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Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Pharmaceutics
presented at Uppsala University in 2003
ABSTRACT
Tavelin, S., 2003. New Approaches to Studies of Paracellular Drug Transport in Intestinal
Epithelial Cell Monolayers. Acta Universitatis Upsaliensis. Comprehensive Summaries of
Uppsala Dissertations from the Faculty of Pharmacy 285. 66 pp. Uppsala.
ISBN 91-554-5582-4.
Studies of intestinal drug permeability have traditionally been performed in the colon-derived
Caco-2 cell model. However, the paracellular permeability of these cell monolayers resembles
that of the colon rather than that of the small intestine, which is the major site of drug
absorption following oral administration. One aim of this thesis was therefore to develop a
new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are
conditionally immortalized with a temperature sensitive mutant of SV40T. These cells
proliferate and form multilayers at 33°C. At cultivation temperatures of 37–39°C, they stop
proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed
functional tight junctions. The barrier properties of the tight junctions such as transepithelial
electrical resistance and permeability to hydrophilic paracellular markers resembled those of
the human small intestine in vivo. These cells lacked functional expression of drug transport
proteins and can therefore be used as a model to study passive drug permeability unbiased by
active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of
the human jejunum for both incompletely and completely absorbed drugs, and the prediction
of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely
absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables
better predictions of drug permeability in the small intestine than does Caco-2.
The paracellular route and its importance for intestinal drug permeability was also the
focus of the second part of this thesis, in which a new principle for tight junction modulation
was developed, based on the primary structure of the extracellular tight junction protein
occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased
the permeability of the tight junctions, but lacked apical effect on the paracellular
permeability. This problem was solved by conjugation of one peptide to a lipoamino acid,
resulting in two diastereomers with different effects. The L-isomer had a sustained apical
effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides
constitute a new class of tight junction modulators that can enhance the paracellular
permeability.
Staffan Tavelin, Department of Pharmacy, Uppsala Biomedical Centre, Box 580,
SE-751 23 Uppsala, Sweden
© Staffan Tavelin 2003
ISSN 0282-7484
ISBN 91-554-5582-4
Printed in Sweden by Eklundshofs Grafiska, Uppsala 2003
CONTENTS
1
PAPERS DISCUSSED
5
2
ABBREVIATIONS
6
3
INTRODUCTION
7
3.1
Drug absorption from the gastro-intestinal tract
7
3.2
Mechanisms of intestinal drug permeability
8
3.2.1
Passive transcellular transport
10
3.2.2
Active transport
11
3.2.3
Paracellular transport
12
3.3
In vitro methods for studying drug permeability
12
3.3.1
Cultured cells
13
3.3.2
Artificial membranes
15
3.4
Methods for modulating drug absorption
16
3.4.1
Composition and regulation of the tight junctions
17
3.4.2
Physiological considerations
19
3.4.3
Methods for modulating paracellular transport
20
4
AIMS OF THE THESIS
22
5
MATERIAL AND METHODS
23
5.1
Drugs and radiolabelled markers
23
5.2
Occludin peptides
23
5.3
Cell culture
23
5.3.1
2/4/A1
23
5.3.2
Caco-2
24
5.4
Hexadecane membranes
24
5.5
Microscopy
24
5.5.1
Immunofluorescence
24
5.5.2
Electron microscopy
25
5.6
Establishment of 2/4/A1-Bcl-2 clones
25
5.7
RT- PCR
25
5.8
Cell attachment
25
5.9
Analysis of toxicity and cell death
25
3
5.9.1
DNA staining
25
5.9.2
Dehydrogenase activity
26
5.9.3
Lactate dehydrogenase leakage
26
5.10 Cell monolayer integrity and drug transport studies
26
5.10.1 Electrophysiological measurements
26
5.10.2 Drug permeability studies
26
5.11 Calculations
27
5.12 Statistics
29
6
30
RESULTS AND DISCUSSION
6.1
Characterization of 2/4/A1 (Papers I–III)
30
6.1.1
Expression of SV40 large T antigen (Paper I)
30
6.1.2
Cell attachment and extracellular matrix (Paper I)
31
6.1.3
Monolayer formation (Papers I and II)
32
6.1.4
Development of tight and adherence junctions (Papers I and III)
32
6.1.5
Transepithelial electrical resistance (Papers I and II)
34
6.1.6
Expression of drug transport proteins (Paper II)
35
6.1.7
Cell death (Papers I and II)
36
6.2
Transport of drugs and hydrophilic markers (Papers I – IV)
37
6.2.1
Hydrophilic markers and average pore radius (Papers I and III)
37
6.2.2
Rapidly absorbed drugs (Paper I)
39
6.2.3
Poorly and intermediately absorbed drugs (Papers I and III)
40
6.2.4
Actively transported drugs (Papers I and II)
41
6.2.5
pH dependent permeability (Paper IV)
42
6.3
Modulation of tight junction permeability (Paper V)
44
7
SUMMARY AND CONCLUSIONS
48
8
ACKNOWLEDGEMENTS
50
9
REFERENCES
52
4
1
PAPERS DISCUSSED
This thesis includes the following papers, which will be referred to in the text by their
Roman numerals:
I. Tavelin, S., Milovic, V., Ocklind, G., Olsson, S., and Artursson, P. A
Conditionally Immortalized Epithelial Cell Line for Studies of Intestinal Drug
Transport, J. Pharmacol. Exp. Ther. 1999, 290, 1212–1221.
II. Tavelin, S., Taipalensuu, J., Hallböök, F., Vellonen, K., Moore, V., and
Artursson, P. 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.
III. Tavelin, S., Taipalensuu, J., Söderberg, L., Morrison, R., Chong, S., and
Artursson, P. Prediction of the Oral Absorption of Low Permeability Drugs
Using Small Intestinal-like 2/4/A1 Cell Monolayers, Pharm. Res. 2003, 20,
397–405.
IV. Tavelin, S., Nagahara, N., and Artursson, P. The Contribution of the Paracellular
Route to The pH-Dependent Epithelial Permeability to Cationic Drugs. In
manuscript.
V. Tavelin, S., Hashimoto, K., Malkinson, J., Lazorova, L., Toth, I., and Artursson,
P. A New Principle for Tight Junction Modulation Based on Occludin Peptides.
Submitted to Mol. Pharmacol.
Reprints were made with permission from the journals.
5
2
ABBREVIATIONS
a-b
apical-to-basolateral
ABC
ATP-binding cassette
b-a
basolateral-to-apical
EDTA
ethylene diamine tetraacetate sodium
EHS
Engelbreth-Holm-Swarm
FA
absorbed fraction after oral administration to humans
FCS
foetal calf serum
FITC
fluorescein isothiocyanate
GI
gastrointestinal
HBSS
Hank’s balanced salt solution
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
LDH
lactate dehydrogenase
MAGUK
membrane-associated guanylate kinase
MDR
multidrug resistance
MRP
multidrug resistance associated protein
Papp
apparent permeability coefficient
PBS
phosphate-buffered saline
Pc
permeability coefficient of the cell monolayer
PDZ
peptide motif of the MAGUK proteins
PEG 4000
polyethylene glycol with an average molecular weight of 4000
P-gp
P-glycoprotein
PLC
phospholipase-C
RMSE
root mean squared error
SD
standard deviation
SV40T
Simian Virus large T antigen
TER
transepithelial electrical resistance
ZO
zonula occludens
Å
Ångström (1×10-10 m)
6
3
INTRODUCTION
The oral route is generally considered the most convenient for administration of
drugs. Drug formulations intended for oral administration are preferred to their nonoral alternatives (e.g. intravenous injections) mainly for reasons such as lower
production cost, better suitability for self-medication, a higher level of patient safety
and better patient compliance. In order to be efficient, an orally administered drug
must meet special criteria. For instance, it must be sufficiently soluble in the gastrointestinal (GI) fluids, withstand acidic and enzymatic degradation in the GI tract and
to a sufficient degree permeate the epithelium of the intestinal mucosa. Given the
crucial importance of these drug properties in drug discovery, several methods have
been developed to study and predict solubility and stability in and absorption from the
GI tract (1-3). As a result of the introduction of high throughput pharmacological
screening and combinatorial synthesis, an immense number of pharmacologically
active compounds have entered the pre-clinical screening phase. Therefore the
methods intended for predicting solubility and permeability have recently been
modified to cope with the increasing demands for high throughput in drug discovery
programmes. For instance, cell-based assays have been automated (4) and computerbased predictions of permeability and solubility have been developed (5, 6).
The work presented in the first part of this thesis deals with the development of a new
cell culture model for studies of intestinal drug permeability. This cell line is
particularly suitable for permeability screening since it has a permeability that is
comparable to that of the human small intestine and it requires only 4–6 days of
cultivation compared with several weeks (for the well-established permeability model
based on Caco-2 cells).
In the development of new drugs, compounds that have poor permeability properties
are generally dropped in favour of compounds with adequate permeability properties.
However, in certain therapeutic areas poor permeability of the active compounds is an
inherent feature. Examples include ȕ-lactam antibiotics (unless they are substrates for
a transport protein) (7), peptides and proteins (8, 9). In order to deliver these drugs via
the oral route, the epithelial barrier of the intestine has to be perturbed in a safe,
reversible and reproducible manner. Although the use of so-called “absorption
enhancers” in oral drug delivery has been exploited to a very limited extent in clinical
practice, the concept still remains attractive. The new attention directed to this area in
recent years is due to the mapping of the basic extracellular components of the tight
junctions, occludin and the claudins (10, 11), and a better understanding of the
dynamic regulation of tight junction permeability (12-20). In addition, studies have
shown that bacteria regulate tight junction permeability with specific enterotoxins (21,
22).
The second part of this thesis deals with the development of a new class of tight
junction modulators based on the primary structure of the transmembrane tight
junction protein occludin.
3.1
Drug absorption from the gastro-intestinal tract
Drug absorption following oral administration is a fairly complex sequential series of
events outlined in Figure 1. The disintegration rate is governed by properties
7
associated with the pharmaceutical formulation as well as physiological factors such
as pH, gastric emptying and luminal content. The dissolution rate is dependent on the
physico-chemical characteristics of the particle and the drug, as well as on luminal pH
and components of the luminal fluids. Not until the drug is in solution may it
permeate the intestinal epithelium. However, chemical and enzymatic degradation, as
well as complex formation may limit the amount of drug in solution.
Depending on the physico-chemical properties of the drug, either the dissolution rate
or the transport rate across the intestinal epithelium may be the rate-limiting step for
drugs to enter the systemic circulation. In this thesis studies of drugs whose
absorption is permeability-limited will be reviewed. For such a review a fundamental
understanding of drug transport across the main rate-limiting barrier, the intestinal
mucosa, is required. This will be discussed in the next section.
Disintegration
Permeability
Dissolution
Liver extraction
'UXJLQV\VWHPLF
FLUFXODWLRQ
'UXJLQVROXWLRQ
Degradation/complexation
Figure 1. Schematic illustration of the sequential events during the transfer of a drug
molecule from a solid dosage form in the GI tract to the systemic circulation.
Dissolution and permeability across the epithelium are the two most important
determinants in the absorption process. Enzymatic and chemical degradation as well
as complexation in the GI tract, efflux and enzymatic degradation in the intestinal
epithelium and enzymatic degradation in the liver may decrease the fraction of drug
entering the systemic circulation.
3.2
Mechanisms of intestinal drug permeability
The intestinal mucosa can be considered as a system of sequential barriers to drug
absorption, the outermost barrier being the mucus layer and the unstirred water layer.
The gel-like structure of the mucus is thought to be a barrier to absorption of highly
lipophilic drugs and some peptides because of the restricted diffusion in this matrix
(23).
8
The absorptive epithelium lining the GI tract follows the folds and villi that increase
the anatomical surface area of the mucosa several-fold in the small intestine (24). The
villi are interspaced with crypts in which the regeneration of intestinal cells occurs. In
between the crypts and the tips of the villi are the basal parts of the villi (Fig. 2). The
properties which are relevant for drug absorption differ between the cells along the
crypt-villus axis. This will be further discussed in section 3.2.3.
Villus tip
Absorptive cells
Average pore radius <6 Å
Epithelial cell lining
Basal part of the villus
Migrating enterocytes
Average pore radius 10-15 Å
Crypts
Cell division
Average pore radius 50-60 Å
(Inaccessible to drug
absorption)
Figure 2. Schematic illustration of the crypt-villus axis showing the properties of the
different regions. The villus tip is believed to be the major site of absorption for high
permeability drugs. The paracellular pathway of this region contains small pores. The
paracellular pathway of the basal parts of the villus contains larger pores, and may
be the site of permeation for incompletely permeable drugs. The crypts are probably
not accessible for drug absorption (25).
The main purpose of the intestinal epithelium is not only to restrict access and in this
way protect the body from harmful agents, but also, to allow selective absorption of
nutrients and secretion of waste products and xenobiotics. The absorptive epithelium
consists of several types of cells including the absorptive enterocytes and mucus
secreting goblet cells, of which the enterocytes are the most abundant (26). The
transport processes across absorptive epithelia are mediated through one or several of
the following: passive transcellular, paracellular, active carrier-mediated and
transcytosis as well as carrier-mediated efflux (Fig. 3). The transcytotic pathway has
very limited capacity, and is only of relevance for the transport of small amounts of
macromolecules that are excluded from the other pathways. It will therefore not be
further discussed in this thesis.
9
1
2
3
4
Figure 3. Schematic illustration of the different transport routes that are relevant for
drug absorption. 1. passive transcellular and 2. paracellular transport, 3. carriermediated efflux, and 4. carrier-mediated active transport. The membrane proteins
responsible for efflux and transport may also be situated in the basolateral membrane
(not shown in the figure). The transcytotic pathway may also transport drugs (not
shown in the figure).
