The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Peter Krajcsi - One of the best experts on this subject based on the ideXlab platform.
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The vesicular Transport Assay: validated in vitro methods to study drug-mediated inhibition of canalicular efflux Transporters ABCB11/BSEP and ABCC2/MRP2.
Current protocols in toxicology, 2012Co-Authors: Krisztina Herédi-szabó, Emese Kis, Peter KrajcsiAbstract:The canalicular membrane of hepatocytes contains several Transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which Transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these Transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated Transport processes is the vesicular Transport Assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a Transport Assay for a known or potential substrate and test small molecule inhibition of the Transporters.
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Current Protocols in Toxicology - The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug‐Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2
Current Protocols in Toxicology, 2012Co-Authors: Krisztina Herédi-szabó, Emese Kis, Peter KrajcsiAbstract:The canalicular membrane of hepatocytes contains several Transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which Transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these Transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated Transport processes is the vesicular Transport Assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a Transport Assay for a known or potential substrate and test small molecule inhibition of the Transporters.
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Comparison of 3 Assay Systems Using a Common Probe Substrate, Calcein AM, for Studying P-gp Using a Selected Set of Compounds
Journal of biomolecular screening, 2010Co-Authors: Péter Szerémy, Peter Krajcsi, Akos Pal, Dóra Méhn, Beáta Tóth, Ferenc Fülöp, Krisztina Herédi-szabóAbstract:The multidrug resistance protein 1 (MDR1) Transporter is the most abundantly investigated adenosine triphosphate (ATP)–Binding Cassette (ABC) Transporter protein. Multiple Assay systems were developed to study MDR1-mediated Transport and possible drug-drug interactions. Yet, as different probe substrates are used in these Assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 Assay systems developed to study MDR1: the cellular dye efflux Assay, the ATPase Assay, and the vesicular Transport Assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 Assay systems. The vesicular Transport Assay proved most sensitive, detecting 18 of 22 interacti...
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Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs
Journal of biomolecular screening, 2008Co-Authors: Emese Kis, Dóra Méhn, Zsuzsanna Rajnai, Enikő Ioja, Krisztina Herédi Szabó, Tünde Nagy, Peter KrajcsiAbstract:The mouse ortholog of the human bile salt export pump (BSEP) Transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the Transporter function. The Transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular Transport Assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep Transports TCDC at a high rate that can be sensitively detected in the ATPase Assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC Transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate Transport was tested in both vesicular Transport and ATPase Assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC 50 values measured in TCDC-stimulated mBsep ATPase Assay and in the human BSEP vesicular Transport Assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase Assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular Transport inhibition Assay. (Journal of Biomolecular Screening 2009:10-15)
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Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein Transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4.
Journal of biomolecular screening, 2008Co-Authors: Krisztina Herédi-szabó, Emese Kis, Éva D. Molnár, Andras Gyorfi, Peter KrajcsiAbstract:MRP2 (ABCC2) is an efflux Transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 Transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this Transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular Transport Assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC Transporters. In this Assay, inside-out membrane vesicles are used, so the test compound can readily access the Transporter. As MRP2 Transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular Transport Assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the Transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular Transport Assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular Transport Assay with its optimal detection and Transport properties.
Krisztina Herédi-szabó - One of the best experts on this subject based on the ideXlab platform.
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The vesicular Transport Assay: validated in vitro methods to study drug-mediated inhibition of canalicular efflux Transporters ABCB11/BSEP and ABCC2/MRP2.
Current protocols in toxicology, 2012Co-Authors: Krisztina Herédi-szabó, Emese Kis, Peter KrajcsiAbstract:The canalicular membrane of hepatocytes contains several Transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which Transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these Transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated Transport processes is the vesicular Transport Assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a Transport Assay for a known or potential substrate and test small molecule inhibition of the Transporters.
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Current Protocols in Toxicology - The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug‐Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2
Current Protocols in Toxicology, 2012Co-Authors: Krisztina Herédi-szabó, Emese Kis, Peter KrajcsiAbstract:The canalicular membrane of hepatocytes contains several Transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which Transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these Transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated Transport processes is the vesicular Transport Assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a Transport Assay for a known or potential substrate and test small molecule inhibition of the Transporters.
