Download In Vitro Models of the Intestinal Barrier

ATLA 29, 649–668, 2001
649
In Vitro Models of the Intestinal Barrier
The Report and Recommendations of ECVAM Workshop 461,2
Eric Le Ferrec,3 Christophe Chesne,3 Per Artusson,4 David Brayden,5
Gérard Fabre,6 Pierre Gires,7 François Guillou,6 Monique Rousset,8
Werner Rubas9 and Maria-Laura Scarino10
3BIOPREDIC, Technopole Atalante Villejean, 14–18 rue Jean Pecker, 35000 Rennes, France;
4Department of Pharmaceutics, Biomedical Centre, Uppsala University, 751 23 Uppsala,
Sweden; 5ELAN Biotechnology Research, Biotechnology Building, Trinity College, Dublin 2,
Ireland; 6Sanofi Synthelabo Recherche, Departement Phamacologie & Métabolisme, 371 rue
du Pr Blayac, 34184 Montpellier, France; 7Rhone Poulenc Rorer, DMPK, Preclinical
Department (in vivo & in vitro), 13 Quai Jules Guesdes, 94403 Vitry Sur Seine, France;
8INSERM U178, Hopital Paul Brousse, 16 Avenue Paul Vaillant Couturier, 94807 Villejuif,
France; 9GENENTECH Inc., 1 DNA Way, South San Francisco, CA 94080, USA; 10Istituto
Nazionale di Ricerca per gli Alimente e la Nutrizione, Via Ardeatina 546, 00178 Rome, Italy
Preface
This is the report of the forty-sixth of a series
of workshops organised by the European
Centre for the Validation of Alternative
methods (ECVAM). ECVAM’s main goal, as
defined in 1993 by its Scientific Advisory
Committee, is to promote the scientific and
regulatory acceptance of alternative methods
which are of importance to the biosciences
and which reduce, refine or replace the use of
laboratory animals. One of the first priorities
set by ECVAM was the implementation of
procedures which would enable it to become
well-informed about the state-of-the-art of
non-animal test development and validation
and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be
best achieved by the organisation of ECVAM
workshops on specific topics, at which small
groups of invited experts would review the
current status of in vitro tests and their
potential uses, and make recommendations
about the best ways forward (1). In addition,
other topics relevant to the Three Rs (reduction, refinement and replacement) concept of
alternatives to animal experiments have been
considered in several ECVAM workshops.
The workshop on in vitro models of the
intestinal barrier was held in Paris, France, on
4–5 March 1999. The principal aims of the
workshop were to seek a consensus on the current models of the intestinal barriers and ways
to screen for the movement of drugs across
this barrier, and to make useful recommendations for the promotion of the Three Rs in this
area. The panel of techniques used for the prediction of the relevant parameters is very
large, including in vivo and in situ methods,
Address for correspondence: BIOPREDIC, Technopole Atalante Villejean, 14–18 rue Jean Pecker, 35000 Rennes,
France.
Address for reprints: ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research
Centre, 21020 Ispra (VA), Italy.
1ECVAM
— The European Centre for the Validation of Alternative Methods. 2This document represents the
agreed report of the participants as individual scientists.
E. Le Ferrec et al.
650
cell culture techniques, and in vivo studies
with human volunteers. It was intended to
compare these different models, and to delineate their strengths and weaknesses in the
various screening situations encountered in
the pharmaceutical industry.
Introduction
The intestinal epithelium is a gatekeeper, i.e. it
controls the entry of nutrients and xenobiotics
(for example, medicines). Knowledge of the
absorption and metabolism of these substances at the intestinal mucosal level is of
particular importance, since the oral bioavailability of a drug is defined as the fraction of an
oral dose that reaches the systemic circulation.
Drug absorption is considered to be a complex transfer process across the intestinal lining, which includes passive diffusion through
the paracellular space and/or membranes of
absorptive cells, vesicular uptake (endocytosis/pinocytosis), and release at the basolateral
space (transcytosis; 2). This transport may or
may not be receptor-mediated (or transportermediated), entailing uptake across the apical
domain with subsequent passive diffusion into
the basolateral space (Figure 1; 3). Each transport mechanism depends on the physicochemical properties of the absorbed compound, such
as its stereochemistry, partition into mem-
branes, molecular weight and/or size, molecular volume, pKa, solubility, chemical stability
and charge distribution. Physiological factors such as gastric emptying, gastrointestinal
motility, intestinal pH, blood flow, lymph flow
(Figure 2), pathological state, drug interactions, nutrition, and mucus dissolution, also
need to be considered when evaluating absorption (4–6).
Upon ingestion, compounds have to be liberated from their dosage form, which includes
dissolution into a complex medium containing
numerous compounds (bile salts, ions, lipids,
cholesterol and enzymes; 7). This medium can
vary considerably, depending on the individual, the intestinal segment, the diet, etc. (8).
Measurements of absolute solubility and dissolution are performed routinely during drug
development. However, knowledge of intestinal drug dissolution is limited, due to the
non-physiological aspects of in vitro systems;
for example, instead of using intestinal fluid,
buffer systems are commonly employed, which
may result in an overestimation or underestimation of the actual events in vivo (9–12).