3.2.1
Passive transcellular transport
Drug transport via the passive transcellular route requires that the solute permeates the
apical cell membrane. Cell membranes are made up of phospholipids arranged in
bilayers that are intermingled with membrane proteins. The composition of
phospholipids and proteins varies from cell type to cell type and may theoretically
give rise to different permeability properties depending on the cell type. In addition,
the intestinal enterocytes have a polarized cell membrane with distinct differences in
membrane composition in the apical and the basolateral membrane (27). It is
generally believed that the apical membrane has a lower permeability than the
basolateral membrane and the former is therefore considered to be the rate-limiting
barrier to passive transcellular drug transport (28, 29). One study suggests that the
transcellular permeability to drugs is higher in the more distal parts of the intestine
than in the small intestine (30). On the other hand, the small intestinal mucosa has an
enlarged anatomical surface area due to its many folds and villi, making the small
intestine the major site for passive transcellular drug absorption. However, the
anatomical surface area of the intestinal epithelium is not necessarily analogous to an
effective absorptive surface area (31). For example, it has been calculated that the
most rapidly absorbed compound known to date, glucose, has an absorptive surface
area that covers only a small part of the total anatomical surface area of the small
intestine (32). By contrast, a low-permeability drug does not distribute rapidly into the
cell membrane and therefore has a longer residence time in the intestinal fluids.
During this time the drug will diffuse down the length of the crypt-villus axis, to
eventually be absorbed over a larger absorptive surface area.
The first step in the absorption process is the permeation of the drug across the apical
membrane. This step requires that the drug is sufficiently lipophilic. Therefore
lipophilic drugs of moderate size are normally transported by the transcellular route.
Most of the rapidly and completely absorbed drugs are absorbed by passive
10
transcellular diffusion. However, if the drug is too lipophilic, in combination with
other physico-chemical properties that favour a strong interaction with the cell
membrane, the drug can become trapped in the apical membrane, resulting in poor
bioavailability (33-35).
3.2.2
Active transport
Transport proteins embedded in the apical cell membrane actively shunt nutrients
such as peptides, amino acids and sugars across the phospholipid bilayer. In order to
restrict access of unwanted solutes via this pathway, these transporters display
substrate specificity. Therefore, in order to utilize this pathway to increase absorption,
the drug has to share some structure similarity with the normal substrate. A limited
number of drugs are substrates for uptake carrier proteins. These include some
cephalosporin antibiotics, cytostatics and angiotensin-converting enzyme (ACE)
inhibitors that are substrates for oligopeptide transporters (36). The oligopeptide
transporters have unusually broad substrate specificity (37), are abundantly expressed
in the small intestine (38) and have therefore been the deliberate target for redesigning pharmaceuticals such as antiviral drugs to make them substrates for this
transport protein (39, 40). Common to all absorption processes involving transport
proteins is that they are saturable. Drugs that are substrates for an active transport
protein can therefore display a non-linear dose-response relationship resulting in a
decreasing absorbed fraction with an increasing dose. In addition, these proteins are
transporters of nutrients, and therefore their capacity is likely to be influenced by food
intake (41). These factors may complicate the oral delivery of drugs that are absorbed
by active mechanisms.
In contrast to transport proteins acting in the absorptive direction, the active efflux
proteins secrete certain drugs that are substrates for these efflux proteins. The most
well-studied efflux proteins belong to the adenosine triphosphate (ATP)-binding
cassette (ABC) super-family of membrane transporters. These include the multi-drug
resistance 1 (MDR1; ABCB1) gene product P-gp and the multi-drug resistance
protein family (MRP; ABCC) (42, 43). More recently the breast cancer related protein
(BCRP, ABCG2) has been identified as potential contributor in actively limiting oral
bioavailability of some drugs (44, 45). The function of the efflux proteins in the
intestine may be to prevent the uptake of toxic substances and also, to eliminate such
substances from the blood (46). The substrate specificity of the most well-studied
efflux protein, P-gp, is broad and only a few structural features of the substrates have
been identified (47-49). Examples of clinical relevance include digoxin and paclitaxel
(46, 50). The substrates for the MRP family of transport proteins are more well
defined and include drugs that have undergone intracellular phase 2 conjugation to
derivatives of glucuronic acid, glutathione and sulfate, as well as some unconjugated
drugs (43).
There are numerous examples of drugs that display significant vectorial transport in
cell-based assays such as the Caco-2 model (see section 3.3.1). Whether or not these
efflux proteins in the intestinal epithelium actually affect the bioavailability of orally
administered drugs that are substrates is under debate (51). One concern, however, is
that the lipophilic drugs generated by high throughput pharmacological screening
appear to be effluxed to a greater extent by these proteins than are drugs that are
11
designed by traditional means. There is therefore a risk that drug efflux and drug-drug
interactions at the level of intestinally located membrane transporters, as well as
interpatient variability will be more pronounced problems in the future.
3.2.3
Paracellular transport
Drugs of small to moderate molecular weights (MWs) can permeate the intestinal
epithelium through the water-filled pores between the cells. This process is known as
paracellular transport, and is generally considered to be a passive process, even if this
pathway appears to be selective for cationic rather than anionic and neutral drugs (52,
53). The paracellular pathway has also been shown to be saturable (54-56), by at least
two separate mechanisms, one of which involves an intracellular process (56). These
examples illustrate the complexity and dynamics of regulation of this pathway, which
was previously considered static (see also section 3.4.1).
The drugs believed to be significantly absorbed by the paracellular route include the
ȕ-receptor antagonist atenolol (57), the H2-receptor antagonists cimetidine, ranitidine
and famotidine (54, 56) and the loop-diuretic furosemide (58). These drugs are all of
moderate MWs (around 200–270) and relatively hydrophilic. Paracellularly absorbed
drugs are usually incompletely absorbed, since the paracellular pores cover only 0.01–
0.1% of the total surface area of the intestine (59, 60) and the size-restricting gate
function of the paracellular pathway significantly decreases the permeability (18). The
paracellular permeability is dynamically regulated and varies both along the path of
the intestine and along the crypt-villus axis (61, 62) (Fig. 2). The average pore radius
of the human small intestine is 8–13 Å (63)), which will limit the paracellular
permeability of drugs >4 Å and restrict those >15 Å (64). The colon is even more
size-discriminating since the paracellular pathway restricts drugs <3.5 Å (65). In a
recent study by Fihn et al., it was shown that the average pore radius of the most
upper part of the villus of the rat small intestine contains small pores (with a radius of
<6 Å) (66), while the basal part of the villus contains larger pores (with a 10–15 Å
radius). Thus the paracellular pathway of the small intestine appears to be a dual-pore
system, and this has also been observed in the human jejunum (67).
The rate-limiting process of paracellular drug transport is therefore the passage
through the narrow pores of the tight junction complex that forms a seal at the most
apical part of the intercellular space. The composition and the regulation of tight
junctions are further discussed in section 3.4.1.
3.3
In vitro methods for studying drug permeability
For reasons of safety but also for reasons of cost, drug absorption studies in humans
are only carried out for a limited number of well-characterized drugs. Studies of drug
absorption in the intestine of the often poorly characterized drug candidates in preclinical studies have therefore traditionally been carried out in experimental animals.
However, the introduction of combinatorial chemistry and high throughput
pharmacological screening in drug discovery has significantly increased the number
of compounds entering the pre-clinical phase, and this has made it impossible to
assess the absorption properties of all these compounds in experimental animals. This
fact has spurred the development and use of in vitro methods to assess drug
12
permeability properties in most drug discovery settings. In vitro methods for drug
permeability are fairly easily adapted to automation, which further increases the
capacity of these methods and at the same time reduces the costs of these otherwise
cost and labour-intensive processes. Also, the insight that drug absorption across
biological barriers is a complex process involving several pathways that cannot easily
be delineated in experimental animals has resulted in the large interest in academic
and industrial institutions in these methods (68).
A prerequisite to the successful use of in vitro methods for drug absorption studies is
that the relationship between drug absorption in the human intestine in vivo and drug
permeability in the in vitro model must be good (but not necessarily quantitative).
Therefore, a great deal of effort has to be put into the characterization of the in vitro
method, into establishing a relationship with data obtained in humans and into
ensuring that the drug permeability in the in vitro method is predictive of the drug
permeability in vivo (1, 69, 70).
The methods range from the determination of partition coefficients into and transport
across isotropic systems to experiments using whole tissues in situ or in vivo. The
methods covered in the papers of this thesis, cultured cells and artificial membranes,
are discussed below.
3.3.1
Cultured cells
In 1990, the human adenocarcinoma cell line Caco-2 was introduced as an
experimental tool for mechanistic studies of intestinal transport (71-73) and a year
later, it was suggested that the Caco-2 model was suitable for screening intestinal drug
permeability and predicting the oral absorption potential of new drug substances (1).
The Caco-2 cells were grown on permeable supports and spontaneously formed
polarized monolayers that resembled that of the intestinal epithelium (Fig. 4).
Apical
Basolateral
Porous filter membrane
Figure 4. Schematic illustration of the chamber used for growing Caco-2 and other
cell cultures for drug transport experiments.
In many respects the Caco-2 cells are therefore functionally similar to the human
small intestinal enterocyte, despite the fact that they originate from a human
colorectal carcinoma (74-76). The methods that are based on cultured cells such as
Caco-2 cells are, however, not only useful for drug absorption screening. It is also
13
possible to extract information about specific transport processes that would be
difficult to obtain in more complex models such as those based on whole tissues from
experimental animals. For instance, the methods enable us to investigate the relative
contribution of passive transcellular and paracellular transport (58), the effect of
charge on paracellular transport (52, 53) and the effect of solvent drag (52).
Furthermore, cell-based assays have been used to investigate the effects of
pharmaceutical excipients that may enhance passive drug transport either by
enhancing the solubility of the compound e.g. (78-80) or by affecting the epithelial
integrity e.g. (81-84) (see also section 3.4.3). Cell culture models have also been used
extensively to study active transport mechanisms and more recently, the interplay
between several transporters e.g. (85, 86), and between drug metabolism and drug
transport e.g. (87, 88), and the relative contribution of passive and active transport
mechanisms to the overall transport of a drug e.g. (58).
However, extensive experience in the use of Caco-2 cells has revealed some
limitations with this in vitro model. For instance, the paracellular route in Caco-2 cells
is tighter than that in the small intestine in vivo. While the average pore radius of the
tight junctions in the human small intestine is around 8–13 Å (63), the corresponding
radius in Caco-2 cells is about 5 Å (89). The lower paracellular permeability of Caco2 cells has been attributed to the colonic origin of the cell line. Some studies suggest
that the Caco-2 cell line has a paracellular permeability that is comparable to the tips
of the villi of the small intestinal mucosa, and that other structural differences
between the cell cultures and the intestine in vivo are the cause of the apparent lower
paracellular permeability in the Caco-2 model (90, 91).
Several studies have searched alternatives to Caco-2 to circumvent the limitations
associated with this cell line. The use of primary cultures appears attractive since
these cells are not transformed. However, primary cultures are difficult to obtain,
demanding to cultivate and require constant renewal since normal intestinal epithelial
cells can only be maintained in culture for about 10 days (92). Most studies have
therefore been carried out using spontaneously transformed or immortalized cell lines.
Examples of cell cultures that have a more leaky paracellular route than Caco-2
include HT29-18-C1, a monolayer forming epithelial cell line of colonic origin (93),
and the small intestinal epithelial cell line, IEC-18 (94). Co-cultures of absorptive and
goblet cell lines with a higher paracellular permeability have been established.
However, these co-cultures express an abnormal phenotype in cell culture (95).
Another disadvantage of the Caco-2 cell line is that it requires several weeks to
stabilize paracellular permeability and expression of drug transport proteins (96). This
problem can be circumvented, at least in part, by using cultures that differentiate
faster, or by optimising the culture conditions of Caco-2 to induce faster
differentiation (to less than 1 week). Alternative short-term cultures for the studies of
passive drug transport include the MDCK (70) and the short-term Caco-2 cultures
(97, 98). However, both MDCK and short-term Caco-2 cells express varying amounts
of functional transporters such as P-gp (usually at a lower level than traditional Caco2) (97, 98). One potential limitation with the short-term Caco-2 cells in drug transport
studies may be that they can be used for only a very short time (approximately 1–2
days) since their phenotype differs from one day to another and the integrity of these
cultures decreases rapidly shortly after reaching maximum transepithelial electrical
resistance (TER) (97).
14
The expression of multiple transport systems in Caco-2 is generally considered an
advantage of this cell line. For instance since, the human origin of Caco-2 can provide
information on human transporters in the well-differentiated enterocyte. The abundant
expression of a variety of transporters in Caco-2 cells also makes it attractive to apply
functional genomics tools, such as cDNA arrays in order to map the expression (99)
and relative abundance of these transporters (44). Such studies have shown that while
the expression of some transporters may correlate with that in vivo (44), the
expression of other may differ from the in vivo situation (100). Moreover, the variety
of transport systems that are expressed in Caco-2 cells may obscure the study of a
specific transporter, especially if the transporter lacks a specific substrate, as in the
case of most efflux transporters of the ABC transporter super-family (101). When
specific membrane transporters are to be studied, simple epithelial expression systems
such as those based on the kidney epithelial cell lines MDCK and LLC-PK1 are
perhaps more suitable e.g. (102). However, differences in transport parameters for the
human ABC transporters MDR1 and MRP2 have been observed between Caco-2 cells
and the expression systems based on the MDCK cell line (103, 104). Differences
between the Caco-2 and MDCK cell lines have also been reported with regard to the
activity of peptide transporters (105). Since MDCK cells originate from the canine
kidney epithelium, it may be speculated that the observed differences are a result of
either the species difference or the different “organ origin” of the cell lines.
