In vitro models for the blood-brain barrier.

The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes.

Sodium caprate (C10), a medium chain fatty acid, is used clinically to enhance rectal absorption of the low molecular weight (MW) drug ampicillin. The main aim of this study was to investigate whether C10 also enhances the permeability of high MW model drugs in a model of the intestinal epithelium. The second aim was to present visual evidence of the route of enhanced transport across the epithelial cell layer. The studies were performed in Caco-2 monolayers cultured on permeable supports. The effects of non-toxic concentrations (< or = 13 mM) of C10 on drug transport across the monolayers was studied using monodisperse 14C-polyethylene glycols (MW 238-502; 14C-PEGs), 125I-Arg5-vasopressin (MW 1,208), 125I-insulin (MW 6,000) and FITC-labelled dextrans (MW 4,400 and 19,600; FD4 and FD20 respectively) as model drugs. Electron and confocal laser scanning microscopy were used to demonstrate transport routes across the epithelium. 10 mM C10 increased the permeability of all 14C-PEGs to approximately the same extent. 13 mM C10 increased the permeability of 125I-Arg8-vasopressin 10-fold. Only small increases in FD4 and FD20 permeabilities were observed. After C10 exposure, both tight junctions with normal morphology and those with dilatations showed an increased permeability to ruthenium red, indicating that C10 enhanced the paracellular transport of molecules with a MW < 1,000. Confocal microscopy showed that C10 increased the transport of FD4 and FD20 by the paracellular route. In conclusion, non-toxic concentrations of C10 can be used to enhance the permeability of drugs of MW up to approximately 1,200. Enhancement of the absorption of molecules larger than 4,000 is quantitatively insignificant. The enhanced permeability occurred via the paracellular pathway.

Mechanisms of absorption enhancement by medium chain fatty acids in intestinal epithelial Caco-2 cell monolayers.

Sodium salts of medium chain fatty acids (MCFAs) enhance the absorption of hydrophilic drugs across the intestinal mucosa, but the mechanism behind the effect is largely unknown. In this study, the dose-dependent effects of the sodium salts of four MCFAs, C6 (caproate), C8 (caprylate), C10 (caprate) and C12 (laurate), on the permeability of the hydrophilic marker molecule [14C]mannitol were studied in monolayers of the human intestinal epithelial cell line, Caco-2, grown on permeable supports. C8, C10 and C12, but not C6, enhanced the permeability of [14C]mannitol in a dose-dependent manner. Comparison of the cellular effects of the MCFAs at concentrations that gave comparable (8.1- to 8.5-fold) absorption enhancement showed that: 1) C8 was active as absorption enhancer only when the tonicity of the medium was increased; 2) absorption enhancement mediated by C10 was related to a redistribution of the cytoskeleton and structural dilatations in the tight junctions; and 3) C12 was without effect on the cytoskeleton and cellular morphology. Studies on C10 under anisotonic conditions showed that deviations from isotonicity enhanced its effect. These results suggest that structurally similar MCFAs display dramatic differences in their mechanism of action. In addition, the effects of osmolality provide an explanation for the previously reported variability in the efficacy of MCFAs as absorption enhancers.

Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells.

Medium chain fatty acids (MCFAs) are used to enhance the permeability of mucosal tissues to hydrophilic drugs, but their mechanism of action is largely unknown. In this study, the absorption-enhancing effects of the sodium salts of two MCFAs, capric acid (C10) and lauric acid (C12), were studied in monolayers of human intestinal epithelial Caco-2 cells. Both MCFAs induced a rapid increase in epithelial permeability to the hydrophilic marker molecule sodium fluorescein. Inhibition of phospholipase C and inhibition or activation of various kinases and buffering of intracellular calcium indicated that the effects on epithelial permeability were mediated through phospholipase C-dependent inositol triphosphate/diacylglycerol pathways. Surprisingly, the inositol triphosphate and diacylglycerol pathways were found to have opposing effects on paracellular permeability. Exposure to the MCFAs also resulted in a concentration dependent reduction of cellular dehydrogenase activity and ATP levels. C10, but not C12, induced redistribution of the tight junction proteins ZO-1 and occludin. These results indicate that the two MCFAs have partially different and more complex mechanisms than previously recognized, which has important implications for their use in vivo.

Sodium caprate elicits dilatations in human intestinal tight junctions and enhances drug absorption by the paracellular route.