Since the intestinal fluid is rich in enzymes
derived from dying enterocytes and/or the
intestinal flora, gastrointestinal stability
includes both physicochemical and enzymic
stability.
The intestinal cell lining is covered with a
viscous and elastic gel produced by the gob-
Figure 1: Routes and mechanisms of transport of molecules across the intestinal
epithelium
1
2
3
4
Apical membrane
Tight
junctions
Basolateral
membrane
Intestinal cells
Paracellular
Transcellular
1) Paracellular; 2) transcellular passive diffusion; 3) transcytosis; 4) carrier-mediated uptake
at the apical domain followed by passive diffusion across the basolateral membrane.
ECVAM Workshop 46: the intestinal barrier
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Figure 2: Factors influencing intestinal absorption
Nutrient intake
Nervous innervation
Paracrine
hormonal control
Gastric motility
Intestinal motility
Dissolution or
binding in mucus
Nervous innervation
Paracrine
hormonal control
Xenobiotic physical and
chemical characteristics
(molecular weight, pKa,
solubility, chemical stability,
lipophilicity etc.)
Physiological factors
(disease, intestinal pH etc.)
Absorption
(Paracellular, transcellular
passive diffusion,
transcellular endocytosis,
transcellular carriermediated transport)
Luminal factors, bacterial
toxins, bile
Number of enterocytes
Metabolism
Secretion
(ions, fluid)
Functional state of
enterocyctes
Blood and lymph flow
Distribution
Elimination
(metabolism, excretion)
From (3).
let cells of the villous epithelium. The
physicochemical properties of this gel can
influence the rate of diffusion from the bulk
to the site of absorption. Furthermore,
metabolic enzymes could be associated with
the mucus layer. Thus, the gel is considered
to be the first in a series of absorptive barriers (13). The enzymic barrier to oral
absorption could consist of several layers,
depending on the properties of a compound.
Also, as well as the enzymes within the
intestinal lumen, there are additional
enzymes located in the absorptive cells
themselves, such as several cytochrome
P450 (CYP) isoforms (Table I). In combination with P-glycoprotein (P-gp), these
enzymes are responsible for the intestinal
first-pass effect.
E. Le Ferrec et al.
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Table I:
Examples of CYP expression as a function of their human intestinal
localisation
CYP
Stomach
Duodenum
Ascending
colon
Transverse
colon
Descending
colon
1B1
2E1
3A4
3A5
+++++
+++
+++
++++
+
++
++++
+++++
++++
++
++
++++
+
+
+
+++
+
+++
++
++
See reference (60).
The number of + signs indicates the relative amount of CYP expressed
The Current Models
The intestinal mucosa is characterised by
the presence of villi that constitute the
anatomical and functional unit for nutrient
and drug absorption (14). The presence of
villi and microvilli provides a massive surface area for absorption (approximately
250m2 in a human). The mucosa consists of
the epithelial layer, the lamina propria (collagen matrix containing blood and lymphatic
vessels) and the muscularis mucosa. Therefore, any xenobiotic entering the bloodstream
has to pass through the epithelial layer, part
of the lamina propria, and the wall of the
respective vessel. It is crucial to select an
appropriate model for understanding the ratelimiting step in the absorption process.
Figure 3: The place of isolated perfused organs in biomedical research
Animal
Experiments
In situ
(animals)
Isolated
perfused organs
In vivo
(human)
Animal
cell culture
Human
cell culture
Monolayer,
multilayer
Monolayer,
multilayer
= predictive for;
= cell/organs derived from.
ECVAM Workshop 46: the intestinal barrier
Three groups of methods have emerged for
investigating the principal mechanisms of
absorption in animals, namely, in vivo, in situ
and in vitro methods. The choice of model
depends totally on the questions to be
answered with respect to the test compound
being studied.
In vivo models
The main advantage of in vivo models is the
integration of the dynamic components of
the mesenteric blood circulation, the mucous
layer and all the other factors that can influence drug dissolution.
The most frequently used animal model is
the rat, since it better reflects the human situation with respect to paracellular space and
metabolism than the dog, which is different
to the human, particularly in relation to
metabolism, and overestimates the absorption of paracellularly restricted compounds
(15). However, oral studies in rats also have
limitations, and tend to provide false-positive
results. It has therefore been stated that
“the only real model for man is man” (16).
Techniques such as “cassette dosing” can
give global information on drug bioavailability, including intestinal barrier passage (17).
This technique is useful when there are a
large number of products to test (high
throughput screening [HTS)], when products with good bioavailability are tested, and
when organotypic in vitro models are not
appropriate.
The disadvantage of in vivo models is that
it is impossible to separate the variables
involved in the process of absorption, i.e. it is
not possible to identify individual rate-limiting factors.
In situ models
The development of stable, vascularly perfused preparations of the small intestine has
provided a powerful research tool for the
investigation of intestinal transport and
metabolism. In this approach, the abdominal
cavity of an anaesthetised animal is exposed
by laparotomy. The intestinal segment into
which the drug solution is introduced can be
either a closed loop or an open loop.