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Comparison of 3 Assay Systems Using a Common Probe Substrate, Calcein AM, for Studying P-gp Using a Selected Set of Compounds
Journal of biomolecular screening, 2010Co-Authors: Péter Szerémy, Peter Krajcsi, Akos Pal, Dóra Méhn, Beáta Tóth, Ferenc Fülöp, Krisztina Herédi-szabóAbstract:The multidrug resistance protein 1 (MDR1) Transporter is the most abundantly investigated adenosine triphosphate (ATP)–Binding Cassette (ABC) Transporter protein. Multiple Assay systems were developed to study MDR1-mediated Transport and possible drug-drug interactions. Yet, as different probe substrates are used in these Assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 Assay systems developed to study MDR1: the cellular dye efflux Assay, the ATPase Assay, and the vesicular Transport Assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 Assay systems. The vesicular Transport Assay proved most sensitive, detecting 18 of 22 interacti...
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Multidrug Resistance Protein 2-Mediated Estradiol-17β-d-glucuronide Transport Potentiation: In Vitro-in Vivo Correlation and Species Specificity
Drug metabolism and disposition: the biological fate of chemicals, 2008Co-Authors: Krisztina Herédi-szabó, Emese Kis, Dóra Méhn, H. Glavinas, György Báthori, Zsuzsa Veres, László Kóbori, O. Von Richter, Katalin Jemnitz, P. KrajcsiAbstract:Multidrug resistance protein 2 (MRP2) is a multispecific organic anion Transporter expressed at important pharmacological barriers, including the canalicular membrane of hepatocytes. At this location it is involved in the elimination of both endogenous and exogenous waste products, mostly as conjugates, to the bile. Estradiol-17β-d-glucuronide (E 2 17βG), a widely studied endogenous substrate of MRP2, was shown earlier to recognize two binding sites of the Transporter in vesicular Transport Assays. MRP2 modulators (substrates and nonsubstrates) potentiate the Transport of E 2 17βG by MRP2. We correlated data obtained from studies of different complexities and investigated the species-specific differences between rat and human MRP2-mediated Transport. We used vesicular Transport Assays, sandwich-cultured primary hepatocytes, and in vivo biliary efflux in rats. Our results demonstrate that the rat Mrp2 Transporter, unlike the human MRP2, Transports E 2 17βG according to Michaelis-Menten type kinetics. Nevertheless, in the presence of modulator drugs E 2 17βG Transport mediated by the rat Transporter also shows cooperative kinetics as potentiation of E 2 17βG Transport was observed in the vesicular Transport Assay. We also demonstrated that the potentiation exists both in rat and in human hepatocytes and in vivo in rats.
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Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein Transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4.
Journal of biomolecular screening, 2008Co-Authors: Krisztina Herédi-szabó, Emese Kis, Éva D. Molnár, Andras Gyorfi, Peter KrajcsiAbstract:MRP2 (ABCC2) is an efflux Transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 Transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this Transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular Transport Assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC Transporters. In this Assay, inside-out membrane vesicles are used, so the test compound can readily access the Transporter. As MRP2 Transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular Transport Assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the Transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular Transport Assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular Transport Assay with its optimal detection and Transport properties.
Cesare Indiveri - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial carnitine/acylcarnitine translocase: insights in structure/ function relationships. Basis for drug therapy and side effects prediction.
Mini reviews in medicinal chemistry, 2015Co-Authors: Annamaria Tonazzi, Lara Console, Nicola Giangregorio, Cesare IndiveriAbstract:The mitochondrial carnitine/acylcarnitine translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of Transport Assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as K + /H + -ATPase inhibitors (omeprazole) or antibiotics (β-lactams) on the carnitine/acylcarnitine translocase, which can explain some of their adverse effects.
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Mitochondrial Carnitine/Acylcarnitine Translocase: Insights in Structure/Function Relationship.
Mini reviews in medicinal chemistry, 2015Co-Authors: Annamaria Tonazzi, Lara Console, Nicola Giangregorio, Cesare IndiveriAbstract:The mitochondrial carnitine/acylcarnitine translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of Transport Assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as KM+/H+-ATPase inhibitors (omeprazole) or antibiotics (β-lactams) on the carnitine/acylcarnitine translocase, which can explain some of their adverse effects.