The effects of the absorption enhancer sodium caprate on human intestinal epithelial cells were investigated using Caco-2 cell monolayers. The effects on epithelial integrity and drug transport are dependent on time and concentration and are decreased by Ca2+, most likely through the formation of Ca2+ soaps. Morphological data indicate that exposure to sodium caprate results in cytoskeletal changes and in structural alterations of the tight junctions in the form of dilatations, while the effects on the apical cell membranes are limited. We conclude that sodium caprate increases the absorption of drugs mainly by the paracellular route.

Mechanism of absorption enhancement in humans after rectal administration of ampicillin in suppositories containing sodium caprate.

PURPOSE: The medium chain fatty acid sodium caprate (C10) is approved as an absorption enhancer but its mechanism of action has not been studied in humans. The aim of this study was to investigate the mechanism of action of C10 in human subjects after rectal administration. METHODS: Twelve healthy human subjects were randomised to receive ampicillin suppositories with (AM-C10) or without (AM) C10. Serum and urine samples were collected and analysed for ampicillin by HPLC. Rectal biopsies were taken before and 25 min (approximate time of maximum serum concentration, Cmax, for ampicillin) and 185 min (during the final part of the elimination phase) after rectal administration of the suppositories. The osmolality of the rectal fluid was also measured. RESULTS: AM-C10 administration increased Cmax, area under the serum concentration-time curve (AUC) and urinary recovery of ampicillin 2.6-, 2.3- and 1.8-fold, respectively, compared to AM. Histological examination of the biopsies showed that AM-C10 exposure resulted in reversible mucosal damage that occurred at the same time as the Cmax for ampicillin while AM prolonged mucosal damage. A reversible increase in rectal fluid osmolality was observed with both treatments. CONCLUSIONS: AM-C10-enhanced absorption of ampicillin coincides with non-specific damage to the rectal mucosa. C10 itself as well as the suppository base and the hyperosmolality of the rectal fluid contributed to this effect. However, the histological damage was reversible with AM-C10, suggesting that C10 also has a protective effect on the rectal mucosa.

Reversible increase in tight junction permeability to macromolecules in rat ileal mucosa in vitro by sodium caprate, a constituent of milk fat.

Intestinal tight junction function is thought to be of importance in the pathogenesis of various diseases. The regulation of uptake of macromolecules via the tight junctions is largely unknown. Effects of luminal sodium caprate (10 mM), a dairy product constituent, and cytochalasin B (30 microM), were studied in rat ileum in vitro in Ussing chambers. Both agents caused a reversible fall in potential difference and increased [51Cr]EDTA permeability. In addition, sodium caprate induced increased permeability to polysucrose (15,000 daltons) and opening of the tight junctions as visualized by transmission electron microscopy. Dose-response patterns suggested mainly dose-dependent differences between the agents. Confocal laser scanning microscopy suggested paracellular permeation of polysucrose. Luminal sodium caprate, a food constituent, can increase tight junction permeability, allowing passage of macromolecules, without affecting epithelial viability. Increased permeability to medium-sized molecules does not necessarily coincide with increased paracellular uptake of protein-sized molecules.

Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease.

BACKGROUND: Crohn's disease is associated with deranged intestinal permeability in vivo, suggesting dysfunction of tight junctions. The luminal contents are important for development of neoinflammation following resection. Regulation of tight junctions by luminal factors has not previously been studied in Crohn's disease. AIMS: The aim of the study was to investigate the effects of a luminal stimulus, known to affect tight junctions, on the distal ileum in patients with Crohn's disease. PATIENTS: Surgical specimens from the distal ileum of patients with Crohn's disease (n=12) were studied, and ileal specimens from colon cancer patients (n=13) served as controls. METHODS: Mucosal permeability to 51Cr-EDTA and electrical resistance were studied in Ussing chambers during luminal exposure to sodium caprate (a constituent of milk fat, affecting tight junctions) or to buffer only. The mechanisms involved were studied by mucosal ATP levels, and by electron and confocal microscopy. RESULTS: Baseline permeability was the same in non-inflamed ileum of Crohn's disease and controls. Sodium caprate induced a rapid increase in paracellular permeability--that is, increased permeation of 51Cr-EDTA and decreased electrical resistance--which was more pronounced in non-inflamed ileum of Crohn's disease, and electron microscopy showed dilatations within the tight junctions. Moreover, sodium caprate induced disassembly of perijunctional filamentous actin was more pronounced in Crohn's disease mucosa. Mucosal permeability changes were accompanied by mitochondrial swelling and a fall in epithelial ATP content, suggesting uncoupling of oxidative phosphorylation. CONCLUSIONS: The tight junctions in the non-inflamed distal ileum of Crohn's disease were more reactive to luminal stimuli, possibly mediated via disturbed cytoskeletal contractility. This could contribute to the development of mucosal neoinflammation in Crohn's disease.