In summary, epithelial cell culture models are not only useful for screening absorption
properties, but a vast number of studies have also shown that these models are
indispensable in the elucidation of and interplay between the drug transport
mechanisms. Caco-2 is the most widely used cell line for drug permeability studies,
but there are alternatives that may be superior to Caco-2 for studies of specific
transport routes. However, these alternative cell cultures are usually much less well
characterized than the Caco-2 culture.
3.3.2
Artificial membranes
As stated in the previous section, epithelial cell cultures are well suited for studies and
screening of drug permeability. However, one disadvantage of cell cultures, which is
difficult to overcome, is the labour and cost-demanding maintenance of cell cultures
and facilities. Also, despite efforts to automate the cell culture assays, cells are rather
fragile for easy adaptation to automated processes. Therefore an alternative method
has been developed for high-capacity screening of permeability properties that are
based on immobilized organic solvents with or without the addition of phospholipids,
or phospholipids alone prepared as vesicles or liposomes.
Experiments using vesicles or liposomes in suspension have been fairly successful in
predicting intestinal drug permeability for diverse data sets of drugs (106, 107).
Chromatographic methods where the stationary phase consists of immobilized
phospholipids or liposomes have also been developed (108). In these methods the
retention time on the column is correlated to the epithelial permeability. These
methods are attractive for screening purposes since they require very little compound,
are easily automated and are adaptable to diverse sets of drugs. However, the
retention time of a drug does not always reflect the transport across a phospholipid
bilayer. Rather, it reflects the interaction with and partitioning into the phospholipid
15
bilayer. Methods that allow the transport across a lipid membrane are therefore an
attractive alternative.
A single phospholipid bilayer, a bilayer lipid membrane (BLM), can be formed by
carefully placing a mixture of phospholipid in an alkane solvent over a small (0.5
mm) hole in a thin sheet of Teflon (109). However, a membrane thus formed is
extremely delicate and is therefore not suitable for routine drug permeability
screening. In order to circumvent the problem of the BLM being too delicate,
permeable supports (similar to those on which epithelial cells are grown) are used to
stabilize the membrane. Successful attempts at achieving a single stabilized lipid
bilayer were reported when a mixture of egg phosphatidylcholine (PC) and
cholesterol in n-decane or n-octane was added to polycarbonate filters with welldefined pores (110). It was stated that the conditions for formation of a single bilayer
have to be carefully controlled to avoid formation of a “lipid plug” in the pores. This
method has also been adapted to high throughput screening, for which permeable
supports impregnated with a mixture of PC in an inert organic solvent were fitted in a
microtiter plate (69). Drug permeability studies in this system resulted in a
relationship, with fraction absorbed in the human intestine, much like that obtained
with Caco-2 cells. However, the data set was selected to exclude low-MW hydrophilic
drugs that might permeate the intestinal epithelium via the paracellular route. Outliers
in this study were drugs known to be actively transported in vivo. Recently this
approach has been taken further by using a lipid composition that corresponds to that
found in the apical cell membrane in vivo (111), or simplified by removing the lipid
component, thus reducing the system to consist of an organic solvent only (112).
These modified systems display relationships to fraction absorbed in humans that are
comparable to the relationship observed in the original paper, and in studies using
Caco-2 cell monolayers. There are potential problems associated with the use of
artificial membranes. For instance, in systems in which thick lipid membranes are
formed drug may be retained in the membrane and result in poor mass balance and an
under-estimation of the permeability coefficient of the drug. However, this can be
overcome by mathematical corrections (113).
3.4
Methods for modulating drug absorption
In principle there are two strategies for modulating the absorption of a poorly
permeable drug. The first involves increasing the transcellular permeability by means
of a pro-drug. The second strategy is to modulate the permeability of the tight
junctions and thus, increase paracellular permeability.
Research on the tight junctions of the intestinal epithelium has received much
attention for three major reasons. Firstly, it has been reported that changes in the
integrity of these junctions contribute to the pathology of inflammatory bowel disease
e.g. (114, 115). Secondly, some bacterial toxins are known to modulate the
permeability of the tight junctions (21, 116) and thirdly, it was found that by
perturbing the tight junctions it is possible to increase the absorption of orally
administered drugs that do not normally permeate the intestinal epithelium. Drugs that
could be given orally together with a tight junction modulator include peptides and
proteins (117-119), and ȕ-lactam antibiotics (7, 120, 121). Substances that are capable
of modulating the permeability of the tight junctions should preferably be active
16
without irreversibly compromising the intestinal integrity and function. Several
endogenous substances and hormones are known to modulate the integrity of the tight
junctions (122-125). Traditionally, however, tight junction modulators used mediate
the effect on the tight junctions via complex intracellular pathways, which may differ
from one situation to another, jeopardizing the safety of this approach e.g. (126, 127).
In order to develop safer tight junction modulators, the composition of the tight
junctions have to be understood.
3.4.1
Composition and regulation of the tight junctions
The seal connecting the most apical part of the cell membrane of two neighbouring
cells is known as the “tight junction complex” and consists of tight junctions and
adherence junctions (128) (Fig. 5). The tight junctions are believed to be the major
barrier to paracellular solute permeability, and are therefore the target of most efforts
to modulate paracellular permeability. However, studies on the modulation of the
adherence junction protein E-cadherin have shown that paracellular permeability can
be modulated by peptides targeted also to this protein (129).
The tight junction is composed of transmembrane and cytosolic proteins that interact
with each other as well as with components of the cytoskeleton. The first identified
transmembrane protein that was identified is occludin (11). This approximately 65
kDa protein has four transmembrane regions and two extracellular loops of similar
size. The cytoplasmatic C-terminus binds to tight junction associated proteins (ZO-1,
ZO-2 and ZO-3) via PDZ binding motifs (130-132), and also directly to the actin
filaments of the cytoskeleton (133). Occludin therefore provides a link between the
extracellular domains of the tight junctions and is part of the regulatory elements of
the junction. The first extracellular loop of occludin is unusually rich in glycine and
tyrosine residues, lacks charged residues (as does also the second loop) (11) and
appears to be necessary for maintaining the barrier function of the tight junctions,
since over-expression of a deletion mutant lacking the N-terminus and the first
extracellular loop has been reported to impair the properties of the tight junctions
(16). Occludin has the capacity to form tight junction filaments when transfected into
fibroblasts (which normally lack tight junctions) (134), but does not appear to be
necessary for tight junction formation since an occludin knock-out mouse has been
reported to have formed apparently normal tight junctions (135). The regulation of
function and localization of occludin are achieved by phosphorylation of serine,
threonine and tyrosine residues (135, 136). However, the phosphorylation patterns are
complicated and there is ambiguity about the effects of the phosphorylation of this
protein (14). Despite evidence that occludin is a constituent of the tight junctions and
that its function appears to be finely tuned by intracellular regulation, its precise
function remains to be elucidated.
17
Transmembrane
proteins of the tight
junctions e.g occludin
and the claudins
Tight junctions
ZO 1
ZO 2
ZO 3
Cytosolic plaque of
the tight junctions
Actin filament of
the cytoskeleton
Ca 2 +
Adherence junctions
Figure 5. Schematic illustration of the composition of the tight junction complex. The
extracellular domain of the tight junctions consists of at least three proteins, occludin,
the claudins and the junctional adhesion molecule (JAM). The cytosolic plaque of the
tight junctions consists of several PDZ and non-PDZ-containing proteins. The PDZcontaining proteins (including the ZO proteins) bind to occludin and the claudins and
link these to the actin filaments of the cytoskeleton. The non-PDZ-containing proteins
(e.g. cingulin, symplekin and the Rab proteins; not shown in the Figure) are
scaffolding proteins that are also involved in vesicular trafficking. The adherence
junctions are situated just below the tight junctions, and are also linked to the
cytoskeleton.
The second class of extracellular tight junction proteins that have been identified is
the claudin family (10). To date 24 members of the claudin family have been
identified and all members are 20–27 kDa proteins with four transmembrane domains
and two extracellular loops (137). The C-terminus contains PDZ-binding motifs to
ZO-1, ZO-2 and ZO-3 (138). When expressed in cells normally lacking tight
junctions, the individual claudins form tight junctional strands that are more well
developed than when expressing occludin (139). The claudins are therefore
considered to constitute the backbone of the tight junctional strands (140). The
individual claudins can interact with different claudins on the apposing cell, giving
rise to a wide variety of combinations (141). Variations in the tightness and ion
selectivity of various epithelia have been attributed to differential expression and
ratios of the different claudins in the various tissues. For example, claudin-2 was
characterized as a determinant of leaky epithelia after over-expression of this claudin
was seen to change the normally tight MDCK I cells to leaky cells (142).
Interestingly, this claudin is absent in the differentiated enterocyte at the tip of the
villus in the intestine, but is present in cells lining the basal parts of the villi, known to
be leakier (143).
Occludin and the claudins are not the only extracellular proteins. For instance, the
junctional adhesion molecule (JAM) was recently identified (144). The JAM has been
associated with the gate function of tight junctions (145) and also with transmigration
of monocytes and dendritic cells (144, 146).
18
The cytosolic tight junction-associated proteins can be divided into two classes, PDZ
and non-PDZ-containing proteins, and build the cytosolic plaque of the tight junctions
(137). Examples of PDZ-containing proteins include ZO-1, ZO-2 and ZO-3 (138).
The N-termini of ZO-1 and ZO-2 bind to the C-termini of occludin and the claudins,
and the C-termini bind to the actin filaments of the cytoskeleton (138). In contrast to
the other two ZO proteins, ZO-3 binds to the actin filaments by its N-terminal, and
both the N- and the C-terminal bind to occludin (131, 133, 138). All three ZO proteins
are phosphoproteins, but the effects of phosphorylation of these proteins remain
unclear (137). The cytosolic tight junction proteins that lack PDZ domains include
cingulin, symplekin and the Rab proteins (137). These proteins are involved in
vesicular trafficking and in scaffolding the transmembrane tight junction proteins to
the cytoskeleton. There are numerous other PDZ and non-PDZ-containing proteins in
the cytosolic plaque of the tight junctions. These are extensively reviewed in
Gonzalez-Mariscal et al. (137).
As mentioned above, many of the components of the tight junctions are regulated by
phosphorylation. This implies that the tight junctions are regulated by intracellular
cascades mediated by second messengers such as the intracellular concentration of
Ca2+, tyrosine kinases and protein kinase C, with an altered phosphorylation pattern of
the tight junction proteins as end-points in these cascades (137). However, it is
difficult to distinguish the individual effects on the tight junction proteins from effects
on other structures of the cell (e.g. the actin cytoskeleton). One example of a distinct
action on a tight junction protein is that histamine has a specific effect on the
phosphorylation of occludin, which leads to increased tight junction permeability
(14).
3.4.2
Physiological considerations
Knowledge of the composition and regulation of the tight junctions is, however, not
sufficient to develop a potent tight junction modulator for use in humans. Obstacles
remain, such as correlation of the initial studies normally performed in cell cultures
under controlled conditions to the far more complex and dynamic environment of the
intestine in vivo. For one, the concentrations of the modulator and drug will be
variable and difficult to predict as they are diluted in the GI fluids e.g. (147). In
addition, the protective mucus layer of the mucosa in vivo may prevent the direct
interaction necessary for the modulator to be effective and the pH and the different
components of the GI fluids such as complexing agents and enzymes may also affect
the result. Further the roles of subepthelial tissues, innervation and local blood flow
cannot be predicted in cell cultures.
Also, one of the major concerns in the use of tight junction modulators in
pharmaceutical products is that of toxicity. Usually acute toxicity, and damage to
epithelial cells are measured and correlated to the effect of the modulator. A toxicity
index (defined as the concentration that causes toxicity divided by the concentration
that results in the desired tight junction modulation) can be used to define the efficacy
of the modulator. Normally the toxicity index is around 2–3 in vitro (148). However,
studies of in vitro acute toxicity may over-predict the toxicity of the modulator in the
intestine in vivo. The reason for this is that the intestinal mucosa is under constant
renewal every 3–4 days. More serious are potential systemic toxicity caused by
19
systemic absorption of the modulator, autoimmune responses and inflammatory
reactions.
A third potential problem is that the effect of the modulator has to be controlled in
such a way that the unwanted absorption of potentially harmful agents such as
bacteria and toxins can be avoided. Therefore, the modulator has to be presented to
the mucosa in close proximity to the drug to ensure that the drug is selectively
absorbed and that the effect of the modulator is controlled and transient.
3.4.3
Methods for modulating paracellular transport
A large number of compounds have been identified that modulate tight junction
permeability. The traditional ones include Ca2+ chelators, surfactants and cationic
polymers. Most of these modulators have been studied in cell cultures such as Caco-2
cells, in order to elucidate their efficacies and mechanisms of action (148). Few
studies have been carried out in vivo and even fewer modulators are used clinically
e.g. (121).
Calcium ion chelators (e.g. ethylenediaminetetraacetic acid (EDTA)) were initially
thought to mediate their effect by reducing extracellular Ca2+ levels and thus,
affecting the Ca2+-dependent proteins of the tight junction complex. However, binding
of extracellular Ca2+ also affect the intracellular levels of Ca2+, which will result in a
series of intracellular events, such as activation of protein kinases and subsequent
altered protein phosphorylation of the tight and adherence junctional proteins (149,
150). Therefore, Ca2+ chelators are believed to have multiple mechanisms of action on
the tight junctions.
