A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial ( Caco-2) cells grown in permeable filter chambers

Karlsson, Johan; Artursson, Per

doi:10.1016/0378-5173(91)90067-x

Cited by 157 publications

(119 citation statements)

References 17 publications

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“…It has been shown in both Caco-2 and PAMPA models that agitation during the permeability experiments can decrease the UWL and thereby increase the apparent permeability of lipophilic drug compounds (Adson et al, 1995;Avdeef et al, 2004;Karlsson and Artursson, 1991). It has been claimed that slight shaking using plate shakers cannot reduce the thickness of the UWL sufficiently to match the in vivo situation , but there are other reports showing that already at 150 rpm the thickness can be considerably reduced down to 220 µm when using an orbital shaker (Adson et al, 1995).…”

Section: Resultsmentioning

confidence: 97%

“…Means to decrease or even minimize this effect have involved e.g. shaking or stirring of the permeation assay setup (Adson et al, 1995;Avdeef et al, 2004;Karlsson and Artursson, 1991). The phospholipid vesicle based method has so far been run without agitation, so the aim of this part of the study was therefore to investigate the effect of agitation.…”

Section: Resultsmentioning

confidence: 99%

“…3.1.1 Influence of agitation on the permeability of drugs and the physical stability of the barrier In the most used permeability models, the Caco-2 model and the PAMPA model, the adherent "fixed" layer of water, the so-called unstirred water layer (UWL) has been found to significantly influence the permeability values (Adson et al, 1995;Kansy et al, 2004;Karlsson and Artursson, 1991). Especially if drug diffusion in water is slow, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…If this phenomenon is not taken into account, absorption parameters can be underestimated. The permeability of highly permeable drugs, such as testosterone, has been shown to depend on the stirring conditions in in vitro permeability assays (Karlsson and Artursson, 1991). So, if not compensating for the UWL-effect the permeability of lipophilic compounds in the UWL could significantly contribute to the resulting permeability values.…”

Section: Introductionmentioning

confidence: 99%

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Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug–membrane interactions and the effect of agitation on the barrier integrity and on the permeability

Flaten¹,

Skar²,

Luthman³

et al. 2007

European Journal of Pharmaceutical Sciences

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Recently we reported on the development and structural characterization of a phospholipid vesicle based barrier useful for medium throughput screening of passive drug permeability.Here, we investigate the physical and functional integrity of the phospholipid vesicle based barriers to agitation by stirring or shaking, and whether agitation affects drug permeability of sulpiride, metoprolol and testosterone. In addition, three drugs (caffeine, naproxen and sulphasalazine) which were shown in a previous study to affect the electrical resistance of the barriers, were investigated for their influence on the permeability of a simultaneously applied hydrophilic marker (calcein), and on the thermotropic phase transition of the phospholipid bilayers using differential scanning calorimetry (DSC).Electrical resistance measurements indicated that the barriers should withstand shaking speeds up to 150 rpm without losing their integrity, but significant release of phospholipids from the membrane barriers to the donor and acceptor chambers was observed under agitation ≥150 rpm. When using agitation up to 100 rpm no increase in permeability was observed for sulpiride, metoprolol and testosterone. The phospholipid vesicle-based barrier thus differ from other permeability models in that agitation does not lead to an increase in permeability, not even for highly lipophilic drugs such as testosterone. This is explained by the different morphology of the vesicle-based barrier which is containing a 100 µm thick layer of mostly aqueous compartments immobilised within a matrix of phospholipids vesicles.Sulphasalazine and naproxen were shown to decrease the electrical resistance and increase the permeability of the hydrophilic marker calcein. The DSC experiments showed that these two drugs probably interact with the head groups of the phospholipids. In contrast, caffeine gave an increase in electrical resistance and a decrease in permeability of calcein. From the DSC experiments no signs of interaction of caffeine with the phospholipid bilayer could be observed.3

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug–membrane interactions and the effect of agitation on the barrier integrity and on the permeability

Flaten¹,

Skar²,

Luthman³

et al. 2007

European Journal of Pharmaceutical Sciences

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“…The unstirred water layer forms a considerable permeability barrier for compounds that traverse the cell monolayer very rapidly (Karlsson and Artursson, 1991). Therefore, we determined the P app values at a low quinidine concentration (0.3 M) in both directions at various stirring rates.…”

Section: Resultsmentioning

confidence: 99%

Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates

Korjamo

Kemiläinen²,

Heikkinen³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Passive permeability and active efflux are parallel processes in transcellular flux. Therefore, the observed kinetics of a transporter substrate depends on both of these factors. The transporter expression has been shown to affect both the apparent K m and V max values. Kinetic parameters can be obtained from various experimental settings, but these do not necessarily reflect the situation in transcellular flux. Kinetic absorption models need reliable estimates of saturable kinetics when accurate in silico predictions are to be made. The effect of increasing P-glycoprotein expression on apparent transport kinetics was studied using quinidine and digoxin as model compounds. The intracellular concentrations of drugs during the transport process were also measured. A dynamic simulation model was constructed to study the observed data. The apparent K m and V max values increased as the P-glycoprotein expression increased. Simulations reproduced the shift in both kinetic parameters as a function of efflux pump expression. In addition, the apparent K m value showed a strong inverse relationship to the passive permeability. In contrast, the apparent V max value reached a maximum at intermediate passive permeability and declined above and below this passive permeability. The true V max and K m values were never reached. The shift in K m was assigned to a decrease in intracellular concentration at the Pglycoprotein interaction site with both experimental and simulation data. In conclusion, the apparent kinetic parameters in transcellular permeability assays depend on passive permeability and efflux pump activity. Therefore, parameters that are obtained from in vitro assays should be cautiously applied to in vivo predictions.

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Caco‐2 Cells as a Model for Intestinal Absorption

2011

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The Caco-2 cell system, a well characterized intestinal in vitro model, makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Permeability values estimated with this model correlate well with human in vivo absorption data for many drugs and chemicals. As a consequence, the use of the Caco-2 cellular model as a permeability assay to predict oral absorption in humans is growing, and its importance is increasing as a screening tool in drug-discovery strategies for the prediction of intestinal drug permeability. Strict observance of the experimental conditions, coupled with careful control of the cell-culture procedures, are crucial for obtaining a meaningful correlation with in vivo data. Because of the intrinsic variability of this absorption model, it is important to highlight that each experimental system must be calibrated with internal reference compounds, to permit data sharing between different laboratories.

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A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial ( Caco-2) cells grown in permeable filter chambers

Cited by 157 publications

References 17 publications

Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug–membrane interactions and the effect of agitation on the barrier integrity and on the permeability

Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug–membrane interactions and the effect of agitation on the barrier integrity and on the permeability

Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates

Caco‐2 Cells as a Model for Intestinal Absorption

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A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial ( Caco-2) cells grown in permeable filter chambers

Cited by 157 publications

References 17 publications

Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug–membrane interactions and the effect of agitation on the barrier integrity and on the permeability

Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug–membrane interactions and the effect of agitation on the barrier integrity and on the permeability

Decrease in Intracellular Concentration Causes the Shift in Km Value of Efflux Pump Substrates

Caco‐2 Cells as a Model for Intestinal Absorption

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Decrease in Intracellular Concentration Causes the Shift in K_m Value of Efflux Pump Substrates