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Adriana O Santos - One of the best experts on this subject based on the ideXlab platform.
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development and application of an ex vivo fosphenytoin nasal Bioconversion permeability evaluation method
European Journal of Pharmaceutical Sciences, 2016Co-Authors: Daniel Antunes Viegas, Marcio Rodrigues, Joana Francisco, Amilca Falcao, Gilberto Alves, Adriana O SantosAbstract:There is an increasing interest in the intranasal delivery of central nervous system-active drugs due to the existence of a direct nose-to-brain connection. However, poor solubility limits the amount of drug that can be administered within an aqueous solution. In the present work, the objectives were to develop an ex vivo Bioconversion/permeability evaluation method and to study the ex vivo Bioconversion of the hydrophilic phosphate ester prodrug fosphenytoin (FOS) to the active drug phenytoin (PHT) and their comparative nasal permeation. Bioconversion/permeability studies were performed in excised porcine nasal mucosa mounted in Ussing chambers. The physical integrity of the tissues was evaluated by measurement of the transepithelial electrical resistance (TEER). The simultaneous quantitative assay of FOS, PHT and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) was developed and validated according to international guidelines using a liquid chromatography analytical method. The FOS Bioconversion rate and PHT and FOS apparent permeability coefficients (Papp) were determined at different time points. FOS Bioconversion was also qualitatively investigated in human nasal mucus. The developed liquid chromatography method combines a fast and inexpensive sample preparation with inactivation of the enzymatic metabolism of the prodrug during sample manipulation and storage. It was linear, precise, accurate, and presented a high analyte recovery. FOS was converted ex vivo to PHT but the metabolite HPPH was not detected. The Bioconversion rate increased with FOS concentration and with time, which suggests a diffusion-limited process. FOS was also converted to its active drug by human nasal mucus. A novel mathematical data analysis method was developed to reduce the bias introduced by variable mucosal TEER in the permeability results. At comparable FOS and PHT concentrations the ln(Papp(PHT)) of both compounds showed little difference, which indicates that the use of a hydrophilic and charged prodrug did not hinder overall drug permeation. At the highest tested FOS concentration it was possible to quantify FOS in the receiver chambers, meaning that at a sufficiently high concentration the FOS permeation rate overcame its Bioconversion rate. The ln(Papp(PHT)) tended to similar equilibrium values as the assay progressed, but with higher FOS concentrations that equilibrium was attained faster. Acidic pH reduced the permeability of both PHT and FOS. The developed Bioconversion/permeability evaluation method will constitute an important tool to select the most promising formulations before proceeding to in vivo studies. Importantly, it allowed the demonstration of phosphatase activity and FOS Bioconversion in nasal mucosa, as well as the prodrug's nasal permeation potential. Furthermore, this study demonstrates the possibility of formulating phosphate prodrugs of poorly soluble central nervous system-active drugs as a strategy to increase the solubilized drug doses administered through the nasal route.
Gilberto Alves - One of the best experts on this subject based on the ideXlab platform.