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nimesulide binding site in the b0at1 slc6a19 amino acid Transporter mechanism of inhibition revealed by proteoliposome Transport Assay and molecular modelling
Biochemical Pharmacology, 2014Co-Authors: Lorena Pochini, Angela Seidita, Cristina Sensi, Mariafrancesca Scalise, Ivano Eberini, Cesare IndiveriAbstract:Abstract The effect of pharmaceutical compounds on the rat kidney B0AT1 Transporter in proteoliposomes has been screened. To this aim, inhibition of the Transport activity by the different compounds was measured on Na+-[3H]glutamine co-Transport in the presence of membrane potential positive outside. Most of the tested drugs had no effect on the Transport activity. Some compounds exhibited inhibitory effects from 5 to 88% at concentration of 300 μM. Among the tested compounds, only the anti-inflammatory drug nimesulide exerted potent inhibition on B0AT1. From dose response analysis, an IC50 value of 23 μM was found. Inhibition kinetic analysis was performed: noncompetitive inhibition of the glutamine Transport was observed while competitive behaviour was found when the inhibition was analyzed with respect to the Na+ concentration. Several molecules harbouring functional groups of nimesulide (analogues) were tested as inhibitors. None among the tested molecules has the capacity to inhibit the Transport with the exception of the compound NS-398, whose chemical structure is very close to that of whole nimesulide. The IC50 for this compound was 131 μM. Inhibition kinetics showed behaviour of NS-398 identical to that of nimesulide, i.e., noncompetitive inhibition respect to glutamine and competitive inhibition respect to Na+. Molecular docking of nimesulide suggested that this drug is able to bind B0AT1 in an external dedicated binding site and that its binding produces a steric hindrance effect of the protein translocation path abolishing the Transporter activity.
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Inactivation by Hg and methylmercury of the glutamine/amino acid Transporter (ASCT2) reconstituted in liposomes. Prediction of the involvement of a CXXC motif by homology modelling.
Biochemical Pharmacology, 2010Co-Authors: Francesca Oppedisano, Michele Galluccio, Cesare IndiveriAbstract:The effect of HgCl, methylmercury and mersalyl on the glutamine/amino acid (ASCT2) Transporter reconstituted in liposomes has been studied. Mercuric compounds externally added to the proteoliposomes, inhibited the glutamine/glutamine antiport catalyzed by the reconstituted Transporter. Similar effects were observed by pre-treating the proteoliposomes with the mercurials and then removing unreacted compounds before the Transport Assay. The inhibition was reversed by DTE, cysteine and N-acetyl-cysteine but not by S-carboxymethyl-cysteine. The data demonstrated that the inhibition was due to covalent reaction of mercuric compounds with Cys residue(s) of the Transporter. The IC of the Transporter for HgCl, methylmercury and mersalyl, were 1.4±0.10μM, 2.4±0.16μM or 3.1±0.19μM, respectively. Kinetic studies of the inhibition showed that the reagents behaved as non competitive inhibitor. The presence of glutamine or Na during the incubation of the mercuric compounds with the proteoliposomes did not exerted any protective effect on the inhibition. None of the compounds was Transported by the reconstituted Transporter. A metal binding motif CXXC has been predicted as possible site of interaction of the mercuric compounds with the Transporter on the basis of the homology structural model of ASCT2 obtained using the glutamate Transporter homologue from as template.
Emese Kis - One of the best experts on this subject based on the ideXlab platform.
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The vesicular Transport Assay: validated in vitro methods to study drug-mediated inhibition of canalicular efflux Transporters ABCB11/BSEP and ABCC2/MRP2.
Current protocols in toxicology, 2012Co-Authors: Krisztina Herédi-szabó, Emese Kis, Peter KrajcsiAbstract:The canalicular membrane of hepatocytes contains several Transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which Transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these Transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated Transport processes is the vesicular Transport Assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a Transport Assay for a known or potential substrate and test small molecule inhibition of the Transporters.
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Current Protocols in Toxicology - The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug‐Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2
Current Protocols in Toxicology, 2012Co-Authors: Krisztina Herédi-szabó, Emese Kis, Peter KrajcsiAbstract:The canalicular membrane of hepatocytes contains several Transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which Transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these Transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated Transport processes is the vesicular Transport Assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a Transport Assay for a known or potential substrate and test small molecule inhibition of the Transporters.
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Multidrug Resistance Protein 2-Mediated Estradiol-17β-d-glucuronide Transport Potentiation: In Vitro-in Vivo Correlation and Species Specificity
Drug metabolism and disposition: the biological fate of chemicals, 2008Co-Authors: Krisztina Herédi-szabó, Emese Kis, Dóra Méhn, H. Glavinas, György Báthori, Zsuzsa Veres, László Kóbori, O. Von Richter, Katalin Jemnitz, P. KrajcsiAbstract:Multidrug resistance protein 2 (MRP2) is a multispecific organic anion Transporter expressed at important pharmacological barriers, including the canalicular membrane of hepatocytes. At this location it is involved in the elimination of both endogenous and exogenous waste products, mostly as conjugates, to the bile. Estradiol-17β-d-glucuronide (E 2 17βG), a widely studied endogenous substrate of MRP2, was shown earlier to recognize two binding sites of the Transporter in vesicular Transport Assays. MRP2 modulators (substrates and nonsubstrates) potentiate the Transport of E 2 17βG by MRP2. We correlated data obtained from studies of different complexities and investigated the species-specific differences between rat and human MRP2-mediated Transport. We used vesicular Transport Assays, sandwich-cultured primary hepatocytes, and in vivo biliary efflux in rats. Our results demonstrate that the rat Mrp2 Transporter, unlike the human MRP2, Transports E 2 17βG according to Michaelis-Menten type kinetics. Nevertheless, in the presence of modulator drugs E 2 17βG Transport mediated by the rat Transporter also shows cooperative kinetics as potentiation of E 2 17βG Transport was observed in the vesicular Transport Assay. We also demonstrated that the potentiation exists both in rat and in human hepatocytes and in vivo in rats.
