Transport across cell monolayer

Molecules with a large molecular weight or size are confined to the transcellular route and its requirements related to the hydrophobicity of the molecule. The transcellular pathway has been evaluated for many years and is thought to be the main route of absorption of many drugs, both with respect to carrier-mediated transport and passive diffusion. The most well-known requirement for the passive part of this route is hydrophobicity, and a relationship between permeability coefficients across cell monolayers such as the Caco-2 versus log P and log D 7.4 or 6.5 have been established [102, 117]. However, this relationship appears to be nonlinear and reaches a plateau at around log P of 2, while higher lipophilicities result in reduced permeability [102, 117, 118]. Because of this, much more attention has recently been paid towards molecular descriptors other than lipophilicity [86, 119-125] (see section 5.5.6.). The relative contribution between the para-cellular and transcellular components has also been evaluated using Caco-2 cells, and for a variety of compounds with different charges [110, 112] and sizes [112] (see Section 5.4.5). 

Substrates are usually identified using transfected MDCK, Caco-2, or endothelial cell lines that express the transporter of interest. These cell types are grown in a monolayer on a membrane separating two chambers of culture medium (i.e., the TransweU Cell Culture Assay, Coming Costar Corp., Cambridge, MA). Drag is administered into one chamber, and drag transport across the monolayer is... 

The setup of this membrane permeability study involves the use of culture inserts that contain Caco-2 cells grown as epithelial layers. A drug candidate is delivered to the apical side of the cell mono-layer (donor) and allowed to incubate for approximately 60 min. Samples from the apical and basolateral (recipient) side are collected for analysis by LC/MS/MS. Membrane permeability is expressed as the percentage of substrate transported across the monolayer from the apical to the basolateral side. 

Figure 6 Directional transport of pravastatin in Oatplb2/Mrp2 double transfectants in the apical direction (A), and comparison of in vivo biliary excretion clearance and in vitro transcellular transport clearance across the double transfectant (B). (A) Transcellular transport across the monolayers of MDCK II cells was determined in the basal-to-apical and the opposite direction. (B) The x axis represents CLint determined in vitro multiplied by /B and the scaling factor, and the y axis represents the in vivo biliary clearance defined for the blood ligand concentrations. The symbol ( ) represents data whose x axis values were corrected for the scaling factor (a = 17.9). The solid line represents the theoretical curve, and the symbol (o), the observed data. Source From Ref. 59.

The SLC transporters mediate either drug uptake or efflux, whereas ABC transporters mediate only unidirectional efflux. Asymmetrical transport across a monolayer of polarized ceUs, such as the epithelial and endothelial cells of brain capillaries, is called vectorial transport (Figure 2-5). Vectorial transport is important in the efficient transfer of solutes across epithelial or endothelial barriers it plays a major role in hepatobiliary and urinary excretion of drugs from the blood to the lumen and in the intestinal absorption of drugs and nutrients. In addition, efflux of drugs from the brain via brain endothelial cells and brain choroid plexus epithelial cells involves vectorial transport. 

Hypoxia (low oxygen tension), which can be induced by a blood phase transport limitation, can lead to a breakdown of the endothelial transport barrier either by a direct effect on the endothelial layer or by an indirect mechanism in which hypoxia up-regulates the production of hyperpermeabilizing cytokines from other cells in the arterial wall. A number of recent studies have shown that hypoxia increases macro-molecular transport across endothelial monolayers in culture due to metabolic stress [40-42]. These studies describe direct effects on the endothelial layer since other cells present in the vessel wall were not present in the cell culture systems. 

The in vitro system we have been using to study the transepithelial transport is cultured Madin-Darby canine kidney (MDCK) epithelial cells (11). When cultured on microporous polycarbonate filters (Transwell, Costar, Cambridge, MA), MDCK cells will develop into monolayers mimicking the mucosal epithelium (11). When these cells reach confluence, tight junctions will be established between the cells, and free diffusion of solutes across the cell monolayer will be markedly inhibited. Tight junction formation can be monitored by measuring the transepithelial electrical resistance (TEER) across the cell monolayers. In Figure 1, MDCK cells were seeded at 2 X 104 cells per well in Transwells (0.4 p pore size) as described previously. TEER and 14C-sucrose transport were measured daily. To determine 14C-sucrose... 

