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S D Krämer

Publications and source records attributed to S D Krämer.

10 recordsLinked to original sources
PubMed2003

No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides.

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism. We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb(3+)-containing liposomes. Upon chelation of Tb(3+) by an aromatic carboxylic acid, the fluorescence of Tb(3+) increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol. In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57). Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.

Animals↗
PubMed2000

P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope.

Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc. Natl. Acad. Sci. USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations. Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture. Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM). The transfected cells grew in multilayers. After reaching confluence they exhibited a complete tight junction (TJ) network. P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture. The lower cell layers were not clearly polarised. P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space. Verapamil, a P-gp inhibitor, significantly decreased efflux. For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected. These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Cell cultures as tools in biopharmacy.

A survey is given on a few selected cell culture models that are used for transport studies. They are characterised for growth, transcellular electrical resistance and cytoarchitecture. The importance of standardisation in view of their use as transport models is documented. Their potential for studies on passive permeation and P-glycoprotein-mediated transport is explored and related to published data. Transport studies are presented that were performed in a two-chamber set-up, the Costar "vertical diffusion system". A series of non-homologous compounds showed similar permeability data (P(app)) in the different cell cultures. The origin of the cell type had no remarkable influence on passive transcellular permeation. MDCK cells, an epithelial cell line of canine kidney origin, are perfectly suited to screen for passive permeation. They have low expression of transporter proteins and low metabolic activity. In general, they probably represent the best-known epithelial cell line with respect to genetics as well as lipid and protein composition. MDCK cells are easy to handle. Transport experiments can be done between 7 and 14 days after seeding, when the stationary growth phase is reached. To screen for P-glycoprotein substrates, efflux and uptake studies were performed with mdr1-transfected MDCK cells (MDR1-MDCK) in a one-chamber system in the presence or absence of verapamil or cyclosporin A as inhibitor. Evidence is presented why the transfected cells, which express large amounts of P-glycoprotein, are not suitable for two-chamber transport studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Permeation studies in vitro and in vivo of potential radiopharmaceuticals with affinity to neuro receptors.

PURPOSE: To check the influence of structural characteristics on their permeation through the blood-brain barrier (BBB), a set of radioactive [99mTc]chelates bearing amine groups was synthesized and tested in vitro as well as in vivo. METHODS: Compounds with different log P and pKa values were obtained by complex forming reactions of [99mTc]pertechnetate with varying substituents. Transport was studied in rats and mice, as well as in an ECV304 cell culture model. RESULTS: In vitro higher permeation was found for compounds with electron attracting substituents in beta-position to the amine group (pKa values 7.4 to 8.3) than for those with more basic amine groups (pKa values > 8.9) even for similar log DH 7.4. In vivo brain uptake between 0.8 and 4.8% of the injected dose (ID) per organ was found for the former, whereas <0.4% ID were present for the latter. CONCLUSIONS: Three structurally diverse classes of [99mTc]chelates showed distinct patterns with regard to brain uptake in vivo and BBB permeability in vitro which could not be predicted by their lipophilicity alone. The close correlation between the data from rats and mice and those obtained with cell cultures render the ECV304 cells an attractive model for the screening of new compounds.

Animals↗
PubMed1998

Towards the predictability of drug-lipid membrane interactions: the pH-dependent affinity of propanolol to phosphatidylinositol containing liposomes.

PURPOSE: Prediction of the pH-dependent affinity of (RS)-[3H]propranolol to mixed phosphatidylcholine (PhC)/phosphatidylinositol(Phl) membranes from the partitioning in the single lipid liposome/buffer systems. METHODS: Partition studies in liposome/buffer systems were performed by means of equilibrium dialysis at 37 degrees C between pH 2 and 11 at a molar propranolol to lipid ratio of 10(-6) to 10(-5) in the membrane. Results. The Phl membrane more strongly attracts the protonated (RS)-[3H]propranolol than the neutral solute, i.e. the partition coefficient of the protonated base (Pi) is 17'430+/-1320, P of the neutral compound (Pn) is 3110+/-1650. In the PhC-liposome system Pi is 580+/-17, Pn 1860+/-20. The partition coefficients show an exponential dependence on the molar Phl fraction in mixed liposomes. The partitioning in mixed PhC/Phl membranes is predictable from Pn and Pi in the single lipid liposome systems. CONCLUSIONS: The negative charge of biological lipid membranes causes strong electrostatic interactions with positively charged solutes. This strong attraction is not predictable from the octanol/buffer partition system, but it is important regarding drug accumulation in the tissue and drug attraction by certain lipids in the vicinity of membrane proteins.

