The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
David S. Hage - One of the best experts on this subject based on the ideXlab platform.
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on column entrapment of alpha1 acid glycoProtein for studies of Drug Protein Binding by high performance affinity chromatography
Analytical and Bioanalytical Chemistry, 2016Co-Authors: Jeanethe Anguizola, Michelle Koke, Abby J Jackson, David S. HageAbstract:An on-column approach for Protein entrapment was developed to immobilize alpha1-acid glycoProtein (AGP) for Drug-Protein Binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the Binding of various Drugs with the entrapped AGP. Site-selective competition studies were also conducted for these Drugs. The results showed good agreement with previous observations for these Drug-Protein systems and with Binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of Drug interactions with AGP or other Proteins.
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entrapment of alpha1 acid glycoProtein in high performance affinity columns for Drug Protein Binding studies
Journal of Chromatography B, 2016Co-Authors: Abby J Jackson, Jeanethe Anguizola, John Vargasbadilla, Giana Rada, Erika L Pfaunmiller, David S. HageAbstract:A slurry-based method was developed for the entrapment of alpha1-acid glycoProtein (AGP) for use in high-performance affinity chromatography to study Drug interactions with this serum Protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in Binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other Drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the Binding of various Drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these Proteins. The same entrapment method could be extended to other Proteins and to the investigation of additional types of Drug-Protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of Drug candidates for their Binding to a given Protein.
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analysis of Drug Protein Binding using on line immunoextraction and high performance affinity microcolumns studies with normal and glycated human serum albumin
Journal of Chromatography A, 2015Co-Authors: Ryan Matsuda, Donald Jobe, Jared Beyersdorf, David S. HageAbstract:A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining Drug-Protein interactions with normal or modified Proteins. Normal human serum albumin (HSA) and glycated HSA were used as model Proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of Protein at 0.05-0.10 mL/min and had a Binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed Proteins were tested for use in various approaches for Drug Binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these Proteins for the Drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar Protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this Drug at Sudlow sites I and II of the adsorbed Proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that Drug-Protein Binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other Drugs or Proteins of interest in pharmaceutical studies or biomedical research.
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analysis of multi site Drug Protein interactions by high performance affinity chromatography Binding by glimepiride to normal or glycated human serum albumin
Journal of Chromatography A, 2015Co-Authors: Ryan Matsuda, Zhao Li, Xiwei Zheng, David S. HageAbstract:Abstract High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea Drug, and normal or in vitro glycated forms of the transport Protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or K a , 9.2–11.8 × 10 5 M −1 at pH 7.4 and 37 °C) and a group of lower affinity regions ( K a , 5.9–16 × 10 3 M −1 ). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the Binding by this Drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R -warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The Binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site Binding by a Drug such as glimepiride to a Protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how Drug–Protein Binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified Proteins.
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development of affinity microcolumns for Drug Protein Binding studies in personalized medicine interactions of sulfonylurea Drugs with in vivo glycated human serum albumin
Analytical Chemistry, 2013Co-Authors: Jeanethe Anguizola, K S Joseph, Omar S Barnaby, Ryan Matsuda, Guadalupe Alvarado, William Clarke, Ronald L Cerny, David S. HageAbstract:This report used high-performance affinity microcolumns to examine the changes in Binding by sulfonylurea Drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of Protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple Drugs and experimental conditions over hundreds of sample application cycles. Both the overall Binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in sit...
Ryan Matsuda - One of the best experts on this subject based on the ideXlab platform.
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analysis of Drug Protein Binding using on line immunoextraction and high performance affinity microcolumns studies with normal and glycated human serum albumin
Journal of Chromatography A, 2015Co-Authors: Ryan Matsuda, Donald Jobe, Jared Beyersdorf, David S. HageAbstract:A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining Drug-Protein interactions with normal or modified Proteins. Normal human serum albumin (HSA) and glycated HSA were used as model Proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of Protein at 0.05-0.10 mL/min and had a Binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed Proteins were tested for use in various approaches for Drug Binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these Proteins for the Drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar Protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this Drug at Sudlow sites I and II of the adsorbed Proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that Drug-Protein Binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other Drugs or Proteins of interest in pharmaceutical studies or biomedical research.
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analysis of multi site Drug Protein interactions by high performance affinity chromatography Binding by glimepiride to normal or glycated human serum albumin
Journal of Chromatography A, 2015Co-Authors: Ryan Matsuda, Zhao Li, Xiwei Zheng, David S. HageAbstract:Abstract High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea Drug, and normal or in vitro glycated forms of the transport Protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or K a , 9.2–11.8 × 10 5 M −1 at pH 7.4 and 37 °C) and a group of lower affinity regions ( K a , 5.9–16 × 10 3 M −1 ). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the Binding by this Drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R -warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The Binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site Binding by a Drug such as glimepiride to a Protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how Drug–Protein Binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified Proteins.
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development of affinity microcolumns for Drug Protein Binding studies in personalized medicine interactions of sulfonylurea Drugs with in vivo glycated human serum albumin
Analytical Chemistry, 2013Co-Authors: Jeanethe Anguizola, K S Joseph, Omar S Barnaby, Ryan Matsuda, Guadalupe Alvarado, William Clarke, Ronald L Cerny, David S. HageAbstract:This report used high-performance affinity microcolumns to examine the changes in Binding by sulfonylurea Drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of Protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple Drugs and experimental conditions over hundreds of sample application cycles. Both the overall Binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in sit...