Hilgers, A. R. Conradi, R. A. Burton, P. S., Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa, Pharm. Res. 7, 902-910 (1990). 

Hidalgo, I. J. Hillgren, K. M. Grass, G. M. Borchardt, R. T., A new side-by-side diffusion cell for studying transport across epithelial cell monolayers, in Vitro Cell Dev. Biol. 28A, 578-580 (1992). 

In vitro studies permit further isolation of parallel transport processes and can provide a reduction in experimental variability. Rate of absorption assessment can be measured as intestinal uptake or flux across an intestinal barrier at both the tissue and cell monolayer levels. Experimental variability is also reduced by the fact that a large number of tissue samples can be used from the same experi-... 

The sole purpose of the filter support and any applied extracellular matrix is simply to provide a surface for cell attachment and thus to provide mechanical support to the monolayer. However, the filter and matrix also can act as serial barriers to solute movement after diffusion through the cell monolayer. The important variables are the chemical composition of the filter, porosity, pore size, and overall thickness. In some cases, pore tortuosity also can be important. It is desired that the filter, with or without an added matrix, provide a favorable surface to which the cells can attach. However, in some cases these properties can also result in an attractive surface for nonspecific adsorption of the transported solute. In these instances, the appearance of the solute in the receiver compartment of the diffusion cell will not be a true reflection of its movement across the mono-layer. Such problems must be examined on a case-by-case basis. 

The summary of Pe values for the steroids as a function of stirring rates is found in Table 11 and their correlations with log PC (n-octanol-water) in Figure 20. The transport kinetics of the relatively hydrophilic hydrocortisone and dexa-methasone are controlled by passive diffusion across the cell monolayer. On the other hand, the Pe values of testosterone and progesterone are highly dependent on stirring rate. The results for testosterone are used to obtain the relationships between the effective permeability coefficients of the ABL on the donor and receiver sides and the stirring rate, using the linear expression (see Eq. (69)]... 

Burton PS, RA Conradi, AR Hilgers, NFH Ho, LL Maggiora. (1992). The relationship between peptide structure and transport across epithelial cell monolayers. J Controlled Release 19 87-98. 

Buur A, N Mprk. (1992). Metabolism of testosterone during in vitro transport across Caco-2 cell monolayers Evidence for beta-hydroxysteroid dehydrogenase activity in differentiated Caco-2 cells. Pharm Res 9 1290-1294. 

Imanidis G, C Waldner, C Mettler, H Leuenberger. (1996). An improved diffusion cell design for determining drug transport parameters across cultured cell monolayers. J Pharm Sci 85 1196-1203. 

P. S. Burton. Caco-2 cell monolayers as a model for drug transport across... 

Lee, K. and D. R. Thakker. Saturable transport of H2-antagonists ranitidine and famotidine across Caco-2 cell monolayers, J. Pharm. Sci. 1999, 88, 680-687... 

Pal, D., C. Udata, and A. K. Mitra. Transport of cosalane-a highly lipophilic novel anti-HIV agent-across caco-2 cell monolayers, J. Pharm. Sci. 

Solvents used to increase solubility for compounds during screening of permeability across the cell monolayers, together with commonly used excipients for formulations, can also affect the barrier as they contain ingredients which enhance drug absorption [100, 151]. There are different mechanisms by which these compounds can modulate the barrier [4, 149, 150] for example, they may increase the tight junctional pathway inhibiting carrier-mediated transport, or cholesterol... 

Neuhoff, S., Zamora, I., Ungell, A.-L., Artursson, P., pH-dependent bidirectional transport of weak basic drugs across Caco-2 cell monolayers, implications for drug-drug interactions, 2002 Pharm. Res. (submitted). 

Twaites, D. T., Basterfield, L., McCleave, P. M. J., Carter, S. M., and Simmons, N. L., Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers, Brit.J. Pharmacol. 2000, 129, 457-464. 

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