The objective of this study was to determine whether nanoemulsion formulations constitute a viabl... more The objective of this study was to determine whether nanoemulsion formulations constitute a viable strategy to improve the oral bioavailability of danazol, a compound whose poor aqueous solubility limits its oral bioavailability. Danazol-containing oil-in-water nanoemulsions (NE) with and without cosurfactants stearylamine (SA) and deoxycholic acid (DCA) were prepared and characterized. Nanoemulsion droplets size ranging from 238 to 344 nm and with surface charges of -24.8 mV (NE), -26.5 mV (NE-DCA), and +27.8 mV (NE-SA) were reproducibly obtained. Oral bioavailability of danazol in nanoemulsions was compared with other vehicles such as PEG400, 1% methylcellulose (MC) in water (1% MC), Labrafil, and a Labrafil/Tween 80 (9:1) mixture, after intragastric administration to rats and after oral administration of NE-SA, a Labrafil solution, or a Danocrine® tablet to dogs. The absolute bioavailability of danazol was 0.6% (PEG400), 1.2% (1% MC), 6.0% (Labrafil), 7.5% (Labrafil/Tween80), 8.1% (NE-DCA), 14.8% (NE), and 17.4% (NE-SA) in rats, and 0.24% (Danocrine), 6.2% (Labrafil), and 58.7% (NE-SA) in dogs. Overall, danazol bioavailability in any nanoemulsion was higher than any other formulation. Danazol bioavailability from NE and NE-SA was 1.8- to 2.2-fold higher than NE-DCA nanoemulsion and could be due to significant difference in droplet size.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1975
Previous studies support the validity of a linear thermodynamic formalism relating the rates of a... more Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na-+ transport and oxygen consumption Jr to the electrical potential difference delta-psi and the affinity A (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na-+ transport. Ouabain, a specific inhibitor of the Na-+ pump, might be expected to diminish the dependence of Jr on delta-psi without affecting A, whereas 2-deoxy-D-glucose, a competitive inhibitor of glucose metabolism should be expected to diminish A. Both inhibitors were used at concentrations adequate to depress Na-+ transport (i.e. short-circuit current Io) to some 50% of control level. Measurements were made of Io and dJr/d(delat-psi), and the apparent value of the affinity Aapp was calculated according to the thermodynamic formulation. Ouabain depressed minus dJr/d(delta-psi) without affecting Aapp whereas 2-deoxy-D-glucose depressed Aapp without affecting minus dJr/d(delta-psi). The demonstration of these effects indicated the utility of the formalism.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1975
Previous studies support the validity of a linear thermodynamic formalism relating the rates of a... more Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na-+ transport and oxygen consumption Jr to the electrical potential difference delta-psi and the affinity A (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na-+ transport. Ouabain, a specific inhibitor of the Na-+ pump, might be expected to diminish the dependence of Jr on delta-psi without affecting A, whereas 2-deoxy-D-glucose, a competitive inhibitor of glucose metabolism should be expected to diminish A. Both inhibitors were used at concentrations adequate to depress Na-+ transport (i.e. short-circuit current Io) to some 50% of control level. Measurements were made of Io and dJr/d(delat-psi), and the apparent value of the affinity Aapp was calculated according to the thermodynamic formulation. Ouabain depressed minus dJr/d(delta-psi) without affecting Aapp whereas 2-deoxy-D-glucose depressed Aapp without affecting minus dJr/d(delta-psi). The demonstration of these effects indicated the utility of the formalism.
The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of h... more The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo-[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3-fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5-10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells.
A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with ... more A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with bovine serum albumin and glutaraldehyde, and then cast as membranes. The properties of immobilized N-acetyl-beta-D-hexosaminidase were studied. The recovery of N-acetyl-beta-D-hexosaminidase after immobilization was unaffected by increasing the concentration of glutaraldehyde, but was decreased by increasing the bovine serum albumin concentration. The immobilized enzyme showed enhanced resistance towards proteolytic and thermal inactivation. While the pH optimum for the soluble enzyme was 4.0, a bimodal pH curve with optima at 3.4 and 5.0 was observed after insolubilization. This bimodality was abolished when the immobilized enzyme was assayed in the presence of M NaCl. The Km values, for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside, of the immobilized isoenzymes of N-acetyl-beta-D-hexosaminidase were larger than those of their soluble counterparts. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage for at least 50 days at 4 degrees.