In situ methods have significant advantages over in vivo models. For example,
bypassing the stomach means that acidic
compounds are not likely to precipitate, so
dissolution rates do not confuse intestinal
drug concentrations and therefore plasma
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levels. Furthermore, in situ instillation
allows the experimenter to assess formulation-independent breakdown in the stomach
under acidic conditions. Although the animal
has been anaesthetised and surgically
manipulated, mesenteric blood flow is intact.
However, caution must be taken with the
choice of anaesthetic, since it has recently
been demonstrated that anaesthesia can
have strong effects on intestinal drug
absorption (18).
An additional consideration when using in
situ techniques is the volume of the luminal
drug solution, because this may change due
to either absorption or secretion of water.
This necessitates the use of non-absorbable
or low-absorbable volume-marker compounds, such as radiolabelled poly(ethylene
glycol) (PEG) 4000, inulin or mannitol, and
fluorescent markers, such as lucifer yellow.
It is noteworthy that the disappearance of
drug from the perfusate does not always
equate with absorption. Thus, it is prudent
to include sampling from the portal vein, in
addition to monitoring the change in perfusate concentration. By moving the sampling location from the portal vein to the
hepatic vein, additional information about
liver first-pass effect can be obtained.
In humans, the intestinal passage of drugs
can be studied by the balloon technique (Loc1-Gut), with drug administration by catheter
and analysis of blood sampling (2, 19). With
this technique, drug passage can be studied
at various intestinal levels. This is a reference technique, but it is expensive to perform and difficult to handle; it cannot be
used routinely during drug development
(20).
With this model, it is also possible to study
compound secretion into the intestinal
lumen after intravenous administration, i.e.
it is feasible to investigate mediation of
export of xenobiotics into the intestinal
lumen by P-gp, multidrug resistance-associated protein (MRP) and lung cancer-associated resistance protein (LRP).
In vitro models
Organotypic models
All intestinal cell types (for example, enterocytes, caliciform cells and lymphocytes) are
present in organotypic models, which are
used to study formulation effects (with the
possible use of parenteral lipid emulsions,
such as Intralipid®, or bovine serum albumin
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for drugs having low solubility), intestinal
metabolism/stability, and regional differences in permeability. Some studies have
shown that permeability to various marker
molecules varies along the intestinal canal.
In general, permeability decreases in the
order: jejunum > ileum > colon (21). The
half-lives of these models are short (1–3
hours).
Everted gut sac
The everted gut sac of the rat small intestine
can be used to determine kinetic parameters
with high reliability and reproducibility (19,
22). Oxygenated tissue culture media and
specific preparation techniques ensure tissue
viability for up to 2 hours. The technique can
be used to study drug transport across the
intestine and into the epithelial cells, provided that sensitive detection methods are
employed (23). Radiolabelled compounds are
most appropriate.
This technique was used in the past to
study the transport of macromolecules and
liposomes but, more recently, it was used
mainly to quantify the paracellular transport
of hydrophilic molecules, and to estimate the
effects of potent enhancers on their absorption (23). The transport of mannitol, a paracellular marker, shows an apparent
permeability (Papp) of 1.5 × 10–5 to 1.7 × 10–5
cm/s. This value is the same as those
reported with low-molecular weight hydrophilic drugs in human perfusion studies. The
toxicities of potential enhancers can be monitored by studying the release of intracellular
enzymes or by histological examination. Molecules that cross the epithelial barrier by a
transcellular route have a much higher permeability, which can also be accurately quantified by using the everted sac system.
This kind of model is suitable for measuring absorption at different sites in the small
intestine (24), and for performing preliminary experiments on the colon (19, 21). It is
also useful for estimating the first-pass
metabolism of drugs in intestinal epithelial
cells. Also, by using this model (everted or
not), it is convenient to study the effect of Pgp on xenobiotic transport through the intestinal barrier.
A potential disadvantage of this approach
is the presence of the muscularis mucosa,
which is not usually removed from everted
sac preparations. Therefore, this model does
not reflect the actual intestinal barrier,
because compounds under investigation pass
E. Le Ferrec et al.
from the lumen into the lamina propria
(where blood and lymph vessels are found)
and across the muscularis mucosa. Thus, the
transport of compounds with a propensity to
bind to muscle cells might be underestimated.
Isolated and perfused intestinal segments
During the last decade, a wide range of isolated organ systems have been developed for
biomedical and pharmaceutical research.
The availability of sophisticated equipment,
increased manual skills, and the routine use
and standardisation of models and protocols,
have led to the increased reproducibility and
validity of experimental results under circumstances that are virtually “true-to-life”.
These methods contribute to the reduction of
animal experimentation. Figure 3 shows the
place of isolated perfused organs in biomedical research, compared with cell cultures of
either human or animal origin. The results
are recognised as predictive of the in vivo situation, including absorption at the organ
level (19, 20, 25). Isolated perfused organs
have the advantage that the scientist works
with an intact organ, where physiological
cell–cell contacts and normal intracellular
matrixes are preserved (25). The major limitation is the short duration of the experiments that are possible, since changes occur
rapidly.
Ussing chambers
Ussing chambers were introduced by Ussing
& Zehran in 1951 (26) for studying the active
transport of sodium as a source of electric
current in short-circuited, isolated frog skin.
Later on, these chambers were extensively
used for the study of ion transport across
many types of membrane.
