Review

Caco-2 cell permeability assays to

measure drug absorption
Richard B van Breemen† & Yongmei Li
†University
of Illinois College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy,
1. Introduction 833 S. Wood Street, Chicago, IL 60612, USA

2. Caco-2 cell monolayers as

Caco-2 cells are a human colon epithelial cancer cell line used as a model of
models of intestinal absorption
human intestinal absorption of drugs and other compounds. When cultured
and metabolism
as a monolayer, Caco-2 cells differentiate to form tight junctions between cells
3. Conclusions to serve as a model of paracellular movement of compounds across the mono-
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12

4. Expert opinion layer. In addition, Caco-2 cells express transporter proteins, efflux proteins,
and Phase II conjugation enzymes to model a variety of transcellular pathways
as well as metabolic transformation of test substances. In many respects, the
Caco-2 cell monolayer mimics the human intestinal epithelium. One of the
functional differences between normal cells and Caco-2 cells is the lack of
expression of the cytochrome P450 isozymes and in particular, CYP3A4, which
is normally expressed at high levels in the intestine. However, Caco-2 cells may
be induced to express higher levels of CYP3A4 by treatment with vitamin D3.
Caco-2 cell monolayers are usually cultured on semipermeable plastic supports
that may be fitted into the wells of multi-well culture plates. Test compounds
are then added to either the apical or basolateral sides of the monolayer.
For personal use only.

After incubation for various lengths of time, aliquots of the buffer in opposite
chambers are removed for the determination of the concentration of test
compounds and the computation of the rates of permeability for each com-
pound (called the apparent permeability coefficients). Although radiolabelled
compounds were used in the original Caco-2 cells monolayer assays, radio-
labelled compounds have been replaced in most laboratories by the use of
liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spec-
trometry (LC-MS-MS). Mass spectrometry not only eliminates the need for
radiolabelled compounds, but permits the simultaneous measurement of mul-
tiple compounds. The measurement of multiple compounds per assay reduces
the number of incubations that need to be carried out, thereby increasing the
throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add
another dimension to Caco-2 assays by facilitating the investigation of the
metabolism of compounds by Caco-2 cells.

Keywords: Caco-2 cells, drug absorption, intestinal absorption, intestinal metabolism,

liquid chromatography-mass spectrometry

Expert Opin. Drug Metab. Toxicol. (2005) 1(2):175-185

1. Introduction

1.1 Role of intestinal absorption assays in drug development

During the last 15 years the synthesis of compounds for drug discovery efforts has
evolved from one at a time to combinatorial synthesis, which enables the simultane-
ous synthesis and purification of hundreds of compounds [1]. Furthermore, high-
throughput screening methods have been developed to facilitate the rapid identifica-
tion of pharmacologically active compounds. However, in vitro activity does not
Ashley Publications guarantee in vivo efficacy. On average, for every new drug entity that reaches the
www.ashley-pub.com market, at least 5000 compounds are screened, and the majority of lead compounds
that emerge from drug discovery programmes fail during development due to

10.1517/17425255.1.2.175 © 2005 Ashley Publications Ltd ISSN 1742-5255 175

 Caco-2 cell permeability assays to measure drug absorption

Table 1. Models of intestinal absorption of pharmaceutical compounds.

Model system Advantages Limitations Reference

Nonbiological models
Immobilised artificial membrane Models transcellular passive diffusion Low throughput. Cannot model effects of [5]
chromatography transporters, vesicular transport, efflux
systems or paracellular diffusion
Parallel artificial membrane High-throughput model of transcellular Cannot model effects of transporters, [6]
permeability passive diffusion vesicular transport, efflux systems or
paracellular diffusion
In silico computer modelling of Inexpensive high-throughput model of Does not model transporters, vesicular [7]
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12

membrane diffusion transcellular passive diffusion transport, efflux systems or paracellular

Biological models
In vivo animal models
In situ intestinal segments
Everted gut sacs
Intestinal mucosa/Ussing
For personal use only.
Encompasses mechanisms of
absorption, efflux and metabolism
Encompasses mechanisms of
absorption, efflux and metabolism
Provides accurate measurement of
intestinal permeability
Provides accurate measurement of
diffusion
Expensive and low throughput. Individual
roles of intestinal absorption and hepatic
metabolism on bioavailability undelineated
Expensive and low throughput. Uses [8]
nonhuman tissue. Requires large quantities
of test compounds
Viability lost rapidly. Fresh tissue required [7]
for each assay. Often damaged during
preparation
Loses viability rapidly. Fresh tissue required [9]

chamber intestinal permeability for each assay. Often damaged during

Isolated membrane vesicle
Caco-2 cell monolayer
Caco-2 cells treated with
vitamin D3 to express
cytochrome P450 3A4
preparation
May be cryopreserved for use as Often damaged during preparation. [7]
needed Enzymes lack apical and basolateral polarity
Models transcellular and paracellular Low throughput. Low expression of [10]
permeability, vesicular transport, active intestinal cytochrome P450 isozymes
transport, facilitated transport and
efflux systems
Same advantages as Caco-2 cell Low throughput [11]
monolayer, but also models intestinal
metabolism