There are different classes of both natural and synthetic surfactants that are known to
modulate tight junction permeability, including bile salts, anionic surfactants,
medium-chain fatty acids and phosphate esters (148). Surfactants are known to
interact with the cell membrane either as solubilizing agents or as agents that extract
proteins and lipids from the membrane (119, 151). It is therefore likely that since
surfactants are known to increase epithelial permeability, they do this by means of
membrane perturbation. However, some surfactants also have distinct intracellular
mechanisms of action that result in increased permeability of the tight junctions. For
instance, the medium-chain fatty acid sodium caprate increases intracellular Ca2+
concentration, causes ATP depletion and triggers a phospholipase C (PLC)-mediated
pathway (127). Interestingly, hexadecylphosphocholine inhibits PLC-ȕ with a
concomitant increase in paracellular permeability (13). These two seemingly
contradictory findings illustrate the complexity of tight junction modulation.
Chitosan is an example of a cationic polymer that has been shown to increase
paracellular permeability by modulating the tight junctions. However, the efficiency
of chitosan is dependent on the degree of acetylation and consequently on charge
density (152). Therefore it appears that the positive charges of the polymer are
directly involved in mediating the mechanism of action of this polymer. Chitosan
requires direct contact with the epithelial cells in order to be effective. This fact limits
the clinical use of this modulator in the intestine, since the mucus layer has been
shown to reduce or abolish the efficiency of chitosan in vivo and in vitro in studies
using mucus-producing cell lines (153). Another problem is that the solubility of
20
chitosan is low at physiologic pH since the amines of chitosan are un-ionized at a pH
of around 7. However, this can be overcome by methylation of the amine groups to
form permanently charged quaternary amines (83).
Recently the use of bacterial toxins as potential tight junction modulators has been
investigated. For instance, the Vibrio cholerae enterotoxin zonula occludens toxin
(ZOT) has been shown to increase paracellular permeability by interacting with a
specific intestinal epithelial surface receptor leading to activation of the second
messenger proteinkinase C, with subsequent activation of a complex intracellular
cascade of events which leads to rearrangement of the actin filaments and subsequent
loosening of the tight junctions (116, 154). The ZOT has also been shown to increase
the absorption of insulin after oral administration to rabbits (118). Interestingly, an
endogenous analogue to ZOT, zonulin, was recently identified. Zonulin interacts with
the same surface receptor and has some structural features in common with ZOT. This
finding suggests that intestinal epithelium regulates its paracellular permeability
through specific mechanisms, for example in response to bacterial infections (155).
An alternative strategy to modulating the diffusion barrier of the tight junctions may
be to directly target the tight junction modulator to the extracellular proteins of the
tight junctions. It can be speculated that such direct intervention would reduce the
acute toxicity of the modulator, since it does not involve an intracellular mechanism.
Some bacterial toxins have as primary targets the extracellular domains of tight
junction proteins. For instance, the C-terminus of Clostridium perfringens enterotoxin
(CPE), which binds to the tight junction proteins claudin 3 and 4, causes an increase
in paracellular permeability (21). It has also been shown that synthetic peptides can
perturb the tight junction barrier. A homologous large peptide corresponding to the
second extracellular loop of occludin has been shown to perturb the paracellular
barrier function (156) and peptides corresponding to the first loop of occludin have
been reported to inhibit tight junction re-sealing following a Ca 2+ switch (157).
Despite the potential advantages of extracellular targeting, several obstacles remain to
be addressed. One is the fact that peptides targeted to the extracellular domains of
tight and adherence junctions appear to be active only from the basolateral side (21,
129). Whether this is an effect of easier accessibility to the junctions from the
basolateral side remains unclear. Another problem is that synthetic peptides targeted
to the tight junctions have a very slow onset of action (hours). One way of solving
these problems may be to identify active analogues (not necessarily peptides) that can
access the tight junctions from the apical side and that have a more rapid onset of
action.
In conclusion, although tight junction modulators are used in clinical practice in rare
cases, their mechanisms are usually non-specific and poorly understood. Thus there is
a need for new approaches to tight junction modulation.
21
4
AIMS OF THE THESIS
The overall aims of this thesis were to develop and evaluate a new cell culture model
that mimics the permeability of the small intestine and to develop a new principle for
tight junction regulation. Specific aims were to:
x
develop a new cell culture model that better mimics the permeability of the
human small intestine for studies of passive drug transport, based on the
intestinal epithelial cell line 2/4/A1 which is conditionally immortalized with a
temperature-sensitive mutant of the growth-promoting oncogene SV40 large T
(Paper I);
x
improve the viability of the 2/4/A1 cell culture model and investigate different
routes of drug transport in this cell line (Paper II);
x
characterize the paracellular route of 2/4/A1 monolayers and compare the
permeabilities of incompletely absorbed oral drugs in 2/4/A1 with those in
Caco-2 monolayers (Paper III);
x
investigate the contribution of the paracellular route to the pH-dependent
permeability of cationic drugs in 2/4/A1 cell monolayers (Paper IV); and
x
investigate whether peptides from the extracellular loops of the tight junction
protein occludin may be used as a new principle for tight junction modulation
(Paper V).
22
5
MATERIAL AND METHODS
5.1
Drugs and radiolabelled markers
Acyclovir, Alprenolol-HCl, atenolol, cimetidine, digoxin, D-raffinose,
fluorescein-conjugated dextran (MW 50,000), glycylsarcosine, lactulose, mannitol,
metolazone, metoprolol-tartrate, mitoxantrone, Na-fluorescein, phenazone,
phosphonoformic acid (foscarnet), pindolol, propranolol-HCl, sulfasalazine, sulpiride,
terbutaline, verapamil and vinblastine were purchased from Sigma (St. Louis, MO).
BMS 189664, BMS 187745, didanosine, metformin, nadolol, pravastatin, and
lobucavir were obtained from Bristol Myers Squibb (Princeton, NJ). Alfentanil was
obtained as Rapifen IV solution from Janssen-Cilag through Apoteket AB
(Stockholm, Sweden). Lucifer yellow was purchased from Molecular Probes (Eugene,
OR). Olsalazine was a gift from Dr. Wenche Rolfsen, Pharmacia & Upjohn (Uppsala,
Sweden). [14C]-Acyclovir, [14C]-ganciclovir, [3H]-glycylsarcosine, [3H]-mitoxantrone,
>14C@-phosphonoformic acid and [3H]-vinblastine were obtained from Moravek
Biochemicals (Brea, CA). >14C@-Creatinine, >14C@-mannitol, >3H@-raffinose, [3H]testosterone and >3H@-vasopressin were obtained from New England Nuclear (Boston,
MA). [3H]-Digoxin and [3H]-lactulose was obtained from American Radiolabeled
Chemicals Inc. (St. Louis, MO). [14C]-Clodronate and unlabeled clodronate (Leiras
Co., Turku, Finland) were gifts from Dr. J. Mönkkönen (University of Kuopio,
Finland). >14C@-PEG (MW 4,000) was obtained from Amersham Biosciences
(Uppsala, Sweden). GF12918 was a gift from GlaxoWellcome (Hertfordshire, UK).
5.2
Occludin peptides
Peptide synthesis was accomplished as described previously (158). The crude
peptides were purified and lyophilized to a final purity of >95%. The purified
peptides were stored in a dessicator at 4˚C. Mass spectra were recorded on a Fisons
VG-TOF spectrometer using matrix assisted laser desorption ionisation time-of-flight
(MALDI-TOF) mass spectrometry, a VG Analytical ZAB-SE instrument using fast
atom bombardment (FAB) ionisation, or a Finnigan MassLab Navigator quadrupole
mass spectrometer using electrospray (ES) ionisation. The stability of the peptides (20
µg/ml) during incubation with Caco-2 cell monolayers was examined for up to 360
minutes. At 60, 120, 180, 240 and 360 minutes, samples (20 Pl) were removed from
the apical and/or basolateral solutions. The samples were analyzed by HPLC.
5.3
5.3.1
Cell culture
2/4/A1
In paper I, the 2/4/A1 cells were cultured at 33°C in RPMI 1640 medium
supplemented with 2% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, 200
Pg/ml geneticin, 1 mg/ml bovine serum albumin, 1 Pg/ml water soluble
dexamethasone, equivalent to 65 ng/ml dexamethasone, 20 ng/ml epidermal growth
factor, 50 ng/ml insulin-like growth factor I and ITS premixTM containing 10 Pg/ml
insulin, 10 Pg/ml transferrin and 10 ng/ml selenic acid. The cells were expanded in 75
cm2 plastic cell culture flasks at 33°C, 5% CO2 and 95% humidity. The medium was
changed every second day and the cells were passaged by trypsinisation at 80%
23
confluence, i.e. approximately every fourth day. Cells cultivated at passage number
30 through 43 were used. For drug transport experiments using 2/4/A1 cells,
permeable supports, 0.45 Pm pore size, 12 mm in diameter, were coated with ECL.
2/4/A1 cells were thereafter seeded at 100,000/cm2 on the ECL coated permeable
filter supports. In paper II – IV, 2/4/A1 cells were expanded as described for in paper
I, with a few changes to the culture medium. The selection marker G418 was
removed, the dexamethasone addition was reduced to 0.5 µg/ml, equivalent to 33.5
ng/ml and the culture medium was supplemented with 4% FCS. For functional
studies, the 2/4/A1 cells were grown in RPMI 1640 supplemented with growth
factors, 6% fetal calf serum (FCS) with or without G418 on permeable supports at
100,000 cells/cm2 coated with EHS matrix.
5.3.2
Caco-2
Caco-2 cells originating from a human colorectal carcinoma were obtained from
American Type Culture Collection (Rockville, MD) and cultivated as described in
detail previously (96). In brief, Caco-2 cells were seeded at a density of 440,000/cm2
on uncoated permeable polycarbonate filters and cultivated for 21 days. Cells at
passage number 95 through 105 were used.
5.4
Hexadecane membranes
The HDM was prepare essentially as described by Whonsland et al (112), with a few
modifications. 71.5 ml 5 v/v% hexadecane in hexane was added to polycarbonate
filter inserts (Transwell Costar, Badhoevedorp, the Netherlands; mean pore size 0.4
mm or 3.0 mm; diameter 12 mm) (total amount of hexadecane: 3.6 ml/insert). The
impregnated inserts where left at room temperature in a fume hood for at least one
hour prior to drug transport studies to ensure complete evaporation of the hexane.
5.5
5.5.1
Microscopy
Immunofluorescence
Cells cultivated permeable supports were washed with PBS and fixed with 3%
paraformaldehyde. The cells were then permeabilized using 0.1% Triton X-100. The
F-actin distribution was visualized using direct fluorescence with
rhodamine-conjugated phalloidin. Tight and adherence junction protein distributions
were studied by indirect immunofluorescence using rabbit polyclonal antibody to
occludin or ZO-1 and a mouse monoclonal Ig antibody to human E-cadherin
respectively. The SV40 large T antigen was stained with mouse monoclonal Ig SV40
large T antigen antibody. The cells were incubated with the primary antibodies,
washed with PBS and incubated with secondary FITC-labeled antibodies. The
samples were washed, mounted and examined under a confocal laser scanning
microscope.
24
5.5.2
Electron microscopy
2/4/A1 and Caco-2 cells cultivated on permeable supports as described above were
fixed in 2.5% glutaraldehyde, then immersed in 1% osmium tetroxide and dehydrated
with ethanol. Thin sections were stained with uranyl acetate plus lead citrate and
examined by transmission electron microscopy.
5.6
Establishment of 2/4/A1-Bcl-2 clones
2/4/A1 cells at 80% confluence were trypsinized and pelleted, harvested by
centrifugation and washed. Two plasmids were added to each fraction; human Bcl-2
cDNA containing plasmid (159) and hygromycin B resistance plasmid (P3’SS).
Electroporation was performed. After electroporation the cells were plated onto
culture dishes and left overnight. Twenty-four hours after transfection, 200 U/ml
hygromycin B was added to the conditioned medium. Clones were immediately
isolated with cloning cylinders when they appeared. The clones were harvested by
local trypsinization followed by successive expansion.
For the characterization of Bcl-2 clones, cells were harvested, washed and used for
the preparation of RNA. RNA was quantified using UV absorption at 260/280 nm,
and RNA integrity was checked by assessing the sharpness of ribosomal RNA bands
on a native 1% agarose gel. Bcl-2 clones were investigated for Bcl-2 expression using
Northern blot analysis, with a 32P-labelled full-length (1.9 Kbp) Bcl-2 cDNA-probe,
and using a human Bcl-2 ELISA kit.
5.7
RT- PCR
For the characterization of transport and efflux systems, approximately 7 u 106 filtergrown 2/4/A1 cells were harvested using ice-cold phosphate-buffered saline solution
and a cell scraper. Cells were kept on ice during the scraping procedure and
subsequently recovered by centrifugation. 2/4/A1 cells and the rat ileal tissue (positive
control) were homogenized, the RNA was extracted and checked for absence of
contaminating genomic DNA as previously described (44). Also, PCR primer-design
and reaction conditions were as previously described (44).
5.8
Cell attachment
For the cell attachment assay, 2/4/A1 cells were seeded on plastic chamber slides
coated with extracellular matrix proteins and were further studied after 24 hours
incubation. The plastic chamber slides were coated with collagen I, fibronectin,
mouse laminin, or a mixture of entactin, collagen IV and laminin (ECL).
5.9
Analysis of toxicity and cell death
5.9.1 DNA staining
2/4/A1 cells were seeded on plastic chamber slides coated with ECL. After 24 hours,
the medium containing non-attached cells was replaced with fresh medium. After four
25
days in culture, the cells were washed, fixed and permeabilized. The cells were
stained with propidium iodide. The slides were examined under a fluorescence
microscope. Cells containing highly condensed chromatin and irregular nuclear DNA
were defined as dying (160). Cells with homogeneous DNA staining throughout a
regularly shaped nucleus were considered normal.