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development and application of an ex vivo fosphenytoin nasal Bioconversion permeability evaluation method
European Journal of Pharmaceutical Sciences, 2016Co-Authors: Daniel Antunes Viegas, Marcio Rodrigues, Joana Francisco, Amilca Falcao, Gilberto Alves, Adriana O SantosAbstract:There is an increasing interest in the intranasal delivery of central nervous system-active drugs due to the existence of a direct nose-to-brain connection. However, poor solubility limits the amount of drug that can be administered within an aqueous solution. In the present work, the objectives were to develop an ex vivo Bioconversion/permeability evaluation method and to study the ex vivo Bioconversion of the hydrophilic phosphate ester prodrug fosphenytoin (FOS) to the active drug phenytoin (PHT) and their comparative nasal permeation. Bioconversion/permeability studies were performed in excised porcine nasal mucosa mounted in Ussing chambers. The physical integrity of the tissues was evaluated by measurement of the transepithelial electrical resistance (TEER). The simultaneous quantitative assay of FOS, PHT and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) was developed and validated according to international guidelines using a liquid chromatography analytical method. The FOS Bioconversion rate and PHT and FOS apparent permeability coefficients (Papp) were determined at different time points. FOS Bioconversion was also qualitatively investigated in human nasal mucus. The developed liquid chromatography method combines a fast and inexpensive sample preparation with inactivation of the enzymatic metabolism of the prodrug during sample manipulation and storage. It was linear, precise, accurate, and presented a high analyte recovery. FOS was converted ex vivo to PHT but the metabolite HPPH was not detected. The Bioconversion rate increased with FOS concentration and with time, which suggests a diffusion-limited process. FOS was also converted to its active drug by human nasal mucus. A novel mathematical data analysis method was developed to reduce the bias introduced by variable mucosal TEER in the permeability results. At comparable FOS and PHT concentrations the ln(Papp(PHT)) of both compounds showed little difference, which indicates that the use of a hydrophilic and charged prodrug did not hinder overall drug permeation. At the highest tested FOS concentration it was possible to quantify FOS in the receiver chambers, meaning that at a sufficiently high concentration the FOS permeation rate overcame its Bioconversion rate. The ln(Papp(PHT)) tended to similar equilibrium values as the assay progressed, but with higher FOS concentrations that equilibrium was attained faster. Acidic pH reduced the permeability of both PHT and FOS. The developed Bioconversion/permeability evaluation method will constitute an important tool to select the most promising formulations before proceeding to in vivo studies. Importantly, it allowed the demonstration of phosphatase activity and FOS Bioconversion in nasal mucosa, as well as the prodrug's nasal permeation potential. Furthermore, this study demonstrates the possibility of formulating phosphate prodrugs of poorly soluble central nervous system-active drugs as a strategy to increase the solubilized drug doses administered through the nasal route.
Daniela Ubiali - One of the best experts on this subject based on the ideXlab platform.
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a comparison between immobilized pyrimidine nucleoside phosphorylase from bacillus subtilis and thymidine phosphorylase from escherichia coli in the synthesis of 5 substituted pyrimidine 2 deoxyribonucleosides
Journal of Molecular Catalysis B-enzymatic, 2013Co-Authors: Immacolata Serra, Teodora Bavaro, Davide A. Cecchini, Simona Daly, Marco Terreni, Alessandra M Albertini, Daniela UbialiAbstract:Abstract Pyrimidine nucleoside phosphorylase from Bacillus subtilis ( Bs PyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli ( Ec TP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2′-deoxyribonucleosides ( 14 – 18 ) by transglycosylation in fully aqueous medium. From the comparative study of the two biocatalysts, no remarkable differences emerged about their substrate specificity, Bioconversion yield, stability in organic cosolvents (DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2′,3′-dideoxynucleosides as substrates, whereas they catalyze Bioconversions involving 5′-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14 – 18 proceeded at a similar conversion (33–68% for Bs PyNP and 25–62% for Ec TP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2′-deoxyuridine ( 14 ) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% ( Ec TP) or 76% ( Bs PyNP) conversion (about 9 g/L).
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A Comparison between Immobilized Pyrimidine Nucleoside Phosphorylase from Bacillus subtilis and Thymidine Phosphorylase from Escherichia coli in the Synthesis of 5-Substituted Pyrimidine 2’-Deoxyribonucleosides
2013Co-Authors: Immacolata Serra, Teodora Bavaro, Davide A. Cecchini, Simona Daly, Alessandra Albertini, Marco Terreni, Daniela UbialiAbstract:Pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli (EcTP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2'-deoxyribonucleosides (14–18) by transglycosylation in fully aqueous medium. From the comparative study ofthe two biocatalysts, no remarkable differences emerged about their substrate specificity, Bioconversion yield, stability in organic cosolvents(DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2',3'- dideoxynucleosides as substrates, whereas they catalyze Bioconversions involving 5-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14–18 proceeded at a similar conversion (33–68% for BsPyNP and 25–62% for EcTP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2'-deoxyuridine (14) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% (EcTP) or 76% (BsPyNP) conversion (about 9g/L
S Huertaochoa - One of the best experts on this subject based on the ideXlab platform.