Proceedings of the Entomological Society of Washington, 2004
Page 1. Adryas, a new genus of trichogrammatidae (Hymenoptera: Chalcidoidea) from the new world t... more Page 1. Adryas, a new genus of trichogrammatidae (Hymenoptera: Chalcidoidea) from the new world tropics John D Pinto and Albert K Owen Proceedings of the Entomological Society of Washington 106:905-922 (2004) http ...
... 44135 Suresh Khandelwal RS Information Systems, Inc. Cleveland, Ohio 44135 ... Simulation). T... more ... 44135 Suresh Khandelwal RS Information Systems, Inc. Cleveland, Ohio 44135 ... Simulation). The simulation is developed within the Numerical Propulsion Simulation System architecture using component elements from earlier models. ...
The present study is to evaluate the feasibility of using human liver S9 fractions and 7-hydroxyc... more The present study is to evaluate the feasibility of using human liver S9 fractions and 7-hydroxycoumarin (7-HC) to screen compounds with potential to inhibit glucuronidation and sulfation. The sole substrate, 7-HC, was incubated at 37C with human S9 fractions along with the necessary cofactors and test compounds. The effect of test compounds as well as organic solvents on 7-HC-glucuronide (7-HC-G) and 7-HC-sulfate (7-HC-S) formation was investigated, and the percent inhibition was calculated if possible. Under test conditions, curcumin showed the strongest inhibition against UGTs, with an approximate apparent IC50 of 5 &muM; ketoconazole and quercetin showed apparent IC50 of 60 and 80 &muM, respectively; apparent IC50 for cyclosporine A, probenecid, 17-&Alpha ethynyl estradiol, or niflumic acid was greater than 100 &muM. For SULT inhibition, curcumin and pentachlorophenol showed inhibition of SULTs with approximate apparent IC50 of 0.02 and 0.01 &muM; ketoconazole showed apparent IC...
The multidrug resistance protein 2 (MRP2 or ABCC2) is an apical ATP- dependent efflux pump that c... more The multidrug resistance protein 2 (MRP2 or ABCC2) is an apical ATP- dependent efflux pump that can affect the pharmacokinetics of anticancer, antimicrobial and statin drugs, and plays a role in clinical drug-drug interactions. The purpose of this study was to establish a fluorescent assay for screening MRP2-mediated drug interactions. The non-fluorescent reagent 5(6)-carboxy-2’, 7’-dichlorofluorescein diacetate (CDCFDA) is known to be hydrolyzed by intracellular esterases to a more hydrophilic, fluorescent form (CDCF) that is actively extruded from the cells by MRP2. Caco-2 cells were grown for 3 weeks to allow the formation of cell monolayers. Bi-directional transport of CDCF, after dosing with 5 µM CDCFDA, was measured in the absence and presence of 3 known MRP2 inhibitors (MK-571, probenecid, and sulfinpyrazone), 4 possible substrates (furosemide, phenobarbital, diclofenac, acetaminophen), a Pgp inhibitor (verapamil), and a BCRP inhibitor (Ko-143). Assays were conducted at 37 °C...
Purpose. Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are predominantly expre... more Purpose. Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are predominantly expressed at the sinusoidal membrane of hepatocytes and play an important role in the hepatic uptake of numerous drugs including statins. Inhibition of OATP1B1/1B3-mediated drug transport may reduce statin liver uptake and/or efficacy, and elevate systemic exposure. Therefore, it is imperative to have reliable assay systems for identifying potential OATP1B1- and OATP1B3-mediated adverse drug reactions. The objective of this study was to establish rapid in vitro inhibition assays for screening OATP1B1- and OATP1B3-mediated drug interactions in stably transfected HEK293 cells using either flow cytometry or a fluorescence microplate reader. Methods. For flow cytometry, HEK293 cells, stably transfected with OATP1B1 or OATP1B3 or the control vector, were cultured as monolayers, trypsinized, and then assayed in suspension; for the fluorescence microplate reader, cells were cultured in 96-well plate...