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Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs
Journal of biomolecular screening, 2008Co-Authors: Emese Kis, Dóra Méhn, Zsuzsanna Rajnai, Enikő Ioja, Krisztina Herédi Szabó, Tünde Nagy, Peter KrajcsiAbstract:The mouse ortholog of the human bile salt export pump (BSEP) Transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the Transporter function. The Transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular Transport Assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep Transports TCDC at a high rate that can be sensitively detected in the ATPase Assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC Transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate Transport was tested in both vesicular Transport and ATPase Assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC 50 values measured in TCDC-stimulated mBsep ATPase Assay and in the human BSEP vesicular Transport Assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase Assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular Transport inhibition Assay. (Journal of Biomolecular Screening 2009:10-15)
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Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein Transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4.
Journal of biomolecular screening, 2008Co-Authors: Krisztina Herédi-szabó, Emese Kis, Éva D. Molnár, Andras Gyorfi, Peter KrajcsiAbstract:MRP2 (ABCC2) is an efflux Transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 Transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this Transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular Transport Assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC Transporters. In this Assay, inside-out membrane vesicles are used, so the test compound can readily access the Transporter. As MRP2 Transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular Transport Assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the Transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular Transport Assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular Transport Assay with its optimal detection and Transport properties.
Albertus G De Boer - One of the best experts on this subject based on the ideXlab platform.
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relationship between permeability status of the blood brain barrier and in vitro permeability coefficient of a drug
European Journal of Pharmaceutical Sciences, 2000Co-Authors: Pieter J. Gaillard, Albertus G De BoerAbstract:Abstract Objective: The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood–brain barrier (BBB) during in vitro drug Transport Assays is essential for an accurate estimation of the permeability coefficient of a drug. Methods: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug Transport, sodium fluorescein (Papp,FLU, Mw 376 Da) and FITC-labeled dextran (Papp,FD4, Mw 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between Papp and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). Results: Papp,FLU and Papp,FD4 in control experiments varied from 1.6 up to 17.6 (10−6cm/s) and 0.3 up to 7.3 (10−6cm/s), respectively; while for individual filters Papp,FLU was 4 times higher than Papp,FD4 (R2=0.97). As long as TEER remained above 131·Ω cm2 for FLU or 122·Ω cm2 for FD4 during the Transport Assay, Papp remained independent from the basal permeability of the in vitro BBB. Below these TEER values, Papp increased exponentially. This nonlinear relationship between basal BBB permeability and Papp was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (PFLU and PFD4) were estimated to be 2.2±0.1 and 0.48±0.03 (10−6cm/s), respectively. In the experimentally challenged experiments, a reliable indication for PFLU and PFD4 could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. Conclusions: The assessment of basal BBB permeability status during drug Transport Assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug Transport Assay are determined.
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Relationship between permeability status of the blood–brain barrier and in vitro permeability coefficient of a drug
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2000Co-Authors: Pieter J. Gaillard, Albertus G De BoerAbstract:Abstract Objective: The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood–brain barrier (BBB) during in vitro drug Transport Assays is essential for an accurate estimation of the permeability coefficient of a drug. Methods: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug Transport, sodium fluorescein (Papp,FLU, Mw 376 Da) and FITC-labeled dextran (Papp,FD4, Mw 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between Papp and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). Results: Papp,FLU and Papp,FD4 in control experiments varied from 1.6 up to 17.6 (10−6cm/s) and 0.3 up to 7.3 (10−6cm/s), respectively; while for individual filters Papp,FLU was 4 times higher than Papp,FD4 (R2=0.97). As long as TEER remained above 131·Ω cm2 for FLU or 122·Ω cm2 for FD4 during the Transport Assay, Papp remained independent from the basal permeability of the in vitro BBB. Below these TEER values, Papp increased exponentially. This nonlinear relationship between basal BBB permeability and Papp was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (PFLU and PFD4) were estimated to be 2.2±0.1 and 0.48±0.03 (10−6cm/s), respectively. In the experimentally challenged experiments, a reliable indication for PFLU and PFD4 could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. Conclusions: The assessment of basal BBB permeability status during drug Transport Assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug Transport Assay are determined.