Adrenergic beta-Antagonists↗
PubMed1998

MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy.

PURPOSE: Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. METHODS: Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. RESULTS: TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (> or = 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. CONCLUSIONS: Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.

Animals↗
PubMed1997

Free fatty acids cause pH-dependent changes in drug-lipid membrane interactions around physiological pH.

PURPOSE: The influence of oleic acid (OA) and phosphatidylethanolamine (PhE) as membrane constituents on the partition behavior of (RS)-[3H]propranolol between unilamellar liposomes and buffer was studied as a function of pH. METHODS: Partition studies were performed by means of equilibrium dialysis at 37 degrees C over a broad pH range at a molar propranolol to lipid ratio in the membrane of 10(-6). RESULTS: As compared to the standard phosphatidylcholine (PhC)-liposome/buffer partition system PhE and OA have an enhancing effect on the apparent partition coefficient (D) of (RS)-[3H]propranolol between pH 6 and 11. Data analysis with Henderson-Hasselbalch equations revealed that the neutral propranolol has a higher affinity to membranes containing net neutral charged PhE than to pure PhC-liposomes. Net negatively charged PhE seems to have no significant influence on the partitioning. Deprotonated OA caused an increase in the true partition coefficient (P) of the protonated propranolol. Neutral OA showed no influence on the partitioning. From the fit D vs pH curves and from zeta potential measurements of the liposomes the intrinsic pKa values of the membranous lipids were calculated as 7.5 to 7.8 for OA and 9.7 to 9.8 for PhE. CONCLUSIONS: Since the pKa of membranous OA is close to the physiological pH and D depends on the ionisation state of OA, small pH changes around the physiological pH may cause large differences in drug-lipid membrane interactions.

Animals↗
PubMed1996

The pH-dependence in the partitioning behaviour of (RS)-[3H]propranolol between MDCK cell lipid vesicles and buffer.

PURPOSE: The pH-dependent partitioning of (RS)-[3H]propranolol between unilamellar vesicles of MDCK cell lipids and buffer was determined. METHODS: Partitioning studies were performed by means of equilibrium dialysis at 37 degrees C between pH 7 and 11 at a molar propranolol/lipid ratio in the membrane of 10(-6). RESULTS: The partition-pH diagram was bell-shaped. The highest apparent partition coefficient was 1797 at pH 9.7, the lowest was 805 at pH 6.9. Curve fitting with a combination of Henderson-Hasselbalch equations revealed an inflection point at the apparent pKa of propranolol, i.e. 9.7, and two additional pKa values at pH 7.7 and 10.0. The first one corresponds to the pKa of free fatty acids (FFA) within lipid bilayers and the other one to the pKa of phosphatidylethanolamine (PhE). The true partition coefficients (P) of the neutral as well as the ionised solute were fitted for each ionisation status of the membrane. The highest P, i.e. 2123, was calculated for neutral propranolol in the membrane with deprotonated FFA and protonated PhE. CONCLUSIONS: The partitioning behaviour of (RS)-[3H]propranolol in a complex membrane/buffer system can be described when considering ionisation changes of drug and lipids.

Buffers↗
PubMedDate not supplied

Physicochemical properties in pharmacokinetic lead optimization.

The ADME (absorption, distribution in the body, metabolism and elimination from the body) profile of a drug determines its pharmacokinetics in the body. Modern drug design includes the modeling of pharmacokinetically favorable behavior. The pharmacokinetic parameters of most interest concern intestinal absorption, blood-brain barrier (BBB) passage and metabolism. Traditionally, experimental parameters such as partition coefficients and chromatographic capacity factors have been used for the estimation of intestinal absorption or BBB passage of newly synthesized compounds. Several studies have shown a sigmoidal relationship between intestinal absorption and lipophilicity. The latter is usually expressed by the apparent partition coefficient log D in a biphasic system at physiological pH or by the affinity to a lipophilic phase determined by chromatographic techniques. In contrast, structure-based descriptors need no experimental investigation of the compound studied. The most relevant descriptors give information on hydrogen-bonding characteristics and molecular volume. In recent years, attempts have been made to recognize substrates for multidrug resistance proteins by their structure characteristics without crucial success. There is evidence that multidrug resistance is not only driven by direct protein-substrate recognition, but also by the behavior of the compound in the lipid environment of the protein.

Animals↗