5.9.2
Dehydrogenase activity
In paper II, the effect of G418 on cell viability was assessed by measuring the
intracellular dehydrogenase activity using the MTT method (161) as described
previously (162). 2/4/A1 cells were seeded in the presence of different concentrations
of G418 in the culture medium and incubated at 39˚C for 48 hours before carrying out
the MTT assay.
In paper V, The effect of the peptides on cell viability was assessed by measuring the
intracellular dehydrogenase activity of Caco-2 cells using the MTT method as
described above. Caco-2 cells were seeded in the presence of different concentrations
of occludin peptide and incubated for 6 hours before carrying out the MTT assay.
5.9.3
Lactate dehydrogenase leakage
The effect of the peptides on membrane integrity was assessed by measuring the
amount of lactate dehydrogenase (LDH) leakage. Caco-2 cell monolayers were
incubated with peptide for 6 hours. LDH leakage into the apical and basolateral
solutions was determined using an LDH release kit.
5.10 Cell monolayer integrity and drug transport studies
5.10.1 Electrophysiological measurements
The transepithelial electrical resistance (TER) of 2/4/A1 and Caco-2 cell monolayers
grown on permeable supports was measured with an Endohm“ tissue resistance
measurement chamber connected to an Evohm resistance meter. The monolayers were
equilibrated for 20-25 minutes before TER measurements.
5.10.2 Drug permeability studies
All experiments were carried out at 37°C in HBSS as described previously (96). The
solutions were preheated to 37°C and the experiments were performed at 37r1°C on a
heating plate. The cell monolayers were washed with preheated HBSS and
equilibrated in the same buffer for 20-25 minutes prior to the transport experiments.
The filters were then transferred to wells containing fresh preheated HBSS. The
compounds were generally dissolved in HBSS 25 mM HEPES at pH 7.4 to a final
concentration of 0.1-5 mM. In paper IV, MES 10 mM (pH 5.0 – 6.5) and HEPES 25
mM (pH 7 – 8.0) was used to control the apical pH. The amount of each particular
compound used was based on its solubility, detection limit, presence of saturable
active transport mechanisms, and effects of the compound on monolayer integrity.
26
The drug solution was added to the donor side of the cell monolayers and the inserts
were placed on a calibrated shaker and kept at 37°C throughout the experiment. At
4 – 6 regular time intervals, samples were taken from the receiver solution and
replaced with an equal amount of HBSS. Radioactive samples were analyzed using a
liquid scintillation counter and the unlabeled samples were analyzed using reversedphase HPLC.
5.11 Calculations
In paper I-III and V, the apparent permeability coefficient (Papp, cm/s) was determined
according to the following equation:
Papp
K ˜ Vr
A
1
where K is the steady state rate of change in concentration in the receiver chamber
(Ct/C0) versus time (s), Ct is the concentration in the receiver compartment at the end
of each time interval, C0 is the initial concentration in the apical chamber at each time
interval (mole/ml), Vr is the volume of the receiver chamber (ml) and A is the surface
area of the filter membrane (cm2).
In paper IV, the apparent permeability coefficients (Papp, cm/s) were calculated using
the non-sink condition analysis that is general and imposes less restriction on the
experimental conditions than the analysis assuming the previously used sink
conditions (77, 163).
CR(t) = [M/(VD + VR)] + [CR,0 - M/(VD + VR)] × e-Papp × A × (1/VD + 1/VR) × t 2
where VD is the volume in the donor compartment, CR,0 is the drug concentration in
the receiver compartment at beginning of the interval and t is the time from the start
of the interval. The sampling procedure necessitates the recalculation of M and CR,0
for each succeeding interval according to mass balance. Papp for model drugs were
then determined by nonlinear regression, minimizing the sum of squared residuals
(™(CRi,obs – CRi,calc)2), where CRi,obs is the observed receiver concentration at the end of
interval i and CR,i,calc is the corresponding concentration calculated according to eq. 1.
Subsequently, the membrane permeability coefficients (Pm) of the drugs were
calculated by determining Papp at two different stirring rates (V, 100 rpm and 500
rpm). Pm was calculated from the slope of the linear relationship V/Papp and V as
described previously (96).
V
Papp
1 ·
1 § 1
˜V
©
P c P f ¹
K
27
3
where Pf is the calculated permeability coefficient of the filter support and K is a
constant.
The sigmoidal equation:
FA =
100
γ
  P 
c

1 + 
  Pc 50  
4
was fitted to the drug transport data. FA is the fraction of a drug absorbed in humans
after oral administration, Pc50 is the Pc at an FA value of 50% and γ is a slope factor.
The equation was fitted to the data by minimizing the unweighted sum of square
residuals.
The molecular hydrodynamic radii of the marker molecules were estimated from the
cubic root of the molecular weight of each compound multiplied by a constant K,
which was given by the ratio between the radius of mannitol (3.6 Å) and the cubic
root of the molecular weight of mannitol (MW 182).
r = K ⋅ 3 MW
5
This approximation of molecular radii assumes that all molecules have the same
hydrodynamic shape in HBSS at pH 7.4.
The average pore radii of the tight junctions in 2/4/A1 and Caco-2 cells were
calculated using the pore-restricted diffusion equation, also named the “Renkin
function”. This equation describes a mathematical model of the diffusion of a solute
in a cylinder (89, 164):
Pp =
ε
⋅ D ⋅ F ( r / R)
δ
6
where Pp is the paracellular permeability of the solute, ε is the porosity of the cell
monolayer, δ is the tortuousity times the path length, D is the diffusion coefficient and
F(r/R) is the Renkin equation where r is the radius of the solute and R is the radius of
the cylinder:
  r 
F (r / R ) = 1 −   
  R 
2
3
5

r
r
r 
⋅ 1 − 2.104 ⋅   + 2.09 ⋅   − 0.95 ⋅   

 R
 R  
 R

ε/δ and R are constant for any given cell monolayer. It is thus possible to get an
estimate of these constants by measuring the permeability for small hydrophilic
molecules and fitting the data to Equation 6.
28
7
5.12 Statistics
The results were expressed as mean values r SD. One-way ANOVA was used to
compare means. A 95% probability was considered significant. The root mean square
error (RMSE), i.e. the standard error of regression, and the coefficient of
determination (r2) were used to measure how well the sigmoidal equation fits the
observations.
Rank correlation coefficients, a measure of the association between two separate
rankings of n items, were obtained using Spearman’s rank coefficient according to:
rs
1
6˜ ¦d2
( n 3 n)
8
where d is the difference of the two ranks associated with each compound and n is the
number of compounds. The value rs is always between -1 and +1. Large rs values
indicate greater association. Small rs values indicate less association.
29
6
RESULTS AND DISCUSSION
6.1
Characterization of 2/4/A1 (Papers I–III)
The basic steps in establishing and characterizing an epithelial cell line as a cell model
for drug transport studies include studies of cell attachment on various supports, cell
proliferation, differentiation, and monolayer integrity and function. The obvious
prerequisite is that the cells form intact monolayers on permeable supports. In some
cases the cell line (e.g. Caco-2) secretes its own extracellular matrix, but other cell
lines (e.g. 2/4/A1, see section 6.1.2) require that an extracellular matrix is added to the
permeable support to promote cell attachment. The cell proliferation rate is an
important factor for practical reasons. The population doubling time of the Caco-2
and 2/4/A1 cell lines are 30-35 h. These growth rates enable the necessary expansion
of the cultures within a reasonable time frame. An example of a cell population
doubling taking too long is that of tsFHI cells, the human analogue to 2/4/A1, which
have a population doubling time of approximately 7 days (165). This makes this
otherwise promising cell line unsuitable for routine drug permeability studies. In this
thesis, the development of a cell culture model based on the tsFHI cells was initially
considered, in order to establish a human counterpart to 2/4/A1. However, this idea
had to be abandoned, due to the slow growth of the tsFHI cells. The integrity of the
cell monolayers can be measured by TER. From a drug delivery point of view, a more
preferable way of measuring the integrity is testing the permeability to various
paracellular markers. For tight epithelial cell monolayers (e.g. Caco-2), the
permeability to a low MW paracellular marker such as mannitol is an adequate
measure of the integrity. For leaky epithelial cell lines such as 2/4/A1, permeability to
a series of paracellular markers that span a range of MWs has to be studied at least
initially, to ensure the size-discriminating capacity of the cell monolayer. Microscopic
evaluation of selected epithelial markers reveals the differentiation pattern of the cell
line.
The properties of the foetal rat intestinal cell line 2/4/A1 depend on culture
temperature, since this cell line was conditionally immortalized by tsSV40T (166).
Therefore the first part of the characterization was to study the temperature-dependent
expression of this oncogene. Since the temperature dependent differential expression
of SV40T influence many properties of 2/4/A1, the rest of the characterization was
also carried out at 33ºC (where the oncogene is fully active), 37ºC (where the
oncogene seems to be partly active) and 39ºC (where the oncogene is inactive).
6.1.1
Expression of SV40 large T antigen (Paper I)
The temperature-dependent expression of SV40T was studied by using an antibody
raised against SV40T. Immunofluorescence microscopy showed that the SV40 large
T antigen was expressed in an immunoreactive form to a varying degree in all cells
(Fig. 6), and that the antigen was relatively evenly distributed in all cell nuclei at
33°C. At 37°C all cells still showed expression of SV40 large T but the staining was
fainter, indicating a lower degree of expression or, more likely, immunoreactivity. At
39°C only some fragments of the nuclei were stained in an irregular pattern. These
results indicate a gradual loss of immunoreactivity of the mutated SV40 large T
30
antigen with an increase in temperature, which is consistent with the reported
temperature sensitivity of this antigen (167).
Figure 6. Immunofluorescence staining of temperature-sensitive SV40 large T antigen
in 2/4/A1 cells after four days of culture at 33°C (A), 37°C (B) and 39°C (C) on ECL.
The immunostaining of the antigen decreased with increasing temperature, consistent
with the temperature sensitivity of the antigen. Bar=10 Pm
6.1.2
Cell attachment and extracellular matrix (Paper I)
When grown at 33°C, 2/4/A1 cells attached readily to cell culture-treated plastics.
When the culture temperature was increased to either 37°C or 39°C, the cells also
attached initially, but after approximately 2-3 days at 37°C and after 24 hours at 39°C
they started to detach. One explanation for this phenomenon may be anoikis, a form
of apoptosis induced by lack of proper cell attachment (168). In order to promote
better cell attachment, a number of commercially available extracellular matrices
were tested. At all temperatures significantly higher numbers of 2/4/A1 cells attached
to fibronectin, laminin and ECL-coated supports than to the uncoated supports or to
supports coated with collagen I and IV. Since preliminary results indicated that
fibronectin reduced the survival of 2/4/A1 cells and since cells grown on laminin
formed diffuse cell borders (data not shown) the ECL-coated supports were selected
for all further studies (Fig. 7).
Figure 7. Attachment of 2/4/A1 cells to plastic (P), collagen I (C I), collagen IV (C
IV), fibronectin (F), laminin (L) and ECL at 33°C (A), 37°C (B) and 39°C (C). At all
temperatures attachment rates were better to fibronectin, laminin and ECL than to
plastic or the collagens. *p<0.05, **p<0.01 vs plastic (calculations by one-way
analysis of variance (ANOVA)). Each bar represents the mean ± standard deviation
(SD) of four experiments.
31
6.1.3
Monolayer formation (Papers I and II)
Morphological analysis using light microscopy showed that the 2/4/A1 cells formed
continuous multi-layers after 4 days of culture grown on ECL-coated supports at
33°C. At 37°C the cells formed monolayers within 24 hours and remained as intact
monolayers for at least 10 days in culture. When seeded onto ECL at 39°C, 2/4/A1
cells initially formed monolayers comparable to those observed at 37°C. However,
during the first 24 hours the monolayers of these cells developed defects and within
the following 24 hours in culture, cell detachment was observed. The cell detachment
increased over the next 4 days in culture, after which <50% of the surface area of the
support was covered with cells.
Examination of the cell nuclei after propidium iodide staining showed that at 33°C the
2/4/A1 cells grew and divided at a high cell density, which is consistent with the
formation of multi-layers observed in the light microscope. The cell nuclei of 2/4/A1
cells grown at 37°C were more evenly distributed, supporting the finding that the cells
form monolayers at this temperature. The cell nuclei had a normal oval morphology at
both 33° and 37°C. In contrast, many of the cells grown at 39°C displayed condensed
and fragmented nuclear DNA, indicating that many cells died at this temperature (Cell
death is further discussed in section 6.1.7).
6.1.4 Development of tight and adherence junctions (Papers I and III)
When the original cell culture procedure was used (Paper I), the tight junction protein
ZO-1 was present in 2/4/A1 cells grown at all three temperatures (Fig. 8A-C). Its
distribution was less well ordered in cells grown at 33°C than in cells grown at 37°C.
At 37°C, ZO-1 formed a continuous network. By contrast, after cultivation of 2/4/A1
cells at 39°C the ZO-1 network became discontinuous, indicating that the cell-to-cell
contacts had loosened. Similarly the protein E-cadherin, which is associated with
adherence junctions, was present at all temperatures (Fig. 8D-F). E-cadherin formed a
diffusely distributed dot-like network at 33°C. At 37°C the dot-like network was more
marked and was located closer to the apical cell membrane and the cell junctions. At
39°C the E-cadherin network at the cell junctions was not as distinct as that observed
at 37°C. Actin filaments (Fig. 8G-I) were found at the cell borders, but also appeared
as stress fibres at 33°C. At 37°C actin had redistributed to the peri-junctional area of
the terminal web. At 39°C the actin network showed a broadening of extracellular
spaces and defects in the monolayer. These findings indicate that 2/4/A1 cells grown
at 37°C form monolayers consisting of polarized epithelial cells with typical
junctional complexes, containing ZO-1 at tight junctions and E-cadherin at areas of
cell-cell contact.