The usefulness of Ussing chambers for
intestinal transport studies has long been
recognised, and they have also been used to
study the intestinal metabolism of xenobiotics (19). In this system, the drug can be
exposed at either the mucosal level (apical
side of enterocytes) or the serosal level (basolateral side of enterocytes). Furthermore, the
simplicity of Ussing chambers makes them
an attractive in vitro model system for studying drug transport.
When properly equipped with electrodes,
Ussing chambers are useful for studying the
effects of compounds on electro-physiological
parameters of the intestinal barrier. This
type of study may add additional information
ECVAM Workshop 46: the intestinal barrier
on the pharmacological behaviour of the test
compound (27).
Cell models
The study of absorption mechanisms is best
performed in a model that contains only
absorptive cells, without the confounding
contributions of mucus, the lamina propria
and/or the muscularis mucosa. Therefore,
much attention is currently paid to the use of
epithelial cell cultures for studies of drug
transport mechanisms. However, the use of
isolated intestinal epithelial cells has been
slow to gain popularity, because they are difficult to culture and have limited viability
(28–35).
The development of human cell culture
systems has been limited by the loss of
important in vivo anatomical and biochemical features. Attention has therefore turned
to the use of human adenocarcinoma cell
lines, such as HT-29 and Caco-2, that reproducibly display a number of properties characteristic of differentiated intestinal cells
(36). In addition, the development of these
cell line models was paralleled by the development of sensitive and automated measurement techniques (for example, liquid
chromatography and mass spectrometry).
The limitations of cell models must not be
overlooked, but they offer the advantage of
relative simplicity, and are suitable for automated procedures and HTS. These cell lines
originated from tumours, and are out of the
in vivo physiological environment; therefore,
extrapolation of the data to the in vivo situation may be difficult (as is true of most in
vitro systems).
Non-intestinal cell systems
Madin Darby canine kidney (MDCK) cells
were isolated from a dog kidney by Madin &
Darby (37). They are currently used to study
the regulation of cell growth, drug metabolism, toxicity and transport at the distal renal
tubule epithelial level. MDCK cells have been
shown to differentiate into columnar epithelial
cells, and to form tight junctions when cultured on semi-permeable membranes.
The use of these cells as a cellular barrier
model for assessing intestinal epithelial drug
transport was discussed by Cho et al. (38).
The results suggested that MDCK cells, like
Caco-2 cells, are suitable for molecular-permeability screening studies. Interestingly,
these cells do not need 3 weeks in culture
655
before they can be used and, unlike Caco-2
cells, they do not express P-gp.
Small-intestine cell lines from fetal and
neonatal rats
Cell lines such as IEC (39) and RIE (40)
have been isolated after the repeated cloning
of epithelial cells from neonatal rat small
intestines. These cell lines show morphological and functional characteristics which
suggest that they are derived from crypt
cells (41). The IEC line was specifically
employed to analyse the role of growth factors in epithelial cell physiology, and for
studies on the specific functions of intestinal
cells (for example, involving amino acids,
glucose and nucleotide transport, or cholesterol synthesis), as well as to perform fundamental studies (36). In contrast, only a few
studies have dealt with the passage of test
compounds.
Caco-2 cells
Caco-2 cells are the most popular cellular
model in studies on passage and transport
(Table II). They were derived from a human
colorectal adenocarcinoma. In culture, they
differentiate spontaneously into polarised
intestinal cells possessing an apical brush
border and tight junctions between adjacent
cells, and they express hydrolases and typical
microvillar transporters.
This cell line was first used as a model for
studying differentiation in the intestinal
epithelium, and later for estimating the relative contributions of paracellular and transcellular passage in drug absorption.
Caco-2 cells, despite their colonic origin,
express in culture the majority of the morphological and functional characteristics of
small intestinal absorptive cells, including
phase I and phase II enzymes, detected
either by measurement of their activities
toward specific substrates, or by immunological techniques.
However, CYP3A, which is present in
almost all intestinal cells, is very weakly
expressed in Caco-2 cells. Treatment with
1α,25-dihydroxyvitamin D3, an inducer of
CYP3A4 at the mRNA level, and transfection
of CYP3A4 cDNA, are two ways of increasing
CYP3A expression levels in Caco-2 cells.
However, these expression levels do not
reach the levels observed in vivo (42, 43).
With regard to phase II enzymes, Caco-2 cells
express N-acetyl transferase and glutathione
transferase activity.
E. Le Ferrec et al.
656
Table II: Characteristics of parental Caco-2 cells
Origin
Human colorectal adenocarcinoma
Growth in culture
Monolayer epithelial cells
Differentiation
14–21 days after confluence in standard culture medium
Morphology
Polarised cells, with tight junctions, apical brush border
Electrical parameters
High electrical resistance
Digestive enzymes
Typical membranous peptidases and disaccharidases of the
small intestine
Active transport
Amino acids, sugars, vitamins, hormones . . .
