evaluated the localization and functional expression of BCRP in the human choriocarcinoma cell line BeWo, an in vitro model of the human trophoblast, and compared it with the expression of P-glycoprotein (MDR1, ABCB1) as the most widely studied placental transporter. In addition, the expression of BCRP at the mRNA level was compared with that of MDR1 in the human term placenta. 2. Western blotting analysis revealed high endogenous expression of BCRP protein in BeWo cells. Using indirect immunofluorescence microscopy, we found that the BCRP transporter appears to be localized predominantly at the apical plasma membrane. Functional studies showed a significant effect of the BCRP inhibitors GF120918 (5 micromol/L) and Ko143 (1 micromol/L) on mitoxantrone accumulation and, thus, confirmed efflux activity of BCRP in BeWo cells. 3. Using absolute mRNA quantification with real-time reverse transcription-polymerase chain reaction, we found high expression of BCRP in BeWo cells, whereas no transcript of MDR1 (P-glycoprotein), the most extensively studied drug transporter, was detected. 4. In the human placenta, BCRP was localized predominantly in the syncytiotrophoblast layer; however, immunopositivity for the BXP-21 antibody was also observed in fetal vessels of the chorionic villi. The number of BCRP transcripts in the human term placenta was found to be more than 10-fold higher compared with the expression of MDR1. 5. In conclusion, we suggest that BeWo cells could serve as a suitable in vitro model to study trans-trophoblast transport of BCRP substrates and that placental BCRP can play an important role in preventing the accumulation of potentially toxic xenobiotics in the trophoblast cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