32
Figure 8. Immunofluorescence staining of ZO-l (A, B, C), E-cadherin (D, E, F) and
F-actin (G, H, I) in 2/4/A1 cells four days after seeding on ECL at 33°C, 37°C and
39°C as described in Paper I. The ZO-1 distribution was visualized using an rabbit
polyclonal antibody to ZO-1 protein, E-cadherin was visualized using a mouse
monoclonal Ig antibody to human E-cadherin and the F-actin distribution was
visualized using direct fluorescence with rhodamine-conjugated phalloidin. In
contrast to the cells grown at 33°C and 39°C, cells at 37°C had a continuous network
of tight and adherence junction-associated proteins. Images were obtained by
confocal laser scanning microscopy. Bar=10 Pm.
When the modified cell culture procedure was used (Paper II), the tight junction
protein occludin was expressed at the transcriptional level in equal amounts in both
undifferentiated 2/4/A1 cells (grown at 33°C) and differentiated 2/4/A1 cells (grown
at 39°C). By contrast, the expression of the claudins depended on the differentiation
level of the 2/4/A1 cells. Claudin 1 was more abundantly expressed in differentiated
2/4/A1 cells grown at 39°C, while claudin 3 was more abundantly expressed in
undifferentiated 2/4/A1 cells grown at 33°C.
Transmission electron microscopy confirmed the findings of the light and
fluorescence microscopy. At 33°C, 2/4/A1 cells grew as multi-layers two to four cells
thick, while at 37°C monolayers of cuboidal cells connected by tight junctions were
observed (Fig. 9). Cells grown at 39°C showed the morphology of dying cells with
irregular and shrunken cell nuclei and defects in the cell monolayers.
33
Figure 9. Transmission electron micrographs of tight junctions and the intercellular
spaces of 2/4/A1 (A) and Caco-2 cells (B). 2/4/A1 and Caco-2 cells were cultivated on
permeable supports at 37°C for 4 and 21 days respectively as described in paper I.
2/4/A1 cells form tight junctions (arrow) and relatively straight lateral spaces at 37°C
but display few microvilli at the apical surface (A). Caco-2 cells form tight junctions
(arrow) with tortuous lateral spaces and display a more differentiated morphology
with numerous microvilli. Magnification 32,000u (A) and 11,000u (B).
In summary, these data show that 2/4/A1 cell monolayers express several components
of functional tight junctions. However, the expression of perhaps the most important
class of tight junction proteins, the claudins, indicates that 2/4/A1 cells preferentially
express claudin 1 while the other investigated claudins are expressed to a lesser extent
or not at all. It is difficult to speculate what may be the consequences of this, but
recent data suggest that the expression of various claudins gives rise to special tissuespecific tightness and selectivity of the tight junctions (169).
6.1.5
Transepithelial electrical resistance (Papers I and II)
In Paper I the studies of the temperature-dependent differentiation showed that 2/4/A1
cells grown on ECL formed monolayers of viable cells only at the intermediate
temperature of 37°C. Therefore, 2/4/A1 cells grown at this temperature were selected
for further functional studies on the integrity and permeability of the cell monolayers.
The TER in 2/4/A1 cells increased with time and reached a plateau value of 25 :˜cm2
after 4 days in culture (Fig. 10A). The TER was maintained for at least 12 days. This
TER value may seem low but lies within the range of TER values obtained in
segments of the rat jejunum (30). By comparison, the TER in Caco-2 cell monolayers
reached a plateau of 234 :˜cm2 and maintained this value for at least 40 days in
culture (Fig. 10B), which is in agreement with previous findings (72). The TER of
confluent 2/4/A1 monolayers was therefore approximately ninefold lower than that of
Caco-2 monolayers. The fact that the TER values of both 2/4/A1 and Caco-2 reach
stable plateaus after 4 and 14 days, respectively, is an advantage, from a practical
viewpoint, over short-term Caco-2 cultures in which the integrity is lost only a few
days after reaching maximum TER. This plateau allows flexibility in planning of drug
transport experiments.
34
C
TER (: x cm2)
60
*
50
40
*
30
20
10
0
37°C 39°C 39°C
-G418 -G418
+FCS
Figure 10. Time-dependent development of transepithelial electrical resistance (TER)
of 2/4/A1 (A) and Caco-2 (B) cell monolayers. The TER of 2/4/A1 monolayers
increased with time and reached a plateau of 25 :˜cm2 after 4 days in culture. This
TER was maintained for at least 12 days. The TER in Caco-2 cell monolayers grown
on uncoated permeable supports (B) reached a plateau of 234 :˜cm2 after 17 days
and maintained this value for at least 40 days in culture. (C) In paper II the TER in
2/4/A1 cell monolayers cultivated under serum-free conditions without G418 at 37˚C
and at 39˚C reached approximately 27 and 37 :˜cm2 after 6 days, respectively.
2/4/A1 cell monolayers cultivated at 39˚C in the presence of 6% FCS without G418
reached approximately 50 :˜cm2 after 6 days and maintained this value for at least
another 8 days in culture (data not shown). Measurements were performed in HBSS
at 37°C. Each point or bar represents mean ± SD of four experiments.
In Paper II the culture conditions for 2/4/A1 were improved by removing the selection
marker (G418) for the SV40T, which had turned out to be toxic to the 2/4/A1 cells at
39°C, and 6% foetal calf serum (FCS) was introduced to the culture medium (see
6.1.7). These changes allowed culture of 2/4/A1 cells also at 39°C. The TER of the
optimised cell cultures grown at 39°C was significantly higher than that of cultures
grown at 37°C (p<0.05), reaching plateau values of 50 :˜cm2 (Fig. 10C). The
increased TER obtained at 39°C was associated with the shift in expression pattern of
the claudins, including an up-regulation of claudin 1 and a down-regulation of claudin
3.
6.1.6
Expression of drug transport proteins (Paper II)
In Paper II the temperature-dependent expression of membrane transporters was
carried out at 33°C and 39°C using reversed-transcriptase polymerase chain reaction
(RT-PCR) to detect mRNA coding for the transport proteins. Special attention was
paid to one of the membrane transporters of relevance for drug absorption, the
oligopeptide transporter PepT1. However, only very faint smeared bands of mRNA
corresponding to PepT1 were observed for the 2/4/A1 cells. The reason for the lack of
PepT1 expression is unknown, but one possible explanation is that epidermal growth
factor (EGF), which is a constituent of the culture medium, has been shown to down-
35
regulate the expression of PepT1 (170). However, EGF could not be removed from
the culture medium without causing loss of integrity.
In contrast to the expression of PepT1, both genes that code for P-gp in rodents,
MDR1a and MDR1b, were expressed in 2/4/A1. Neither MRP2 nor MRP3 mRNA
was detected in 2/4/A1 samples. RT-PCR was a valuable tool for detecting transcripts,
but had to be confirmed at the functional level, since a proper expression of mRNA
does not always correlate with proper function of the corresponding protein.
Functional assays for PepT1, P-gp, MRP2 and BCRP are discussed in section 6.2.4.
6.1.7
Cell death (Papers I and II)
In Paper I the cells died soon after seeding to permeable supports at 39°C and the
dying cells contained highly condensed chromatin and irregular nuclear DNA.
However, cells grown at an intermediate temperature of 33°C and 37°C were viable.
In Paper II the cause of the cell death at 39°C was confirmed to be apoptosis, as seen
by the fragmentation pattern of DNA at this temperature. The finding that the cell
death of 2/4/A1 cells at 39°C was caused by apoptosis agrees with observations in
other cell lines immortalized by tsSV40T (171-174). Thus, cell death by apoptosis
appears to be an inherent feature of these cell lines. The apoptosis in tsSV40Texpressing cell lines has been attributed to the release of the p53 tumour suppression
protein upon inactivation of SV40T at 39°C (171). Despite the fact that apoptosis may
have been an inherent feature of cells immortalized with tsSV40T, attempts were
made to overcome apoptosis and stabilize the 2/4/A1 cells at 39°C. First, the antiapoptotic gene Bcl-2 was over-expressed in 2/4/A1 cells.
Successful inhibition of apoptosis by over-expression of Bcl-2 in cell lines
immortalized by tsSV40T had previously been shown in kidney tubule (175) and
hippocampal neuronal (176) cell lines. Therefore in Paper II a plasmid-containing
human Bcl-2 cDNA was introduced to 2/4/A1 cells by means of electroporation in
order to inhibit the apoptosis in this cell line. Northern blots of the resulting clones
showed that three out of 37 clones expressed Bcl-2 at different levels. The one clone
expressing the highest levels of Bcl-2 mRNA (2/4/A1/Bcl-2) also showed expression
at the protein level as seen by enzyme-linked immunosorbent assay (ELISA). Visual
inspection in the confocal microscope showed that each 2/4/A1/Bcl-2 cell covered a
large surface area and displayed a flat rather than cuboidal or columnar morphology at
33°C, 37°C and 39°C. The continuous 2/4/A1/Bcl-2 cell monolayers reached TER
values of only 16 :˜cm2 when grown for 4 days at 39°C.
In another attempt to overcome apoptosis, extracellular factors that induce apoptosis
were identified in the serum-free cell culture medium for 2/4/A1 cells, which was
used in Paper I. Thus, it was found that the toxicity of SV40T selection marker G418
turned out to be temperature-dependent. A concentration-dependent reduction of the
intracellular dehydrogenase activity with an IC50 value of 18 µg/ml was observed for
2/4/A1 cells at 39°C, while G418 was non-toxic for 2/4/A1 cells at 33°C or 37°C
(IC50 >400 µg/ml). For this reason G418 was removed from the cell culture medium.
Serum deprivation may induce apoptosis (172); consequently, increasing amounts of
FCS were added to the culture medium. At serum concentrations of >6% intact cell
monolayers of fully viable cuboidal cells were obtained that survived for at least 14
days at 39°C.
36
In summary, these results show that over-expression of Bcl-2 inhibited apoptosis and
allowed the formation of intact cell monolayers. However, the undesirable change in
morphology and the comparably low barrier function of the 2/4/A1/Bcl-2 cells make
these cells unsuitable for studies of drug transport. In contrast, the modified culture
protocol allowed formation of intact 2/4/A1 cell monolayers at 39°C that were used in
all further studies in Papers II-IV.
6.2
Transport of drugs and hydrophilic markers (Papers I – IV)
Once 2/4/A1 was established as and could be cultivated on permeable supports, the
main focus of the first part of this thesis was to study and evaluate this cell line as an
epithelial cell model for drug permeability studies. In paper I, the integrity to
hydrophilic markers was studied, and a relationship between FA after oral
administration to humans and Pc was established for a diverse set of passively
transported drugs. With the improved cell culture procedure presented in paper II, the
permeability to hydrophilic drugs was marginally reduced compared to in paper I,
while the permeability to lipophilic drugs remained unchanged. The focus of paper II
was to study active drug transport. In paper III, the average pore radius of 2/4/A1 was
assessed by studying the permeability of a set of small hydrophilic markers. The
performance of 2/4/A1 in predicting the intestinal permeability to incompletely
absorbed drugs was also studied. In paper IV, by varying apical pH, the 2/4/A1 cell
monolayers were used in parallel to artificial membranes and Caco-2 to disclose the
contribution of the paracellular route to the observed pH dependent drug permeability.
6.2.1
Hydrophilic markers and average pore radius (Papers I and III)
In Paper I, the permeability to the low-MW markers mannitol, fluorescein and Lucifer
yellow, and the high-MW markers PEG 4 000 and dextran 50 000 decreased with
increasing size of the marker molecule, and was comparable to that obtained for
hydrophilic markers in the human small intestine (61, 177). The permeabilities ranged
from 15.5 r 2.09˜10-6 cm/s for the smallest marker molecule mannitol (MW 182) to
0.40 r 0.03˜10-6 cm/s for the largest marker molecule, dextran (MW 50,000), or
38-fold. By comparison, for Caco-2 monolayers, the Papp values ranged only 5-fold.
Thus, the permeability of confluent 2/4/A1 monolayers to the hydrophilic marker
molecules depends more strongly on molecular weight than that of Caco-2
monolayers, despite that the values were significantly higher in 2/4/A1 than in the
Caco-2 monolayers (8-62 times) (Fig. 11).
37
Figure 11. Molecular weight-dependent permeability (Papp) of 2/4/A1 (A) and Caco-2
cell monolayers (B) to the hydrophilic marker molecules mannitol (M) (MW 182),
fluorescein (F) (MW 376), Lucifer yellow (L) (MW 450), PEG (P) (MW 4 000) and
dextran (D) (MW 50 000) at 37°C. Note the 100-fold difference in scale on the y-axis
in the two figures. Each bar represents the mean ± SD of six experiments.
In Paper III the transport of low-MW hydrophilic marker molecules (MW 60–504)
was studied in 2/4/A1 and Caco-2 monolayers in order to determine the average pore
radius of these cell monolayers. The permeability to the paracellular marker
molecules of 2/4/A1 and Caco-2 monolayers differed both qualitatively and
quantitatively (Fig. 12). Although both cell models discriminated the paracellular
markers in a size-dependent manner, the Pc values were 10–190-fold higher in 2/4/A1
than in Caco-2 cell monolayers and the fitted curve was more shallow in 2/4/A1 than
in Caco-2 cell monolayers. The resulting average pore radii in the two cell models
were 9.0r0.2 Å and 3.7r0.1 Å for 2/4/A1 and Caco-2 cell monolayers, respectively.