Membrane ionic transport
Na+/K+ ATPase, H+/K+ ATPase, Na+/H+ exchange,
Na+/K+/Cl– co-transport, apical Cl– channels
Membrane non-ionic transporters Permeability-glycoprotein, multidrug resistant associated
protein, lung cancer associated resistance protein
Receptors
Vitamin B12, vitamin D3, epidermal growth factor, sugar
transporters (GLUT1, GLUT3, GLUT5, GLUT2, SGLT1)
In addition, many groups have demonstrated the presence of P-gp activity in Caco2 monolayers, at levels higher than those
found in the human colon in vivo (44, 45).
The interpretation of transport data is therefore confusing, and is not always in agreement with in vivo observations, even when
P-gp is blocked by specific inhibitors. Other
membrane transporters, i.e. MRP and LRP,
are also expressed (Table II).
Interestingly, TC7 cells, isolated after the
exposure of Caco-2 cells to methotrexate,
express CYP3A at a higher level than their
parental counterparts (Table III; 46). TC7
cells offer marked advantages over parental
Caco-2 cells, because they express CYP3A,
actively transport taurocolic acid, and have
lower levels of P-gp compared with the parent Caco-2 cells. Several studies have demonstrated that TC7 cells are a good alternative
to the Caco-2 parental line for drug transport
studies (49).
Caco-2 cells grow as a monolayer and differentiate on a semi-permeable membrane.
Thus, separating the apical compartment
from the basolateral compartment, which
correspond to the intestinal lumen side and
the serosal side, respectively, is possible (Figure 4). The complete morphological and
functional differentiation of Caco-2 requires
3 weeks in culture under the conditions
described in Table IV.
Before using this model, various controls
(summarised in Table V) have to be performed.
Calculation of the permeability coefficient
and interpretation of results
In most cases, three parameters are determined: the apparent permeability (Papp), the
flux, and the effective permeability (Peff).
The coefficient of permeability is used in in
vitro techniques, and the values are expressed as cm/second and are calculated by
the following equation:
ECVAM Workshop 46: the intestinal barrier
Table III: Expression of mRNAs of
various CYPs in Caco-2 and TC7
cells
CYP
Caco-2
TC7
+
+
+
+
+
–
+
–
+
–
+
–
+
+
+
–
1A1
2B6
2C8/19
2D6
2E1
3A4
3A5
3A7
See reference (61).
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Papp =
dQ
VdC
=
dT ⋅ A ⋅ C0
dT ⋅ A ⋅ C0
Equation 1
where V = sample volume (ml), dC = concentration variations, dQ = quantity variations, dT = time variations, C0 = the initial
concentration in the donor compartment,
and A = exposed surface (cellular monolayer
in cm2). In the above formula, dQ/(dT ⋅ A)
represents the mass transfer per unit time
and unit surface across the monolayer.
The effective permeability is used in in situ
and organotypic techniques, and the values
are calculated as follows:
Peff =
(Cin – Cout) ⋅ Qin
Cout ⋅ 2πRL
Equation 2
where Cin and Qin = the concentration and
the quantity of compound at the entry of the
Table IV: Culture conditions for use of Caco-2 cells in passage assays
Inactivated serum in the medium on the apical site
20%
Inactivated serum in the medium on the basolateral site
20%
Coating
Type 1 collagen (not required
with 0.4µM filters)
Not added under basal culture
conditions
Additive
Non-essential amino acids
Glucose
Glutamine
Antibiotics
1%
25mM
2mM
Streptomycin (100µg/l) (optional)
Penicillin (100mU/ml) (optional)
CO2
10% or 5%
pH
Usually 7.4 at both sides or 7.4 in
basolateral side and 6.5 in apical
side (57)
Cell density at seeding
2.5 × 105 – 4 × 105 cells/cm2
Number of passages
25–100
E. Le Ferrec et al.
658
Figure 4: A schematic respresentation of culture of Caco-2 cells on a microporous
filter
apical side
microporous
filter
cell monolayer
basolateral side
intestinal segment, Cout = the concentration
at the exit, R = the radius, and L = the
length of the intestinal segment.
The accuracy of measurements naturally
depends upon the precision of dQ (Equation
1) or Cout (Equation 2). Moreover, Co (Equa-
tion 1) and Cin (Equation 2) are limited by
the solubility of the drug, the analytical sensitivity, and the effects of high drug concentrations on epithelial integrity.
The classical method for studying transport from the apical to the basolateral sides
Table V: Main controls for cell monolayers
Transepithelial electrical
resistance (TEER)
Cell monolayer integrity. TEER measurement, depending
on the filter area, can reveal a toxicity or an opening of tight
junctions induced by the drug. For CaCo-2 cells, TEER
values are 260–420Ω/cm2 (62–71);
human intestine: 12–69 (64), rat ileum: 35 ± 4.9 (62);
rat colon: 100 ± 26 (62).
Differentiation markers
By determination of sucrase-isomaltase, aminopeptidase and
alkaline phosphatase activities
Morphological differentiation
By electron microscopy
Permeability-glycoprotein (P-gp)
expression
By Western blotting or measurement of P-gp activity and by
using immunohistochemistry
Absence of contamination by
mycoplasma
Detection by standard microbiological methods
Integrity of the cell monolayer by Mannitol, PEG 4000, lucifer yellow
measuring permeability of test
compounds
ECVAM Workshop 46: the intestinal barrier
659
Figure 5: A schematic representation of the method used for measurement of
transmembrane passage (apical [A] to basolateral [B]) of a drug in Caco2 cells
transfer of support at different times
support
donor
compartment (A)
receiver
compartment (B)
relies on the transfer of the cell culture support at different times, as described in Figure
5.