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whole cell Bioconversion of valencene to nootkatone by yarrowia lipolytica using a three phase partitioning bioreactor
Journal of Chemical Technology & Biotechnology, 2016Co-Authors: D M Palmerincarreno, Olga Miriam Rutiagaquinones, Abhishek Dutta, Carlos O Castilloaraiza, Jose Verde R Calvo, Gloria Trejoaguilar, S HuertaochoaAbstract:BACKGROUND Low permeability of substrates across the cell membrane, cofactor regeneration and product inhibition are some drawbacks of (+)-nootkatone Bioconversion. The aim of this work was to evaluate and enhance the Bioconversion of (+)-valencene to (+)-nootkatone with Yarrowia lipolytica in a partitioning bioreactor using orange essential oil as the dispersed phase. RESULTS Preliminary experiments in shake flasks allowed enhancing (+)-nootkatone Bioconversion to obtain favorable operating conditions (0.2% w/v of CTAB, 2.0 mmol L−1 of niacin and 11.5 g L−1 of biomass) to produce 420.9 mg L−1. Bioreactor experiments in a two-phase system using 0.2% (w/v) of CTAB, 2.0 mmol L−1 of niacin and 22.5 g L−1 of biomass produced a maximum (+)-nootkatone concentration of 619.8 mg L−1 which was around the product inhibition concentration. Nevertheless, the partitioning three-phase system using orange essential oil overcame product inhibition, obtaining concentrations up to 852.3 mg L−1. CONCLUSIONS This is the first report of a wild type Y. lipolytica with the enzymatic machinery to carry out this Bioconversion. The multiphase partitioning bioreactor concept seems to have good potential for enhancing the productivity of (+)-nootkatone. The Bioconversion approach presents an attractive way to produce and recover (+)-nootkatone in situ using a natural (+)-valencene source. © 2015 Society of Chemical Industry
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screening of microorganisms for Bioconversion of valencene to nootkatone
Lwt - Food Science and Technology, 2015Co-Authors: D M Palmerincarreno, Olga Miriam Rutiagaquinones, J Verde R Calvo, Arely Pradobarragan, S HuertaochoaAbstract:Abstract The production of (+)-nootkatone, highly appreciated by fragrance and flavour industries, can be performed by whole-cell Bioconversion from the sesquiterpene (+)-valencene, a compound readily available in orange essential oil. The aim of this work was to screen for microorganisms that convert (+)-valencene to (+)-nootkatone using different Bioconversion systems. The screening was conducted using six different microorganisms, and Bioconversion experiments were set up on surface culture using serological flasks containing PDA at 30 °C. It was observed that Botryodiplodia theobromae 1368, Yarrowia lipolytica 2.2ab, and Phanerochaete chrysosporium oxidised (+)-valencene to (+)-nootkatone, reaching (+)-nootkatone concentrations of 231.7 ± 2.1, 216.9 ± 5.8 and 100.8 ± 2.6 mg L −1 , respectively. Different Bioconversion conditions were also tested—aqueous, organic, and biphasic—all resulting in similar (+)-nootkatone production. Both B . theobromae 1368 and Y . lipolytica 2.2ab showed substrate inhibition above 4.2 × 10 −2 and 0.13 g of (+)-valencene (g of biomass) −1 , respectively, in aqueous phase experiments. Furthermore, B . theobromae 1368 and Y . lipolytica 2.2ab showed product inhibition when concentrations reached above 17.02 and 34.78 mg of (+)-nootkatone (g of biomass) −1 , respectively. The experimental method presented will be useful for ongoing studies on the selection and operation of the proper bioreactor at different Bioconversion conditions.
