A liposomal fluorescence assay to study permeation kinetics of drug-like weak bases across the lipid bilayer

Eyer, Klaus; Paech, Franziska; Schuler, Friedrich; Kuhn, Phillip; Kissner, Reinhard; Belli, Sara; Dittrich, Petra S.; Krämer, Stefanie-Dorothea

doi:10.1016/j.jconrel.2013.10.037

Cited by 50 publications

(122 citation statements)

References 28 publications

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“…For solutes, there is a strong correlation between passive in vitro permeability and log D 7.4 (between −4.50 and 3.48) and molecular size (between 146 and 671 Da) . By analogy, the permeation rate of weak bases into liposomes increases with increased lipophilicity of the compound . Recently, the existence of a passive transcellular diffusion component in the intestinal absorption process has been challenged .…”

Section: Bioavailability Absorption and Permeabilitymentioning

confidence: 99%

Direct In Vivo Human Intestinal Permeability (Peff) Determined with Different Clinical Perfusion and Intubation Methods

Dahlgren

Roos

Sjögren

et al. 2015

Journal of Pharmaceutical Sciences

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Regional in vivo human intestinal effective permeability (Peff ) is calculated by measuring the disappearance rate of substances during intestinal perfusion. Peff is the most relevant parameter in the prediction of rate and extent of drug absorption from all parts of the intestine. Today, human intestinal perfusions are not performed on a routine basis in drug development. Therefore, it would be beneficial to increase the accuracy of the in vitro and in silico tools used to evaluate the intestinal Peff of novel drugs. This review compiles historical Peff data from 273 individual measurements of 80 substances from 61 studies performed in all parts of the human intestinal tract. These substances include: drugs, monosaccharaides, amino acids, dipeptides, vitamins, steroids, bile acids, ions, fatty acids, and water. The review also discusses the determination and prediction of Peff using in vitro and in silico methods such as quantitative structure-activity relationship, Caco-2, Ussing chamber, animal intestinal perfusion, and physiologically based pharmacokinetic (PBPK) modeling. Finally, we briefly outline how to acquire accurate human intestinal Peff data by deconvolution of plasma concentration-time profiles following regional intestinal bolus dosing.

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Section: Bioavailability Absorption and Permeabilitymentioning

confidence: 99%

Direct In Vivo Human Intestinal Permeability (Peff) Determined with Different Clinical Perfusion and Intubation Methods

Dahlgren

Roos

Sjögren

et al. 2015

Journal of Pharmaceutical Sciences

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“…The cell-derived vesicles were immobilized by adsorbed biotinylated BSA, an avidin linker and a cholesterol-PEG-biotin moiety (M w 3,400 Da, Nanocs Inc.), which tethers the vesicles without disturbing the domain of the membrane facing towards the fluid (25,31). Next, all valves of the device were closed and the test solutions were added to the respective fluidic channels.…”

Section: Validation Of the Microfluidic Calcein Am Assaymentioning

confidence: 99%

Cell-Free Microfluidic Determination of P-glycoprotein Interactions with Substrates and Inhibitors

et al. 2014

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The membrane protein P-glycoprotein (P-gp) plays key roles in the oral bioavailability of drugs, their blood brain barrier passage as well as in multidrug resistance. For new drug candidates it is mandatory to study their interaction with P-gp, according to FDA and EMA regulations. The vast majority of these tests are performed using confluent cell layers of P-gp overexpressing cell lines that render these tests laborious. In this study, we introduce a cell-free microfluidic assay for the rapid testing of drug- P-gp interactions. Cell-derived vesicles are prepared from MDCKII-MDR1 overexpressing cells and immobilized on the surface of a planar microfluidic device. The drug is delivered continuously to the vesicles and calcein accumulation is monitored by means of a fluorescence assay and total internal reflection fluorescence (TIRF) microscopy. Only small amounts of compounds (~10 μl) are required in concentrations of 5, 25 and 50 μM for a test that provides within 5 min information on the apparent dissociation constant of the drug and P-gp. We tested 10 drugs on-chip, 9 of which are inhibitors or substrates of P-glycoprotein and one negative control. We benchmarked the measured apparent dissociation constants against an alternative assay on a plate reader and reference data from FDA. These comparisons revealed good correlations between the logarithmic apparent dissociation constants (R(2) = 0.95 with ATPase assay, R(2) = 0.93 with FDA data) and show the reliability of the rapid on-chip test. The herein presented assay has an excellent screening window factor (Z'-factor) of 0.8, and is suitable for high-throughput testing.

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“…There are also significant challenges to investigating this behaviour experimentally. Eyer et al 14 proposed a liposomal fluorescence assay method by which the permeation of weak basic drug-like solutes across the lipid membrane can be determined. However, details of membrane crossing mechanisms at an atomistic level are still missing experimentally 14 .…”

Section: Introductionmentioning

confidence: 99%

Calculating Kinetic Rates and Membrane Permeability from Biased Simulations

et al. 2018

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We present a simple approach to calculate the kinetic properties of lipid membrane crossing processes from biased molecular dynamics simulations. We demonstrate that by using biased simulations, one can obtain highly accurate kinetic information with significantly reduced computational time with respect to unbiased simulations. We describe how to conveniently calculate the transition rates to enter, cross and exit the membrane in terms of mean first passage times. To obtain free energy barriers and relaxation times from biased simulations only, we constructed Markov models using the Dynamic Histogram Analysis Method (DHAM). The permeability coefficients that are calculated from the relaxation times are found to correlate highly with experimentally evaluated values. We show that more generally, certain calculated kinetic properties linked to the crossing of the membrane layer (e.g., barrier height and barrier crossing rates) are good indicators of ordering drugs by permeability. Extending the analysis to a 2D Markov model provides a physical description of the membrane crossing mechanism. Figure 1. Representation of the system used in the molecular dynamics simulations: a drug molecule (in brown at center of image) interacts with and passes through a lipid membrane which is surrounded by water.

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A liposomal fluorescence assay to study permeation kinetics of drug-like weak bases across the lipid bilayer

Cited by 50 publications

References 28 publications

Direct In Vivo Human Intestinal Permeability (Peff) Determined with Different Clinical Perfusion and Intubation Methods

Direct In Vivo Human Intestinal Permeability (Peff) Determined with Different Clinical Perfusion and Intubation Methods

Cell-Free Microfluidic Determination of P-glycoprotein Interactions with Substrates and Inhibitors

Calculating Kinetic Rates and Membrane Permeability from Biased Simulations

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