Joanna Szymuraoleksiak - One of the best experts on this subject based on the ideXlab platform.
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capillary electrophoresis frontal analysis versus equilibrium dialysis in dexamethasone sodium phosphate serum albumin Binding studies
Electrophoresis, 2012Co-Authors: Anna Gonciarz, Kamil Kus, Malgorzata Szafarz, Maria Walczak, Agnieszka Zakrzewska, Joanna SzymuraoleksiakAbstract:Plasma Protein Binding of Drugs may have significant effect on its pharmacodynamic, toxicological and pharmacokinetic properties, since only the free Drug can pass across biological membrane and get to its specific site of action. Many Drugs show a high affinity to albumin which is the most abundant plasma Protein. In the present study capillary electrophoresis in the frontal analysis mode (CE/FA), as promising technique for assessment of Drug-Protein interaction was used. The free Drug concentration was measured from height of the frontal peak and calculated based on the external Drug standard in absence of Protein. With a known concentration of total Drug, the percentage of Protein bound Drug was determined. The Binding parameters were also estimated based on the equilibrium dialysis experiment which is considered to be a reference method. This study was designed to examine the interaction of dexamethasone sodium phosphate (DXM) with BSA and HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate buffer, I = 0.17). Using fixed, at physiological level, HSA and BSA concentrations and increasing DXM concentrations, the number of Binding sites (n) and Binding constant (K(a) ) was calculated from both nonlinear regression fitting and Scatchard Plot. Despite some differences, it can be concluded that the CE/FA is comparable with equilibrium dialysis, but since the first one offers advantages such as low sample consumption, short analysis time, and high separation efficiency, it can be used in high-throughput screening of Drug Protein Binding at the early stage of Drug discovery. Interspecies differences in Binding of a Drug to albumins have been observed and it should be taken into account in interpretation of the results.
Jeanethe Anguizola - One of the best experts on this subject based on the ideXlab platform.
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on column entrapment of alpha1 acid glycoProtein for studies of Drug Protein Binding by high performance affinity chromatography
Analytical and Bioanalytical Chemistry, 2016Co-Authors: Jeanethe Anguizola, Michelle Koke, Abby J Jackson, David S. HageAbstract:An on-column approach for Protein entrapment was developed to immobilize alpha1-acid glycoProtein (AGP) for Drug-Protein Binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the Binding of various Drugs with the entrapped AGP. Site-selective competition studies were also conducted for these Drugs. The results showed good agreement with previous observations for these Drug-Protein systems and with Binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of Drug interactions with AGP or other Proteins.
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entrapment of alpha1 acid glycoProtein in high performance affinity columns for Drug Protein Binding studies
Journal of Chromatography B, 2016Co-Authors: Abby J Jackson, Jeanethe Anguizola, John Vargasbadilla, Giana Rada, Erika L Pfaunmiller, David S. HageAbstract:A slurry-based method was developed for the entrapment of alpha1-acid glycoProtein (AGP) for use in high-performance affinity chromatography to study Drug interactions with this serum Protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in Binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other Drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the Binding of various Drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these Proteins. The same entrapment method could be extended to other Proteins and to the investigation of additional types of Drug-Protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of Drug candidates for their Binding to a given Protein.
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development of affinity microcolumns for Drug Protein Binding studies in personalized medicine interactions of sulfonylurea Drugs with in vivo glycated human serum albumin
Analytical Chemistry, 2013Co-Authors: Jeanethe Anguizola, K S Joseph, Omar S Barnaby, Ryan Matsuda, Guadalupe Alvarado, William Clarke, Ronald L Cerny, David S. HageAbstract:This report used high-performance affinity microcolumns to examine the changes in Binding by sulfonylurea Drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of Protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple Drugs and experimental conditions over hundreds of sample application cycles. Both the overall Binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in sit...
K S Joseph - One of the best experts on this subject based on the ideXlab platform.
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development of affinity microcolumns for Drug Protein Binding studies in personalized medicine interactions of sulfonylurea Drugs with in vivo glycated human serum albumin
Analytical Chemistry, 2013Co-Authors: Jeanethe Anguizola, K S Joseph, Omar S Barnaby, Ryan Matsuda, Guadalupe Alvarado, William Clarke, Ronald L Cerny, David S. HageAbstract:This report used high-performance affinity microcolumns to examine the changes in Binding by sulfonylurea Drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of Protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple Drugs and experimental conditions over hundreds of sample application cycles. Both the overall Binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in sit...
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detection of heterogeneous Drug Protein Binding by frontal analysis and high performance affinity chromatography
Journal of Chromatography A, 2011Co-Authors: Zenghan Tong, K S Joseph, David S. HageAbstract:Abstract This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous Binding in biomolecular interactions, using the Binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of Binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the Binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of Binding heterogeneity. The methods developed in this work for the detection of Binding heterogeneity are not limited to Drug interactions with HSA but could be applied to other types of Drug–Protein Binding or to additional biological systems with heterogeneous Binding.