The objective of this study was to determine whether nanoemulsion formulations constitute a viabl... more The objective of this study was to determine whether nanoemulsion formulations constitute a viable strategy to improve the oral bioavailability of danazol, a compound whose poor aqueous solubility limits its oral bioavailability. Danazol-containing oil-in-water nanoemulsions (NE) with and without cosurfactants stearylamine (SA) and deoxycholic acid (DCA) were prepared and characterized. Nanoemulsion droplets size ranging from 238 to 344 nm and with surface charges of -24.8 mV (NE), -26.5 mV (NE-DCA), and +27.8 mV (NE-SA) were reproducibly obtained. Oral bioavailability of danazol in nanoemulsions was compared with other vehicles such as PEG400, 1% methylcellulose (MC) in water (1% MC), Labrafil, and a Labrafil/Tween 80 (9:1) mixture, after intragastric administration to rats and after oral administration of NE-SA, a Labrafil solution, or a Danocrine® tablet to dogs. The absolute bioavailability of danazol was 0.6% (PEG400), 1.2% (1% MC), 6.0% (Labrafil), 7.5% (Labrafil/Tween80), 8.1% (NE-DCA), 14.8% (NE), and 17.4% (NE-SA) in rats, and 0.24% (Danocrine), 6.2% (Labrafil), and 58.7% (NE-SA) in dogs. Overall, danazol bioavailability in any nanoemulsion was higher than any other formulation. Danazol bioavailability from NE and NE-SA was 1.8- to 2.2-fold higher than NE-DCA nanoemulsion and could be due to significant difference in droplet size.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1975
Previous studies support the validity of a linear thermodynamic formalism relating the rates of a... more Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na-+ transport and oxygen consumption Jr to the electrical potential difference delta-psi and the affinity A (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na-+ transport. Ouabain, a specific inhibitor of the Na-+ pump, might be expected to diminish the dependence of Jr on delta-psi without affecting A, whereas 2-deoxy-D-glucose, a competitive inhibitor of glucose metabolism should be expected to diminish A. Both inhibitors were used at concentrations adequate to depress Na-+ transport (i.e. short-circuit current Io) to some 50% of control level. Measurements were made of Io and dJr/d(delat-psi), and the apparent value of the affinity Aapp was calculated according to the thermodynamic formulation. Ouabain depressed minus dJr/d(delta-psi) without affecting Aapp whereas 2-deoxy-D-glucose depressed Aapp without affecting minus dJr/d(delta-psi). The demonstration of these effects indicated the utility of the formalism.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1975
Previous studies support the validity of a linear thermodynamic formalism relating the rates of a... more Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na-+ transport and oxygen consumption Jr to the electrical potential difference delta-psi and the affinity A (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na-+ transport. Ouabain, a specific inhibitor of the Na-+ pump, might be expected to diminish the dependence of Jr on delta-psi without affecting A, whereas 2-deoxy-D-glucose, a competitive inhibitor of glucose metabolism should be expected to diminish A. Both inhibitors were used at concentrations adequate to depress Na-+ transport (i.e. short-circuit current Io) to some 50% of control level. Measurements were made of Io and dJr/d(delat-psi), and the apparent value of the affinity Aapp was calculated according to the thermodynamic formulation. Ouabain depressed minus dJr/d(delta-psi) without affecting Aapp whereas 2-deoxy-D-glucose depressed Aapp without affecting minus dJr/d(delta-psi). The demonstration of these effects indicated the utility of the formalism.
The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of h... more The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo-[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3-fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5-10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells.
A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with ... more A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with bovine serum albumin and glutaraldehyde, and then cast as membranes. The properties of immobilized N-acetyl-beta-D-hexosaminidase were studied. The recovery of N-acetyl-beta-D-hexosaminidase after immobilization was unaffected by increasing the concentration of glutaraldehyde, but was decreased by increasing the bovine serum albumin concentration. The immobilized enzyme showed enhanced resistance towards proteolytic and thermal inactivation. While the pH optimum for the soluble enzyme was 4.0, a bimodal pH curve with optima at 3.4 and 5.0 was observed after insolubilization. This bimodality was abolished when the immobilized enzyme was assayed in the presence of M NaCl. The Km values, for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside, of the immobilized isoenzymes of N-acetyl-beta-D-hexosaminidase were larger than those of their soluble counterparts. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage for at least 50 days at 4 degrees.