When samples are removed from the
receiver chamber and replaced with control
medium, a correction must be made to
account for the corresponding dilution. The
absence of effects of the drug on permeability characteristics and/or electrical parameters has to be verified (TEER measurement).
A linear relationship between the A-to-B
flux and the B-to-A flux at several concentration levels is an indication of a passive diffusion mode. A tight correlation between the
flux (or Papp) and TEER is of significance in
implying the paracellular route of drug
transport. When the flux can be saturated,
the occurrence of an energy-dependent
and/or transporter-mediated transport is
likely. In this case, the Michaëlis–Menten
principles can be applied (for example, Km,
Vmax). Many pharmacological tools exist that
can explain transport mechanisms, for example, P-gp involvement, paracellular passage
(Table VI).
Comparison of the models
Consistently good absorption is a key factor
in selecting new drug candidates for development. In the discovery stage, drug absorption studies can be performed only in
laboratory animals and/or in in vitro systems
where the absorption process can be characterised both qualitatively and quantitatively.
The various experimental protocols for
predicting the fraction absorbed in humans
from permeability coefficients have their
own advantages and limitations (Table VII).
The choice of model depends on compound
availability, the stage of the project, and the
questions to be answered. Drug absorption
consists of passage through the epithelial
layer and uptake into the bloodstream. In
some models, such as the Ussing chamber or
the everted gut sac, the drug must traverse
all the intestinal wall, part of which might be
rate-limiting in vitro, but not involved in
vivo, thus leading to an underestimation of
transport values. Cell lines, such as Caco-2,
can be considered to be good models that
mimic the physiological situation, as the
drug traverses only the epithelial layer. However, it must be noted that one of the great
differences between the in vivo situation and
cell lines is the absence of mucus, which
might limit absorption, especially that of
lipophilic drugs. Moreover, cell monolayers
contain only one cell type. Co-cultures consisting of cell lines with enterocytic markers
(such as Caco-2 cells) and cell lines with
mucus secretory functions (such as HT29MTX) have been proposed (47).
Caco-2 monolayers have emerged as a suitable model for studying drug absorption
(Table VIII). Most studies with Caco-2 monolayers were performed to determine whether
a drug is actively or passively transported
across the intestinal epithelium, and to pro-
E. Le Ferrec et al.
660
vide new insights into the regulation of drug
transport.
However, the predictions of absorption in
humans based on permeability data obtained
with Caco-2 monolayers from different laboratories are not satisfactory. For example,
Artursson & Karlsson (48) have compared the
passage of 20 structurally unrelated drugs in
Caco-2 cell monolayers with the extent of
absorption in humans after oral administration, and concluded that drugs having a complete absorption in humans were found to
have a high permeability coefficient (Papp ≥ 1
× 10–6cm/s) in Caco-2 cells, whereas poorly
absorbed drugs had a low permeability coefficient (Papp < 1 × 10–7cm/s). However, similar
studies (49, 50) did not lead to the same
results (Table IX). The reasons for such a discrepancy between laboratories in the reported
permeability values are not clear.
Such results indicate that, although Caco2 monolayers are a useful model for ranking
drugs according to their permeability, they
cannot be used to quantitatively predict
human absorption in vivo. As an example,
Gan et al. (51) reported that the Caco-2 permeability coefficient of ranitidine was 1 ×
10–7cm/s (i.e. corresponding to a low permeability), whereas the human bioavailability
of this drug is good (50–70%).
Other attempts have been made to compare in vitro and in vivo drug permeability
Table VI: Pharmacological agents for use in passage studies
Mechanism
Agents
P-gp involvement
Inhibitor
MRP involvement
LRP involvement
Verapamil
≤ 0.5mM in apical (A)
and basolateral (B) sides
Quinidine
0.5–1mM in A and B
Cyclosporin A
50µM in A and B
Substrate
Rhodamine 123
1mM
Inhibitor
As for substrates
Substrate
Leucotriene C4,
S-2,4-dinitrophenyl
glutathione, PAH,
doxorubicin,
etoposide,
vinblastine,
methotrexate
Increase the transport
from the apical side to
the basolateral side
(if the drug is added
on the apical side)
Efflux of rhodamine at
the apical side
Inhibitor
Substrate
Paracellular
transport
(by action on
tight junctions)
Effects
Anthracycline
EGTA, cytochalasine
Increase the transport,
if paracellular
P-gp = permeability glycoprotein, MRP = multidrug resistance associated protein, LRP =
lung cancer associated resistance protein.
It is a reference model.
A difficult technique with local anaesthesia at the time
All physiological factors that influence passage are present. of catheter introduction.
Allows studies in humans.
Not used either in development or routinely.
Good correlation with pharmacokinetic studies.
All cell types and the mucous layer are present.
A relatively fast and inexpensive technique.
Can be used for mechanism of absorption or formulation
studies.
Drug absorption and passage at specific intestinal sites
are possible.
The test drug can be added on either the apical or the
basolateral side.
Metabolism studies are possible.