A
B
2/4/A1
Average pore radius: 9.0 Å
7
30
20
10
0
40
Pc x 107 (cm/s)
6
Pc x 106 (cm/s)
40
2
4
3
Caco-2
Average pore radius: 3.7 Å
30
20
10
0
5
Radius (Å)
2
3
4
5
Radius (Å)
Figure 12. Determination of average pore radii in 2/4/A1 and Caco-2 cell
monolayers. The permeability coefficients of urea (MW 60), creatinine (MW 113),
erythritol (MW 122), mannitol (MW 182), foscarnet (MW 126), clodronate (MW 244)
and lactulose (MW 382) were determined in 2/4/A1 (A) and Caco-2 (B) cell
monolayers. The effective hydrodynamic radii of the molecules were determined using
Equation 5. The two curves represent the best fit to Equation 6 in the respective cell
model (n = 4).
38
The seemingly contradictory results obtained in Paper I and III were in fact a result of
the choice of markers in the two studies. In Paper I the molecular radii of the markers
spanned the average pore radius of 2/4/A1, while they were all equal to or larger than
the average pore radius of Caco-2. This explains the stronger size selectivity of
2/4/A1 in Paper I. Accordingly in Paper III the molecular radii of the markers spanned
the average pore radius of Caco-2, while they were all smaller than the average pore
radius of 2/4/A1. This explains the stronger size selectivity of Caco-2 in Paper III.
Consequently, 2/4/A1 is more selective for the molecular radius of large molecules
and Caco-2 is more selective for that of smaller molecules.
6.2.2
Rapidly absorbed drugs (Paper I)
In Paper I, Pc values for a series of 17 drugs and markers were determined in 2/4/A1
cell monolayers and compared with those obtained in Caco-2 cells (Fig. 13). Out of
the 17 drugs five drugs have reported FA values >90%. This cut-off value constitutes
the limit between completely and incompletely absorbed drugs according the
Biopharmaceutical classification system (BCS) (178). These drugs are relatively
lipophilic (ClogP 0.41–2.86) and are therefore presumably absorbed via the
transcellular route. Any difference between the Pc values in 2/4/A1 and in Caco-2
cells for the rapidly transported drugs would disclose differences in the phospholipid
barrier between the cell lines. However, only small differences could be seen between
the Pc values in the two cell lines. By fitting a sigmoidal equation to the experimental
data, cut-off values could be estimated, and the resulting cut-off value for drugs with a
FA >90% was identical in 2/4/A1 and Caco-2 cells (Pc >55 x 10-6 cm/s). Therefore the
transcellular route in 2/4/A1 and in Caco-2 cell monolayers was considered to be
comparable, and differences in Pc values were attributed to differences in either
functional expression of active drug transport proteins or in the paracellular route.
Figure 13. Relationship between the absorbed fraction of structurally diverse sets of
orally administered drugs and permeability coefficients obtained in 2/4/A1
monolayers (diamonds), Caco-2 monolayers (circles) (5, 179) and after in vivo
perfusion of the human jejunum (triangles) (180). Sigmoidal relationships were
obtained between fraction absorbed and the permeability coefficients in all models.
39
6.2.3
Poorly and intermediately absorbed drugs (Papers I and III)
In paper I, the remaining 12 compounds had FA values <90%. The ClogP values of
these ranged from –8.09 – 4.50 and the molecular weights ranged from 113 – 504. In
contrast to the subset of drugs that were completely absorbed, the Pc values for the
incompletely absorbed drugs were 40 - 300 times higher in 2/4/A1 than in Caco-2
(Fig. 13). The sigmoidal relationship between FA and Pc for 2/4/A1 cells is thus
steeper than for Caco-2. Despite this difference, the distinction between completely
and incompletely absorbed drugs was identical in the two models. Based on the
observations that the permeability of the hydrophilic markers were 8 – 62 fold higher,
and that TER was 9 fold lower in 2/4/A1 compared to Caco-2, the difference in
permeability to the incompletely absorbed compounds was attributed to the higher
permeability of the paracellular spaces in 2/4/A1. Most encouragingly, the sigmoidal
relationship between FA and permeability was similar to that found after perfusion of
the human jejunum in vivo. The quantitative overlap in permeability between 2/4/A1
cell monolayers and the human jejunum warranted a further examination of the
properties of the 2/4/A1 cell monolayers.
In Paper III, the aim was to investigate if the 2/4/A1 cell monolayers could predict the
intestinal permeability of incompletely absorbed drugs, using Caco-2 cell monolayers
as a reference. In particular, drugs that have a FA of less than or equal to 80% were
included in this study. This cut-off value was chosen since it constitutes the border
between high and low permeability drugs in drug discovery settings (181). In total, 13
drugs were investigated in this part of the study. Eight of these drugs were categorized
as sparingly absorbed (FA = 0 to 20%) and 5 drugs were categorized as
intermediately absorbed (FA = 20 to 80%). These drugs were used to establish a
relationship between FA and Pc in 2/4/A1 and Caco-2. The relationships were then
used to predict FA for an additional set of drugs (defined as an ad hoc test set) (Fig.
14).
B
75
50
50
FA (%)
FA (%)
A
75
25
25
0
-5.50
-4.75
-5.00
-5.25
Log P c 2/4/A1 (cm/s)
-4.50
0
-8
-6
-7
Log Pc Caco-2 (cm/s)
-5
Figure 14. Sigmoidal relationships between FA and permeability coefficients in
2/4/A1 (A) and Caco-2 (B) cell monolayers. Open symbols represent drugs in the
training set and solid symbols represent drugs in the ad hoc test set. The two curves
represent the best fit to equation 4 for the training set.
The selection of these compounds was based on the observation that their FA values
are particularly difficult to predict using Caco-2 cell monolayers. The average errors
in the prediction of FA for the drugs in the test set were RMSEtest=15.6 and 21.1 for
40
2/4/A1 and Caco-2 respectively. These results suggest that 2/4/A1 cell monolayers
predict the FAs of incompletely absorbed drugs better than Caco-2 cell monolayers,
despite the more permeable paracellular pathway in this cell model. Analysis of the
ranking of the predicted FA values from the Pc values in the two cell culture models
supports this conclusion. Spearman’s rank coefficient was significantly higher
(p<0.05) for FA versus Pc in 2/4/A1 (0.90) than it was for FA versus Pc in Caco-2
(0.75) for the incompletely absorbed drugs.
6.2.4
Actively transported drugs (Papers I and II)
In Paper I preliminary studies on active efflux in 2/4/A1 cell monolayers grown at
37°C using celiprolol and ciprofloxacin as P-gp and non-P-gp substrates, respectively,
showed that the transport in the apical-to-basolateral (a-b) direction equalled that in
the basolateral-to-apical (b-a) direction. Since these two drugs are known to display
vectorial transport in Caco-2 cell monolayers, it was hypothesised that functional
expression of the responsible transport proteins was low or even absent in the 2/4/A1
cell monolayers.
$
%
20
7.5
n.s.
15
n.s.
10
5
Pc X 106 (cm/s)
Pc X 106 (cm/s)
In Paper II a more thorough investigation of the functional expression of membrane
transporters relevant for drug absorption was carried out. Following analysis at the
transcriptional level, transport studies using typical substrates were performed. These
studies showed that no significant H+-coupled active transport of the PepT1 substrate
glycylsarcosine (glysar) occurred in the 2/4/A1 cells (Fig. 15A). By contrast, a clear
concentration-dependent H+-coupled active transport of glysar was observed in Caco2 cells (Fig. 15B) which are known to express PepT1 (182, 183).
5.0
*
2.5
*
0
0.48 PM 50 PM
0.0
50 mM
0.48 PM 50 PM
50 mM
Figure 15. Transport of the PepT1 substrate glysar in 2/4/A1 (A) and Caco-2 (B) cell
monolayers. No significant dose-dependent decrease in the permeability coefficient
for glysar was observed in 2/4/A1 cell monolayers (p >0.05) (A), while a dosedependent decrease in the permeability coefficient for glysar could be observed in
Caco-2 cell monolayers (p <0.05) (B). Each bar represents the mean ± SD of four
experiments. * and n.s. denote significant (p <0.05) and non-significant difference,
respectively, compared with transport at 0.48 µM glysar.
41
Further, there was no vectorial transport in the b-a direction across 2/4/A1 cell
monolayers for any of the substrates for the efflux systems investigated (P-gp, MRP2
and BCRP) (Fig. 16). In Caco-2 cell monolayers, on the other hand, all the substrates
displayed several-fold higher permeability coefficients in the b-a than in the a-b
direction. Since Caco-2 cells express multiple drug efflux systems with overlapping
substrate specificity, the efflux in more specific epithelial expression systems was also
studied. Stable MDCK II and LLC-PK1 cell clones that over-expressed each of the
efflux systems were used for this purpose. All of the typical substrates displayed
higher efflux ratios (b-a/a-b) in the expression systems than in the corresponding
untransfected cell lines.
*
20
MDCK-MRP2
MDCK
Caco-2
0
*
n.s.
2/4/A1
MDCK-MDR1
MDCK
10
Caco-2
*
10
0
*
LLC-BCRP
*
20
30
n.s.
2/4/A1
*
*
40
LLC-PK1
*
20
0
C Mitoxantrone
30
50
*
6
P c x 10 (cm/s)
*
30
10
Vinblastine
B
60
Caco-2
Digoxin
2/4/A1
A
40
Figure 16. Permeability coefficients in the a-b (open bars) and b-a (closed bars)
directions for the P-gp substrate digoxin (A), the MRP2 substrate vinblastine (B) and
the BCRP substrate mitoxantrone (C) in 2/4/A1, Caco-2 (A–C), MDCK II (A and B),
MDCK-MDR1 (A), MDCK-MRP2 (B), LLC-PK1 (C) and LLC-BCRP (C) cell
monolayers. The P-gp inhibitor GF120918 (2 µM) was added in the studies of
vinblastine transport in MDCK II and MDCK-MRP2. Each bar represents the mean ±
SD of three to six experiments. * and n.s. denote a significant (p <0.05) and nonsignificant difference between a-b and b-a transport, respectively.
Therefore, despite 2/4/A1 cells expressing some drug transporters at the
transcriptional level, the cells lacked functional expression of the investigated
transport proteins. The reasons for this may be that the mRNA is not translated into
protein, that the protein is not functional, or that it is not properly sorted into the
apical cell membrane. This apparent drawback can, however, be advantageous in
certain cases. For instance, when structure-permeability relationships are established
for the passive drug transport routes, these data are not biased by active transport
phenomena, which facilitates the design of the drug transport experiments.
6.2.5
pH dependent permeability (Paper IV)
The aim of Paper IV was to quantify the contribution of the paracellular route to the
permeability across epithelial cell monolayers. In a previous study the ionized form of
the drug was reported to have contributed significantly to the permeability in Caco-2
cell monolayers at pH values where the fraction un-ionized (fu) of the drug was <0.1
42
(77). In this study the pH-dependent permeability of one rapidly transported drug,
alfentanil, and one slowly transported drug, cimetidine (shown to be transported by
the paracellular route in vivo), was investigated in three models of the intestinal
epithelium (hexadecane-impregnated membranes (HDMs), the Caco-2 model and
2/4/A1 cells) (Fig. 17). These models vary in their permeability to slowly transported
drugs, presumably owing to differences in their paracellular transport routes. The
transport studies were carried out at apical pH values ranging from 5.0 to 8.0
(representing the pH interval of the GI). In this pH interval the fu of alfentanil varied
from 0.031 to 0.969 and the fu of cimetidine, from 0.016 to 0.941.
Hexadecane
membrane
Non-paracellular
Caco-2
2/4/A1
230 Ω⋅cm2
50 Ω⋅cm2
Tight-paracellular
Leaky-paracellular
: Passive paracellular
: Passive transcellular
Figure 17. Schematic illustrations of the membrane models used in Paper IV.
Sigmoidal relationships were obtained between the membrane permeability (P m) and
pH in all models, but the relationships were qualitatively different (Fig. 18). These
differences became evident when the permeability to the ionized (P mi) and the
unionized (P mu ) forms of alfentanil and cimetidine were determined from the linear
relationships obtained between P m and fu. In HDM, no contribution from the ionized
forms of the drugs was observed, whereas significant contribution was observed in the
two cell models. The selectivity of the permeability to the unionized form over the
ionized form was higher in Caco-2 than in 2/4/A1 for alfentanil (73.8 vs 18.4), as well
as for cimetdine (10.1 vs 1.5). Thus the permeability properties of HDM, Caco-2 and
2/4/A1, with regard to the ionized form of at least hydrophilic drugs can be
categorized as follows: HDM – no contribution, Caco-2 – small contribution, and
2/4/A1 – large contribution. A reasonable assumption is that the difference in
selectivity between Caco-2 and 2/4/A1 is a consequence of the difference in the
relative contribution from the paracellular pathways.
43
A
B
C
Pm (10-7 cm/s)
1.00
10.0
0.75
7.5
0.50
5.0
0.25
2.5
0.00
4
5
6
7
pH
8
Cimetidine in 2/4/A1
Cimetidine in Caco-2
Cimetidine in HDM
9
0.0
400
4
350
300
5
7
6
8
9
250
4
5
pH
6
7
8
9
pH
Figure 18. Sigmoidal relationships between Pm for cimetidine and pH were obtained
in all models. The values are presented as mean ± SD, n = 4.