Proceedings of the Entomological Society of Washington, 2004
Page 1. Adryas, a new genus of trichogrammatidae (Hymenoptera: Chalcidoidea) from the new world t... more Page 1. Adryas, a new genus of trichogrammatidae (Hymenoptera: Chalcidoidea) from the new world tropics John D Pinto and Albert K Owen Proceedings of the Entomological Society of Washington 106:905-922 (2004) http ...
... 44135 Suresh Khandelwal RS Information Systems, Inc. Cleveland, Ohio 44135 ... Simulation). T... more ... 44135 Suresh Khandelwal RS Information Systems, Inc. Cleveland, Ohio 44135 ... Simulation). The simulation is developed within the Numerical Propulsion Simulation System architecture using component elements from earlier models. ...
The present study is to evaluate the feasibility of using human liver S9 fractions and 7-hydroxyc... more The present study is to evaluate the feasibility of using human liver S9 fractions and 7-hydroxycoumarin (7-HC) to screen compounds with potential to inhibit glucuronidation and sulfation. The sole substrate, 7-HC, was incubated at 37C with human S9 fractions along with the necessary cofactors and test compounds. The effect of test compounds as well as organic solvents on 7-HC-glucuronide (7-HC-G) and 7-HC-sulfate (7-HC-S) formation was investigated, and the percent inhibition was calculated if possible. Under test conditions, curcumin showed the strongest inhibition against UGTs, with an approximate apparent IC50 of 5 &muM; ketoconazole and quercetin showed apparent IC50 of 60 and 80 &muM, respectively; apparent IC50 for cyclosporine A, probenecid, 17-&Alpha ethynyl estradiol, or niflumic acid was greater than 100 &muM. For SULT inhibition, curcumin and pentachlorophenol showed inhibition of SULTs with approximate apparent IC50 of 0.02 and 0.01 &muM; ketoconazole showed apparent IC...
The multidrug resistance protein 2 (MRP2 or ABCC2) is an apical ATP- dependent efflux pump that c... more The multidrug resistance protein 2 (MRP2 or ABCC2) is an apical ATP- dependent efflux pump that can affect the pharmacokinetics of anticancer, antimicrobial and statin drugs, and plays a role in clinical drug-drug interactions. The purpose of this study was to establish a fluorescent assay for screening MRP2-mediated drug interactions. The non-fluorescent reagent 5(6)-carboxy-2’, 7’-dichlorofluorescein diacetate (CDCFDA) is known to be hydrolyzed by intracellular esterases to a more hydrophilic, fluorescent form (CDCF) that is actively extruded from the cells by MRP2. Caco-2 cells were grown for 3 weeks to allow the formation of cell monolayers. Bi-directional transport of CDCF, after dosing with 5 µM CDCFDA, was measured in the absence and presence of 3 known MRP2 inhibitors (MK-571, probenecid, and sulfinpyrazone), 4 possible substrates (furosemide, phenobarbital, diclofenac, acetaminophen), a Pgp inhibitor (verapamil), and a BCRP inhibitor (Ko-143). Assays were conducted at 37 °C...
Purpose. Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are predominantly expre... more Purpose. Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are predominantly expressed at the sinusoidal membrane of hepatocytes and play an important role in the hepatic uptake of numerous drugs including statins. Inhibition of OATP1B1/1B3-mediated drug transport may reduce statin liver uptake and/or efficacy, and elevate systemic exposure. Therefore, it is imperative to have reliable assay systems for identifying potential OATP1B1- and OATP1B3-mediated adverse drug reactions. The objective of this study was to establish rapid in vitro inhibition assays for screening OATP1B1- and OATP1B3-mediated drug interactions in stably transfected HEK293 cells using either flow cytometry or a fluorescence microplate reader. Methods. For flow cytometry, HEK293 cells, stably transfected with OATP1B1 or OATP1B3 or the control vector, were cultured as monolayers, trypsinized, and then assayed in suspension; for the fluorescence microplate reader, cells were cultured in 96-well plate...
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