Drug cell monolayers

Since experimental determination of intestinal absorption is quite demanding, Caco-2 cell monolayers have been successfully used to model passive drug absorption. Several models for the prediction of Caco-2 permeability using PSA were developed, including those of van de Waterbeemd et al. [5] and Palm et al. [22] who found that relationships between Caco-2 permeability and PSA is stronger than with Clog D, Krarup et al. [23] who used dynamic PSA calculated for water accessible molecular surface and Bergstrom et al. [24]. 

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). 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

The use of vesicle cell membranes, isolated cells, and cell monolayers and intestinal tissue studies has provided valuable correlations with in situ and in vivo drug absorption in animals as well as correlations with drug absorption in clinical studies. Most prominent among the literature sources establishing correlations between in vitro tissue and cellular systems with drug absorption in humans are the work of Dowty and Dietsch [73], Lennernas et al. [74], and Stewart et al. [75],... 

Methods for quantifying both the transcellular diffusion and concurrent metabolism of drugs and the unusual transcellular diffusion of membrane-interactive molecules coupled with the influence of protein binding are described in detail. To demonstrate the utility of cultured cell monolayers as a tool for basic science investigations, a subsection is devoted to the elucidation of rate-determining steps and factors in the passive diffusion of peptides across biological membranes. The chapter concludes with a discussion on the judicious use of in vitro cell monolayer results to predict in vivo results. 

The steady-state flux of drug solute across the cell monolayer-filter support system (Fig. 5) is... 

Upon examination of the columns of Pe and PM values for each drug in Table 16, one discovers that the influence of PF is minimal. Therefore, Pe = PM, i.e., permeation of the drugs across the ABL/cell monolayer/filter barriers is governed by the cell monolayer. The remaining questions are (1) To what extent is the transmonolayer diffusional process of uncharged and cationic species gated by the transcellular and paracellular routes and (2) What are the governing factors ... 

Figure 28 The biophysical model for passive diffusion and concurrent intracellular metabolism of a drug for a simple A-to-B reaction process. Concentration-distance profiles are depicted in the aqueous boundary layer and intracellular domain for the drug and metabolite. The bottom diagram depicts the direction of the fluxes of drug and metabolite viewed from the donor and receiver sides of the cell monolayer. Details of basic assumptions are found in the text.

The flux of 3H-labeled PNU-78,517 across MDCK cell monolayers shows the characteristic disparity between the kinetics of disappearance from the donor solution and appearance in the receiver sink (Fig. 32). Drug uptake is rapid and exponential with time and approaches a quasi-equilibrium state in contrast, the concomitant efflux of drug into the receiver is slow and linear. While maintaining a 3% bovine serum albumin (BSA) concentration in the donor and varying the BSA concentration between 0.5 and 5% in the receiver, the results show that the... 

It is noteworthy that the kinetics viewed from the donor and receiver sides are seemingly independent of each other. As discussed in the next section, this prompts the employment of experimental strategies to understand the events of drug uptake and efflux mechanistically and independently through drug uptake studies with cell monolayers cultured on flat plastic dishes, drug efflux from the... 

The total flux of free and BSA-bound drug across the ABL over the mono-layer followed by partitioning of free drug into the apical membrane and rapid distribution throughout the cell monolayer (see Fig. 31) is described by... 

Given the low permeability of the antioxidant across MDCK cell monolayers and its large membrane partition coefficient, efflux kinetic studies using drug-loaded cell monolayers cultured on plastic dishes could yield useful information when coupled with the following biophysical model. The steady-state flux of drug from the cell monolayer is equal to the appearance rate in the receiver solution ... 

Table 19 Comparison Between Permeability Coefficients of the Apical and Basolateral Membranes from Independent Efflux Kinetics from Drug-Loaded MDCK Cell Monolayers...

The identification and characterization of cell culture systems (e.g., Caco-2-cells) that mimic in vivo biological barriers (e.g., intestinal mucosa) have afforded pharmaceutical scientists the opportunity to rapidly and efficiently assess the permeability of drugs through these barriers in vitro. The results generated from these types of in vitro studies are generally expressed as effective permeability coefficients (Pe). If Pe is properly corrected to account for the barrier effects of the filter (PF) and the aqueous boundary layer (PAbl) as previously described in Section II.C, the results provide the permeability coefficient for the cell monolayer... 

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. 

Fig. 4.1. Relationship between the absorbed vivo perfusion of the human jejunum (B) (data fraction (FA) of structurally diverse sets of compiled from publications by Lennernas orally administered drugs and permeability laboratory [63, 115]). Sigmoidal relationships coefficients obtained in Caco-2 cell monolayers were obtained between FA and the (A) (data compiled from publications by permeability coefficients in both models.

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

B. Sjostrom et al. Correlation of gene expression of ten drug efflux proteins of the atp-binding cassette transporter family in normal human jejunum and in human intestinal epithelial caco-2 cell monolayers, /. Pharmacol. Exp. Ther. 2001, 299, 164-170... 

Tavelin, S., J. Taipalensuu, F. Hallbook, K. Vellonen, V. Moore, and P. Artursson. An Improved Cell Culture Model Based on 2/4/A1 Cell Monolayers for Studies of Intestinal Drug Transport. Characterization of Transport Routes., Pharm. Res. 2003, 20, 373-381. 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

Many academic and industrial laboratories have shown that the drug permeability measured in Caco-2 cell monolayers can be used to predict the oral absorption of drugs in humans. Various datasets have therefore been used to establish correlations between Caco-2 permeability and the fraction absorbed orally in humans [85, 86, 96]. Taken together, these studies show good predictability, though with a relatively wide variation in the appearance of correlation profiles between different laboratories [86]. These studies emphasize the need to establish correlations and standardization procedures in each laboratory. 

Fig. 5.3. Relationship between the oral fraction basolateral Papp values were determined at absorbed in humans and apparent permeability drug concentrations of 10-500 pM at pH 6.5/ coefficients obtained in Caco-2 cell monoalyers 7.4 or 7.4/7.4 on the apical/basolateral sides of at two different pH conditions. Mean apical to the cell monolayers.

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). 

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... 

Passive diffusion through the lipid bilayer of the epithelium can be described using the partition coefficient between octanol/water (log P) and A log P (the difference between the partition into octanol/water and heptane/ethylene glycol or heptane/ octanol) [157, 158], The lipophilicity of the drug (log P) (or rather log D at a certain pH) can easily be either measured or calculated, and is therefore generally used as a predictor of drug permeability. Recently, a method using artificial membrane permeation (PAMPA) has also been found to describe the passive diffusion in a similar manner to the Caco-2 cell monolayers [159]. 

W., Hilgendorf, C., Spahn-Langguth, H., Wunderli-Allenspach, H., Merkle, H. P., Langguth, P., P-glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers the influence of culturing conditions and drug exposure on P-gp expression levels, J. Pharm. Sci. 1998, 87, 757-762. 

Brayden, D. J., Human intestinal epithelial cell monolayers as prescreens for oral drug delivery, Pharm. News 1997, 4, 11-15. 

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). 

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