A human and animal model.
Human intestinal
perfusion
Intestinal gut sac
(rat)
Ussing chamber
(human, rat)
Measurement techniques must be sensitive, since the
drugs are diluted in the diffusion chambers.
Cell viability is limited.
The drug must cross the whole intestinal wall.
Availability of human tissue is limited.
Not used for screening.
Not a perfused model.
The drug must cross the whole intestinal wall.
It is an animal model.
Used in development, but not routinely.
The increase of luminal hydrostatic pressure during the
experiment can influence intestinal permeability.
It is an animal model.
Integrates passage and metabolism aspects.
All physiological factors that influence passage are present.
Studies of absorption in particular sites of the
intestine are possible.
Studies of direct effects of the drug on intestinal
absorption are possible.
In situ technique
(rat)
Limitations
Advantages
Techniques
Table VII: Advantages and limitations of the various models
Relatively fast and simple method.
A flexible model.
Can be used for mechanism transport studies.
The test drug can be exposed at the apical or the
basolateral side.
Human cells.
Can be used for drug screening testing.
Express CYP3A.
Growth faster than that of Caco-2 cells.
Need less glucose than Caco-2 cells.
Fast and simple method.
Can be used for screening testing.
Can be used for measurement of passive diffusion.
Do not express P-gp.
Caco-2
cells
TC7
cells
MDCK
cells
Cell
culture
P-gp = permeability glycoprotein; MDCK = Madin Darby canine kidney.
Advantages
Techniques
Table VII: continued
Not an intestinal model.
It is an animal model.
Physiological factors that influence passage are not present
(mucous, bile salts, cholesterol).
A static model.
Cells have a tumoral origin.
A model with only one cell type.
Influence of P-gp difficult to estimate.
Limitations
ECVAM Workshop 46: the intestinal barrier
663
Table VIII: Examples of mechanistic and drug absorption studies using Caco-2
cells
Route of passage Factor influencing drug absorption
Model used References
Paracellular
Molecular size
Flexibility of drug geometric structure
Caco-2
Caco-2
(65)
(62)
Transcellular
Lipophilicity
Hydrogen bond
Caco-2
Caco-2
(63, 71)
(66, 67)
(Table X). Comparison of the permeability
coefficients of a series of drugs by using
Caco-2 cells and the double-balloon technique in the human jejunum (52, 53), showed
that the permeability of drugs with complete
absorption differed by two-fold to four-fold
between the in vitro and in situ models,
whereas the permeability of drugs with poor
absorption differed as much as 30-fold to 80fold. Thus, measurement of permeability by
using Caco-2 cells allows only a qualitative
comparison. Moreover, correlations between
the different techniques are better when the
mode of passage is passive diffusion (6, 25,
50, 54). When a receptor-mediated molecular
transport is involved, the calculation of correlation coefficients is difficult, or even
impossible.
Because the properties of Caco-2 monolayers can vary with time in culture, passage
number and culture medium composition, it
is important to include a reference drug
when screening for the permeability of test
drugs.
Table IX:
It should also be borne in mind that monolayers constitute a two-dimensional system,
whereas the intestinal mucosa is a threedimensional one, i.e. it is not flat, but convoluted because of villi and folds. Depending on
the actual available surface area for absorption, the estimates are made difficult by the
calculation of the permeability coefficient,
where the surface area of the tube (the balloon technique) is used. The actual surface
might be larger; hence, the reported permeability would be too high.
Since in vitro models cannot give quantitative predictions of drug absorption in
humans, another possibility is to use animal
models (49). This is based on the assumption
that the membrane permeability of drugs is
not species-dependent (55). Since the composition of the plasma membrane of intestinal
epithelial cells is similar across species, the
permeability of drugs (simple diffusion)
across the wall of the gastrointestinal tract
could be expected to be similar (Table X).
Drug absorption in humans can be extrapo-
Permeability limit values proposed by several authors using Caco-2
cells (56)
Apparent permeability in Caco-2 cells
Low permeability
limit values
High permeability
limit values
Reference
≤ 1 × 10–7cm/second
< 1 × 10–5cm/second
≥ 1 × 10–6cm/second
> 7 × 10–5cm/second
> 3 × 10–5cm/second
(48)
(64)
(50)
20
20
20
12
12
10
16
12
TC7 vs Caco-2 cells (68)
Caco-2 cells vs human
oral route (48, 68, 69)
TC7 cells vs human
oral route (68)
“Rat” Ussing chamber vs
“human” Ussing chamber (70)
“Rat” Ussing chamber vs
oral route in humans (70)
“Rat” in situ perfusion vs
“human” in situ perfusion (53)
“Rat” in situ perfusion vs
human oral route (50)
“Rat” intestinal gut sac vs
human oral route (50)
For preliminary screening.
For small organic molecules.
High correlation exists between the two models for passively absorbed
molecules. The two models can be used to predict absorption in humans.
A marker should be included in the “rat model” to follow viability.
The “rat” Ussing chamber technique is especially useful for screening
substances having local pharmacological and transporter-mediated effects.
Caco-2 and TC7 cells are used for the prediction of passive human passage.
Caco-2 and TC7 cells are used for the prediction of passive human passage.