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biconversion of nootkatone by botryodiplodia theobromae using a membrane aerated biofilm reactor
Revista Mexicana De Ingenieria Quimica, 2014Co-Authors: D M Palmerincarreno, Olga Miriam Rutiagaquinones, J R Verdecalvo, S HuertaochoaAbstract:El objetivo de este trabajo fue evaluar la Bioconversion de (+)-valenceno a (+)-nootkaton por B. theobromae usando un reactor de biopelicula de membrana aireada (MABR) en un sistema de dos fases liquidas con aceite esencial de naranja como fase organica. En el sistema de fase acuosa, se logro una tasa de produccion de (+)-nootkaton de hasta 3.98 mg L-1 h-1, obteniendo una concentracion de producto final de 398.08 mg L 1 con una Bioconversion de 62 %. Tambien se estudio un sistema de dos fases liquidas, utilizando aceite esencial de naranja como fase dispersa, y se alcanzo una concentracion final de (+)-nootkaton en la fase organica de 310.37 mg L-1, con una Bioconversion de 30.5 % y una tasa de produccion de 2.46 mg L-1 dia-1. El menor rendimiento obtenido mediante el sistema de dos fases fue probablemente debido a las limitaciones de transferencia de masa. El presente trabajo es el primer reporte utilizando un MABR para la Bioconversion de (+)-valenceno a (+)-nootkaton. Se necesitan estudios adicionales sobre los productos de Bioconversion y la optimizacion de las condiciones de operacion del reactor de biopelicula para mejorar la Bioconversion
Daniel Antunes Viegas - One of the best experts on this subject based on the ideXlab platform.
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development and application of an ex vivo fosphenytoin nasal Bioconversion permeability evaluation method
European Journal of Pharmaceutical Sciences, 2016Co-Authors: Daniel Antunes Viegas, Marcio Rodrigues, Joana Francisco, Amilca Falcao, Gilberto Alves, Adriana O SantosAbstract:There is an increasing interest in the intranasal delivery of central nervous system-active drugs due to the existence of a direct nose-to-brain connection. However, poor solubility limits the amount of drug that can be administered within an aqueous solution. In the present work, the objectives were to develop an ex vivo Bioconversion/permeability evaluation method and to study the ex vivo Bioconversion of the hydrophilic phosphate ester prodrug fosphenytoin (FOS) to the active drug phenytoin (PHT) and their comparative nasal permeation. Bioconversion/permeability studies were performed in excised porcine nasal mucosa mounted in Ussing chambers. The physical integrity of the tissues was evaluated by measurement of the transepithelial electrical resistance (TEER). The simultaneous quantitative assay of FOS, PHT and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) was developed and validated according to international guidelines using a liquid chromatography analytical method. The FOS Bioconversion rate and PHT and FOS apparent permeability coefficients (Papp) were determined at different time points. FOS Bioconversion was also qualitatively investigated in human nasal mucus. The developed liquid chromatography method combines a fast and inexpensive sample preparation with inactivation of the enzymatic metabolism of the prodrug during sample manipulation and storage. It was linear, precise, accurate, and presented a high analyte recovery. FOS was converted ex vivo to PHT but the metabolite HPPH was not detected. The Bioconversion rate increased with FOS concentration and with time, which suggests a diffusion-limited process. FOS was also converted to its active drug by human nasal mucus. A novel mathematical data analysis method was developed to reduce the bias introduced by variable mucosal TEER in the permeability results. At comparable FOS and PHT concentrations the ln(Papp(PHT)) of both compounds showed little difference, which indicates that the use of a hydrophilic and charged prodrug did not hinder overall drug permeation. At the highest tested FOS concentration it was possible to quantify FOS in the receiver chambers, meaning that at a sufficiently high concentration the FOS permeation rate overcame its Bioconversion rate. The ln(Papp(PHT)) tended to similar equilibrium values as the assay progressed, but with higher FOS concentrations that equilibrium was attained faster. Acidic pH reduced the permeability of both PHT and FOS. The developed Bioconversion/permeability evaluation method will constitute an important tool to select the most promising formulations before proceeding to in vivo studies. Importantly, it allowed the demonstration of phosphatase activity and FOS Bioconversion in nasal mucosa, as well as the prodrug's nasal permeation potential. Furthermore, this study demonstrates the possibility of formulating phosphate prodrugs of poorly soluble central nervous system-active drugs as a strategy to increase the solubilized drug doses administered through the nasal route.