Permeability of Hydrophilic

Supervisors: Vladan Milovic Professor Per Artursson

SUMMARY

Investigations of the integrity and transport characteristics of 2/4/A1 cells
have been done in this report. The cell line was isolated from rat fetal
intestinal epithelial cells and transfected with thermolabile SV40 large T
antigen.

These cells proliferated at 33 °C, but eliminated the antigen and ceased
proliferating at a non-permissive temperature (39°C). At 39°C 2/4/A1 cells
started to differentiate but simultaneously the cells also underwent massive
cell death.

When cultured at 37°C these cells formed confluent and tight monolayers that
seemed to have paracellular transport characteristics similar to that of the
human intestine. Transmission electron microscopy confirmed the development of
multilayers at 33°C, monolayers at 37°C and defects in the cell layer due to
apoptosis at 39°C.

Different immunostainings of ZO-1, E-cadherin and vinculin confirmed formation
of tight and adherence junctions. Transepithelial resistance reached a plateau
of 25-35 Ohm.cm2, which was similar to the small intestine. In transport studies
2/4/A1 cell line monolayers selectively restricted the permeation of hydrophilic
permeability markers proportional to molecular weight and discriminated more
accurately between the molecules of intermediate molecular weight compared to
Caco-2 cells.

These results indicated that 2/4/A1 cells could be used as a model for
hydrophilic drug absorption.

INTRODUCTION

The small intestine plays a crucial role in the absorption of drugs and
nutrients. Exogenous substances cross a series of barriers during the process
of intestinal absorption: (1) the aqueous boundary/mucus layer, (2) a single
layer of epithelial cells, and (3) the lamina propria, which contains the blood
and lymph vessels that then transport the absorbed drugs to other parts of the
body (Artursson 1991).

The cell monolayer is comprised of two parallel barriers: the cell membrane and
the tight junctions. Most drugs are absorbed by a passive diffusion across the
cell membrane by the transcellular route, or across the tight junctions between
the cells - the paracellular route. Drug transport can also be carrier mediated,
when the drug utilizes transporters located in the cellular membrane.
Transcytosis is another kind of active transport, in which macromolecules can be
transported across the intestinal epithelial cell in endocytosed vesicles.

The hydrophilic and charged drugs are absorbed after passing through the
paracellular route, the water-filled channels between the cells (Artursson
1991). Rates and extent of the paracellular transport are, therefore, highly
influenced by the structure and size of the tight junctions as well as by the
size of the molecules. Only small and hydrophilic drugs can pass between the
cells rapidly and completely; permeation of larger molecules can be limited
proportionally to their size and lipophilicity (Hillgren et al. 1995).

Simple assay methods are needed for drug absorption studies. Excised intestinal
tissue, isolated cells, membrane vesicles and in vivo models have distinct
limitations, which have been previously discussed in detail (Audus et al. 1990;
Artursson 1991; Hillgren et al. 1995). The most suitable method for the study of
drug intestinal transport appeared to be the use of cultured intestinal
epithelial cells. This model has several advantages over conventional drug
absorption models: (a) it is less time-consuming; (b) it enables rapid
evaluation of methods for improving drug absorption; (c) it allows an
opportunity to use human rather than animal tissues; (d) it can minimize
expensive and sometimes controversial animal studies.

Human colorectal carcinoma cell line Caco-2 is nowadays the most widely used and
the best explored model for drug intestinal transport (Hidalgo et al. 1989;
Artursson 1990; Artursson & Karlsson 1991). This cell line displays spontaneous
enterocytic differentiation in culture and forms a polarized monolayer with
apical brush borders and well differentiated tight junctions (Hidalgo, 1989).
Drug transport studies across the Caco-2 cell monolayers showed a satisfactory
correlation with other in vitro absorption models, e.g. rat intestinal segments
(Artursson et al. 1993) and in vivo drug absorption (Lennernäs et al., 1995),
although a considerable variability has been reported, being related to
heterogenity, a number of sub populations, and number of passages (Walter &
Kissel, 1995).

Caco-2 cells however, form monolayers that resemble colonic rather than small
intestinal epithelial cells. Due to its well-formed tight junctions, Caco-2 cell
monolayers have a transepithelial electrical resistance of 260 Ohm.cm2 which is
similar to the transepithelial electrical resistance of the colon rather than of
the small intestine (Hillgren et al. 1995). Therefore, there is a need to
investigate drug intestinal transport in a model which has apparent transport
characteristics corresponding to the human intestine, and several studies have
been attempted to characterize a cell line that can be used for this purpose.

A novel intestinal epithelial cell line (2/4/A1) is derived from the rat fetal
intestinal epithelial cells conditionally immortalized with thermolabile SV40
large T antigen, pzipSVtsa58 (Paul et al. 1993). According to the original
report, these cells form more leaky