Based on morphological and biochemical parameters and also on transport
characteristics, it appears that TC7 cells are a reliable alternative to Caco-2
parental lines for transport studies.
The Papp correlation coefficient calculated by these two techniques is 0.79.
Moreoever, comparison of the Papp values calculated in MDCK and Caco-2
cells in relation to the human oral route is 0.58 and 0.54, respectively,
indicating that both cells are suitable.
Comment
+
+++
+++
+++
++
+++
+++
+++
++
Correlation
poly(ethylene
glycol) (PEG),
furosemide,
propranolol,
atenolol,
metoprolol,
terbutaline,
enalapril,
L-dopa,
D-mannitol
D-Glucose,
Currently
tested
compounds
+ = limited correlation, ++ = median correlation, +++ = high correlation. MDCK = Madin Darby canine kidney. Papp = apparent permeability.
55
Caco-2 vs MDCK cells (55)
Technique
Number of
compounds
tested
Table X: Correlations between various techniques
ECVAM Workshop 46: the intestinal barrier
Class II
low solubility
high permeability
Class I
high solubility
high permeability
Class IV
low solubility
low permeability
Class III
high solubility
low permeability
solubility
Figure 7: In vitro/in vivo correlation
permeability
permeability
Figure 6: Biopharmaceutical classification system (57)
665
correlationb
Class IV
no expected
correlation
Class III
low or no
correlation
solubility
a If
the dissolution rate is slower than the
gastric emptying rate, otherwise limited or
no correlation.
See ref. 57.
b
lated reasonably well from animal data,
when information on first-pass metabolism
is also available (56). Bioavailability, however, differs substantially among species,
presumably as a result of species differences
in the scale of first-pass metabolism.
The United States Pharmacopeial Convention (USP) has proposed two schemes for
predicting in vivo and in vitro correlations.
Figure 6 illustrates in vivo/in vitro correlations for molecules classified according to
solubility and permeability. Figure 7 illustrates correlations between in vivo and in
vitro data. It shows that highly permeable
molecules (classes I and II) exhibit a high in
vivo/in vitro correlation, whereas poorly permeable molecules (classes III and IV) show a
low in vivo/in vitro correlation.
The USP proposes the selection of reference molecules with known Papp values
(methotrexate: 1.2 × 10–6cm/s; propranolol
hydrochloride: 28 × 10–6cm/s; testosterone:
73 × 10–6cm/s). If the Papp values obtained
with Caco-2 cells are the same as the Papp
values given by the USP (± 20%), the model
can be considered to be valid (57). However,
because the USP defines rigid limits, such
recommendations are not accepted by all
users. Many researchers prefer to use a collection of laboratory values, with internal
specifications and criteria of acceptance for
each assay system. Another approach chosen
by some researchers is to delineate the
correlationa
If in vivo and in vitro dissolution rates are
similar (57).
physicochemical properties that favour intestinal absorption, and they have developed
computational methods for their prediction.
The best-known method is probably the
“rule of five” designed by Lipinski and coworkers (58) from an analysis of 2245 drugs.
As implemented in their system, the “rule of
five” generates an alert (an indication of possible absorption problems) for compounds,
when: 1) there are more than five hydrogenbond donors (expressed as the sum of
hydroxyl and primary amine groups); 2) the
molecular mass is over 50; 3) the Log P is
over 5 (or MlogP is over 4.15); 4) there are
more than ten hydrogen-bond acceptors
(expressed as the sum of nitrogen and oxygen atoms); and 5) compound classes that
are substrates for biological transporters are
exceptions to the rule.
The “rule of five” method was originally
proposed because many of the large numbers
of hit compounds selected by HTS did not
possess “drug-like” properties. Obviously,
the growing number of publications in this
field indicate that methods for predicting
“drug-likeness” are already having a major
impact on the design and selection of compounds in pharmaceutical companies. The
routine use of experimental absorption systems in the pre-screening of compounds is
providing valuable data, and should permit
E. Le Ferrec et al.
666
the development of improved models for
drug absorption. The value of such experimental and theoretical systems will become
apparent when drug development times are
reduced.
5.
6.
Conclusions and Recommendations
1. Knowledge on P-gp expression in human
intestine and cell lines should be
improved.
2. Cell lines other than Caco-2 (for example,
HT29, TC7, 2/4/A1) should be more fully
investigated.
3. The absence of mucus in cell lines is a
problem. Co-culturing cell lines expressing enterocytic markers (for example,
Caco-2 cells) with cell lines exhibiting
mucus secretory properties (for example,
the HT-29 cell line [59]) could permit this
limitation to be overcome.
4. Internet sites and software should be created and developed to facilitate communication among researchers concerning the
intestinal barrier, and also to improve in
silico predictions.
5. Simple and powerful cell-culture systems
should be commercialised (for example,
dynamic systems, three-dimensional models, automated systems).
6. Suitable conditions permitting the safe
and ethical preservation, transport and
use of human intestinal fragments (from
tissue banks) should be established.
7. Comparison of data from different laboratories on drug passage across the intestinal barrier remains unstandardised. A
consensus should therefore be sought on
the optimal experimental conditions
(medium, reference substances, etc.), and
harmonised with the FDA and USP texts.
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