In summary, the results of Paper IV show that 2/4/A1 cell monolayers can be used to
disclose the dominant passive transport route. The tight Caco-2 cell monolayers may
result in under-estimation of the paracellular route for small hydrophilic drugs.
6.3
Modulation of tight junction permeability (Paper V)
Paper V constitutes the second part of this thesis. In this paper, a new approach to
tight junction modulation was taken. The aim of this study was to investigate whether
peptides from the extracellular loops of the tight junction protein occludin could be
used as a new principle for tight junction modulation by an extracellular mechanism.
Peptides homologous to the extracellular sequences of human occludin were
synthesized and tested for their ability to modulate the tight junction permeability in
Caco-2 cell monolayers. First, the two peptides corresponding to the two complete
extracellular loops of occludin were investigated. In the presence of the first loop
peptide (OP90-135), the permeability to the paracellular marker mannitol increased
approximately 3-fold after two hours. No increase in permeability could be observed
after incubation with the second loop peptide (OP196-243) (Fig. 19).
44
3
7
Papp x 10 (cm/s)
4
2
1
0
Control OP90-135 OP196-243
Occludin peptide
Figure 19. After exposure to OP90-135 for two hours, the permeability to mannitol in
Caco-2 cell monolayers increased 3-fold compared to control. The permeability to
mannitol did not change after exposure to OP196-243. The values are presented as
mean ± SD, n = 4
Next, the effects of two shorter peptides were studied. Two active peptides
corresponding to the N-terminus of the first extracellular loop were identified. The 24
aa long peptide (OP90-113) was approximately 10 fold more potent than the 14 aa long
peptide (OP90-103). Further truncation of the 14 aa peptide resulted in peptides that
were inactive. These peptides were non-toxic as judged by the lactate dehydrogenase
leakage and dehydrogenase activity assays. However, the peptides were fully active
only when added to both the apical and basolateral sides of the Caco-2 cell
monolayers, and the onset of action of these peptides was slow (hours).
Since tight junction modulators will be added to the apical (serosal) side of epithelial
barriers in vivo, the reason for the lack of apical effect had to be identified. The
stability of the occludin peptides showed that significant degradation occurred when
the peptides were added to the apical but not when they were added to the basolateral
side (Fig. 20). This degradation was most likely mediated by peptidases situated in the
apical membrane. Another possible reason for the lack of apical effect could be that
the accessibility to the tight junctions is more size-restricted from the apical side than
from the basolateral side. The problem with size-restricted accessibility may have
been further pronounced by the reported tendency of full-length OPs to form
aggregates (134).
45
Fraction remaining (%)
110
100
90
OP90-103
OP90-103
OP90-103
OP90-103
OP90-103
80
70
60
50
40
0
1
2
3
5
4
6
BL
AP
AP + Inh
BL + Inh
7
Time (hours)
Figure 20. Degradation of OP90-103 after incubation with Caco-2 cells. After 6 hours,
approximately 50% of the OP90-103 was degraded on the apical side, while no
degradation could be observed for the basolateraly exposed OP90-103. The apical
degradation of OP90-103 could be inhibited by a peptidase inhibitor cocktail (bestatin,
0.29 mM; diprotin A, 1 mM and captopril, 1 mM; (184)). The values are presented as
mean ± SD, n = 4.
In order to increase accessibility of the OPs to the tight junctions from the apical side,
a lipoamino acid derivative of the shortest active peptide, OP90–103, was synthesized.
This conjugate has several potential advantages as compared with the free peptide.
Firstly, the conjugation of a lipoamino acid of sufficient length (185, 186) would
insert into the cell membrane and concentrate the peptide at the cell surface; secondly,
it would release the active peptide through the action of brush border peptidases (i.e.
at the site adjacent to the tight junctions); thirdly, it would limit degradation of the
released peptide by apical peptidases (158); and fourthly, it would possibly also
reduce aggregation.
The conjugation resulted in a racemic mixture of two diastereomers. After isolation of
the isomers, the stability of D- and L-C14-OP90–103 added to the apical side of the
Caco-2 cell monolayers was examined. The L-isomer released OP90–103 15 times faster
(5.01±0.11 nmol/hr) than did the D-isomer (0.33±0.60 nmol/hr). Only intact OP90–103
was released, indicating that the lipoamino acid protected the released OP90–103 from
further degradation by the apical peptidases. Thus the conjugation solved the
degradation problem.
Encouragingly, each of the isomers increased the permeability approximately 15-fold
compared to control already 20 minutes after apical addition to Caco-2 cell
monolayers. The permeability was further increased to approximately 40-fold
compared to the control after 40 minutes for the L-isomer, while the effect of the Disomer gradually returned to base line levels (Fig. 21A). The increase in permeability
could be efficiently inhibited by peptidase inhibitors, indicating that release of native
peptide from the conjugate was required for effect. The effect of the two isomers on
TER was in close agreement with that observed for the effect on mannitol
permeability with a rapid decrease in TER for both isomers after 10 minutes. The
exposure of the L-isomer resulted in a further decrease that reached a plateau after
approximately 20 minutes and at this time, the TER was reduced to 30% of control.
The effect of the D-isomer on TER was reversible and returned to values comparable
to controls within 20 minutes (Fig. 21B). C14-OP90-103 did not cause acute toxicity to
Caco-2 cells at concentrations as high as 1 mM as judged by measurements of the
46
intracellular dehydrogenase activity. There was some concern that the free lipoamino
acid could mediate some of the observed effect. However, the lipoamino acid
presented alone or in combination with unconjugated peptide was inactive.
A
B
500
40
L-C14-OP90-103
2
TER (:˜cm 2)
Papp x 107 (cm/s)
50
30
20
D-C14-OP90-103
10
Contr.
0
0
50
100
400
Control
300
200
D-C14 -OP90-103
100
150
0
200
L-C14-OP90-103
0
30
60
90
120
Time (min)
Time (min)
Figure 21. (A) Time dependent studies showed that both L- and D-C14-OP90-103
induced a rapid 15-fold increase in mannitol permeability. The activity was further
increased to approximately 40-fold after 30 minutes for the L-isomer, while the effect
reversed to base line levels with time for the D-isomer. (B) The effects on TER of the
two isomers were in close agreement with those observed for mannitol permeability,
with a rapid initial decrease in TER for both isomers followed by a return to control
levels for the D-isomer while the L-isomer maintained a reduced TER for at least two
hours. Values are presented as mean ± SD (n = 4).
In summary, in this study peptides originating from the first extracellular loop of
occludin were shown to modulate the paracellular permeability of Caco-2 cell
monolayers. A rapid apical effect was obtained after coupling of a lipoamino acid to
the N-terminus of the peptide. The resulting D-isomer displayed a rapid and transient
effect. The short duration of action limits unrestricted permeation of potentially toxic
agents and also reduces the risk of harmful autoimmune responses. For sustained
effect, the lipopeptide must rapidly release intact peptide by a mechanism mediated
by apical peptidases, and this was the case only for the L-isomer. C14-OP90-103
represents a prototype of a new class of tight junction modulators that act on the
extracellular domains of tight junction protein occludin.
47
7
SUMMARY AND CONCLUSIONS
The results of the first part of this thesis show that the conditionally immortalized rat
epithelial cell line 2/4/A1 can be used as a model epithelium for studies of passive
drug transport. After 4 – 6 days on permeable supports, the integrity of the 2/4/A1 cell
monolayers reaches a stable plateau that is maintained for more than a week. This
plateau is reached much quicker for 2/4/A1 than for traditionally cultivated Caco-2
cell monolayers, which require approximately three weeks to stabilize. Both the
passive transcellular and paracellular permeability of this cell line resembles those of
the human small intestine, which will enable better quantitative predictions of
intestinal drug permeability for both rapidly and slowly permeable drugs, than has
been possible with Caco-2. 2/4/A1 cell monolayers lack the functional expression of
several transport proteins that are relevant for intestinal drug absorption. Although an
obvious disadvantage in studies of active drug transport, this feature may be an
advantage in studies of structure permeability relationships for the passive transport
routes. The 100-fold higher permeability for low-permeable drugs enables the use of
more robust analytical techniques such as HPLC-UV since the transported amounts
are larger in 2/4/A1 than in Caco-2, which often requires LC-MS analysis for low
permeable drugs. Despite the approximately 100-fold higher permeability for slowly
transported drugs in 2/4/A1 compared to that in Caco-2, the 2/4/A1 predicts and ranks
the intestinal permeability to incompletely absorbed drugs better than does Caco-2.
2/4/A1 can also be used to study the influence of charge on the intestinal
permeability.
The results of the second part of the thesis show that peptides, 14 and 24 aa in length,
from the N-terminus of the first extracellular loop of occludin modulate the tight
junction permeability in Caco-2 cells. The native peptides increased the tight junction
permeability in a dose-dependent and non-toxic manner, but had to be added to the
basolateral side of the cell monolayers. One reason for the lack of apical effect of
these peptides was that they were degraded by apical peptidases. Another reason may
have been that the peptides had limited access to tight junctions from the apical side.
To solve the problem with the lack of apical activity, a lipoamino acid was conjugated
to the N-terminus of the peptides. This conjugation inhibited degradation of the native
peptide, and made the peptide active from the apical side. The two diastereomers of
the conjugate had distinctly different kinetics of effect. The L-isomer rapidly released
the native peptide, which resulted in a sustained effect. The D-isomer released the
native peptide more slowly, resulting in a transient effect. In conclusion, the concept
of conjugated peptides that act on the extracellular domains of the tight junctions
constitutes a new prototype for tight junction modulation.
The specific conclusions of this thesis are:
x depending on the culture conditions 2/4/A1 cells cultivated at 37ºC and 39ºC
form monolayers on matrix coated permeable supports. These monolayers
require 4 – 6 days to stabilize, and remain stable for at least another week.
x 2/4/A1 cell monolayers express the essential tight junction proteins and
develop functional tight junctions.
48
x 2/4/A1 cell monolayers express several drug transporting membrane proteins
at the transcriptional level, but there is no evidence for functional expression
of these proteins in this cell line. The 2/4/A1 cell model can therefore be used
for prediction of passive intestinal drug permeability.
x 2/4/A1 cell monolayers qualitatively and quantitatively predict the fraction
absorbed after oral administration to humans for a diverse set of passively
absorbed drugs. 2/4/A1 monolayers rank the intestinal permeability of
incompletely absorbed drugs better than Caco-2.
x 2/4/A1 cell monolayers can be used to study the influence of molecular charge
on the drug permeability. Studies of pH dependent drug permeability in
2/4/A1 can be performed to disclose whether the dominating passive transport
route is trans- or paracellular.
x Native peptides homologous to the N-terminus of the first extracellular loop of
occludin modulate the tight junction permeability of Caco-2 cell monolayers
in a slow and inefficient way. Conjugation of a lipoamino acid to an occludin
peptide resulted in a more efficient tight junction modulator. The L-isomer of
the conjugate rapidly released the native peptide, as a result of peptidasemediated removal of the lipoamino acid. This results in a sustained increase in
tight junction permeability. The D-isomer releases the native peptide more
slowly, and resulted in a transient increase in tight junction permeability.
49
8
ACKNOWLEDGEMENTS
The studies in this thesis were carried out at the Department of Pharmacy, Uppsala
University.
I wish to express my gratitude to the following people who contributed to this thesis:
My supervisor professor Per Artursson, for sharing your knowledge, for always
aiming at high quality and for never stopping to teach me how to write papers.
Professor Göran Alderborn for providing good working facilities.
Dr. Jan Taipalensuu (co-author) for sharing your enthusiasm (and your knowledge in
molecular biology).
Dr. Kei Hashimoto (co-author) for sharing your creative thinking and for pushing the
occludin paper to new heights.
Naoki Nagahara (co-author) for your hard and fruitful work on the pH paper.
Dr. Vladan Milovic (co-author) for your significant contribution to the 2/4/A1
research area.
Dr. Finn Hallböök (co-author) for supervising me when I did the transfections.
Johan Gråsjö, Dr. Lucia Lazorová and Dr. Göran Ocklind (co-authors) for help and
advice with mathematics and statistics, with cell culture and transport experiments,
and microscopy.
Magnus Köping-Höggård, my roommate, for pleasant company and creative
discussions, for your positive attitude and for your inspiring self-discipline.
Dr. Patric Stenberg and Dr. Raouf Ghaderi for most(ly) pleasant company.
Susanne Olsson for teaching me how to cultivate those tricky 2/4/A1 cells back then
when I was a freshman.
Christel Bergström for providing me with the calculated descriptors and for
supervising our summer students.
Ulla Wästberg-Galik for making practical matters around the writing as smooth as
possible.
Eva Nises Ahlgren for your help and support.
George Farrants and Proper English for skillful linguistic assistance.
Dr. Richard Morrison, Dr. Vanessa Moore and Dr. Saeho Chong (co-authors) for
stimulating discussions and valuable comments.
50
Kati-Sisko Vellonen, Lauri Söderberg (co-authors) and Outi Haarakangas for skillful
technical assistance.
Past and present members of the cell group for being great colleagues and for trusting
me with the incubators (22°C!).
All my other colleagues at Galeniken for creating the good atmosphere.
Marja-Liisa for being my love and for your support and encouragement. I especially
want to thank you for handling all the practical matters in Umeå during the time I was
writing this thesis, and for taking care of the cats.
Financial support from Bristol-Myers Squibb Company, AstraZeneca, The Swedish
Academy of Pharmaceutical Sciences, CD Carlsons stiftelse, The Swedish Medical
Research Council, Centrala försöksdjursnämnden, and Stiftelsen Forskning utan
Djurförsök is gratefully acknowledged.
51
9
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