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Immobilized liposome and biomembrane partitioning chromatography of drugs for prediction of drug transport
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
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1998 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 164, no 1-2, 129-137 p.Article in journal (Refereed) Published
Abstract [en]

Drug partitioning into lipid bilayers was studied by chromatography on liposomes and biomembranes immobilized in gel beads by freeze–thawing. The drug retention volume was expressed as a capacity factor, Ks, normalized with respect to the amount of immobilized phospholipid. Log Ks values for positively charged drugs on brain phosphatidylserine (PS)/egg phosphatidylcholine (PC) liposomes decreased as the ionic strength was increased, increased as the PS:PC ratio or the pH was increased and varied linearly with the temperature. Log Ks values for beta-blockers, phenothiazines and benzodiazepines on egg phospholipid (EPL) liposomes correlated well with corresponding values on red cell membrane lipid liposomes (r2=0.96), and on human red cell membrane vesicles containing transmembrane proteins (r2=0.96). A fair correlation was observed between the values on EPL liposomes and those on native membranes of adsorbed red cells (r2=0.86). Compared to the data obtained with liposomes, the retentions of hydrophilic drugs became larger and the range of log Ks values more narrow on the vesicles and the membranes, which expose hydrophilic protein surfaces and oligosaccharides. Lower correlations were observed between drug retention on EPL liposomes and egg PC liposomes; and between retention on liposomes (or vesicles) and immobilized artificial membrane (IAM) monolayers of PC analogues. Absorption of orally administered drugs in humans (literature data) was nearly complete for drugs of log Ks values in the interval 1.2–2.5 on vesicles. Both vesicles and liposomes can thus be used for chromatographic analysis of drug–membrane interaction and prediction of drug absorption.

Place, publisher, year, edition, pages
1998. Vol. 164, no 1-2, 129-137 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-89998DOI: 10.1016/S0378-5173(97)00398-0OAI: oai:DiVA.org:uu-89998DiVA: diva2:161998
Available from: 2002-10-03 Created: 2002-10-03 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
Open this publication in new window or tab >>Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction.

Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo.

Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.

An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 43 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 755
Keyword
Biochemistry, Affinity, Binding, Biomembrane, Biotin, Chromatography, Cytochalasin B, Dissociation constant, Drug absorption, Equilibrium, Glucose, GLUT1, Immobilization, Immobilized biomembrane affinity chromatography, Immobilized liposome chromatography, Interaction, Liposome, Membrane protein, Membrane vesicle, Partitioning, Phospholipid bilayer, Proteoliposome, Quantitative, Red blood cell, Solute, Specific, Streptavidin, Wheat germ agglutinin, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-2668 (URN)91-554-5413-5 (ISBN)
Public defence
2002-10-25, lecture hall B41, Biomedical Center, Uppsala, 10:15
Opponent
Available from: 2002-10-03 Created: 2002-10-03 Last updated: 2013-06-12Bibliographically approved
2. Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter
Open this publication in new window or tab >>Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.

Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 50 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 868
Keyword
Biochemistry, Affinity, Aromatic amino acids, Binding, Biomembrane, Biotin, Chromatography, Cytochalasin B, Dihydrocytochalasin B, Dissociation constant, Drug absorption, Ethanol, Equilibrium, Glucose, GLUT1, Immobilization, Immobilized biomembrane affinity chromatography, Immobilized liposome chromatography, Interaction, Liposome, Membrane protein, Membrane vesicle, Partitioning, Phospholipid bilayer, Proteoliposome, Quantitative, Red blood cell, Specific, Streptavidin, Tyrosine, Tryptophan, Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-3525 (URN)91-554-5692-8 (ISBN)
Public defence
2003-09-26, B:41, BMC, Husarvägen 3, 13:15
Opponent
Supervisors
Available from: 2003-09-04 Created: 2003-09-04 Last updated: 2013-06-10Bibliographically approved

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