unacceptable pharmacokinetic, metabolic, and toxicity
profiles. A large proportion of new drug entities that are
tested in clinical trials, therefore, fail due to unexpected toxic-
ity or pharmacokinetics [2]. Thus, it is important to have relia-
ble and accurate pharmacokinetic and metabolic data
available as early as possible during drug discovery in order to
select only those drug candidates for development that are
most likely to be useful as drugs.
As a result of the high failure rate of compounds emerging
from drug discovery programmes, many pharmaceutical com-
panies have incorporated pharmacokinetics and toxicity evalua-
tion in the early screening phase of the drug discovery process.
Furthermore, due to time constraints and the large numbers of
compounds produced by combinatorial chemistry and high-
throughput screening, the demand for in vitro assays that can
be used to evaluate the biological systems that affect pharma-
cokinetics of drug candidates both qualitatively and quantita-
tively has risen dramatically. As oral delivery is the most
convenient form of administration of pharmaceutical agents in
terms of patient compliance, oral administration is preferred
when possible. However, intestinal absorption is a formidable
barrier that restricts the oral bioavailability of many potential
new drugs. To address the need for in vitro assays of intestinal
permeability of potential new drugs as part of the drug develop-
ment process, a variety of approaches has been developed and
are summarised in Table 1. Among these approaches, the
Caco-2 cell monolayer assay of intestinal permeability has
emerged as one of the standard in vitro tools [3,4].
1.2 Drug absorption by the human intestine
The small intestine is the primary organ responsible for the
absorption of nutrients, and serves as a physical and biological
barrier to digestive enzymes and ingested foreign
substances [7]. Among the many factors influencing intestinal
absorption of drugs, their dissolution rate, solubility and
intestinal permeability are the most influential [12]. The disso-
lution rate and solubility determine how fast the drug
achieves its maximum concentration in the luminal intestinal

176 Expert Opin. Drug Metab. Toxicol. (2005) 1(2)


Facilitated
transport
Endocytosis
Paracellular
pathway
Active transport
Efflux
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Transcellular
pathway
Drug-metabolising
enzymes
Figure 1. Routes of drug absorption at the intestinal brush border. Differentiated Caco-2 cell monolayers exhibit all of these routes
of drug absorption. Although Caco-2 cells exhibit phase conjugation enzymes, such as glucuronyl transferases and sulfotransferases,
they usually express abnormally low levels of the cytochrome P450 isozymes CYP3A and -1A. However, Caco-2 cell lines can be treated
to express higher levels of human CYP3A.
fluid and are constants that are easily measured in vitro. How-
For personal use only.
ever, the determination of intestinal permeability is a complex
biological process that is much more challenging to measure.
Intestinal absorption of orally administered drugs occurs
via three pathways: passive diffusion, carrier-mediated or
-limited transport, and vesicular transport [13]. The more
complex biological models, such as the Caco-2 cell assay,
encompass all of these pathways, but the nonbiological
models currently in use do not (Table 1 and discussion
below). Passive diffusion can be divided further into paracel-
lular passive diffusion and transcellular passive diffusion
(Figure 1). In paracellular passive diffusion, chemicals are
transported across the epithelium through the aqueous,
extracellular route. Differences in drug concentration, electri-
cal potential and hydrostatic pressure between the luminal
and blood sides of the intestinal epithelium create gradients,
which are the driving forces for extracellular passive
diffusion. The tight junctions between cells are the major
barrier to paracellular passive diffusion. Similarly, electro-
chemical gradients can also drive transcellular transport. As
the surface of a cell membrane is much larger than the sur-
face of tight junctions (99.9 versus 0.01%) [14], compounds
using the transcellular pathway tend to have higher absorp-
tion rates than compounds that cross the epithelium via the
paracellular pathway. It should be noted that the Caco-2 cell
monolayer model may be used to measure intestinal permea-
bility that results from both transcellular and paracellular
absorption, whereas simpler membrane models only predict
transcellular passive diffusion.
Although some drugs are not able to pass through entero-
cytes by passive diffusion or carrier-mediated pathways, they
can still cross the intestinal epithelium through vesicular
Expert Opin. Drug Metab. Toxicol. (2005) 1(2)
van Breemen & Li
Transcytosis
Gap
junction
transport processes. There are three types of vesicular trans-
port pathways, including fluid-phase endocytosis, receptor-
mediated endocytosis, and transcytosis (Figure 1) [13]. Some
peptides and proteins are absorbed through fluid-phase endo-
cytosis [15]. In this process, the plasma membrane forms vesi-
cles or pinosomes that engulf the dissolved molecules and
move inwardly. The dissolved molecules in the vesicles and
pinosomes are transported to endosomes, which eventually
fuse with lysosomes. Macromolecules can be absorbed by the
process called receptor-mediated endocytosis, in which
macromolecules bind to the receptors on the membrane and
the receptor–ligand complexes cluster together to form clath-
rin-coated pits. Next, a process called sorting leads to the
destruction of the ligand in the lysosomes [13]. However, the
ligand might bypass the lysosomes and be released from the
basolateral side of enterocytes in a process called transcytosis.
Only biological models of intestinal absorption, such as the
Caco-2 cell monolayer assay, encompass vesicular transport as
a mechanism of predicting the intestinal absorption of drugs.
The intestinal mucosa contains various transporter
proteins, such as transporters of di- or tripeptides, large
neutral amino acids, bile acids, nucleosides and monocarboxy-
lic acids, and some of these mediate the absorption of drugs
(Figure 1) [16-18]. Some transporters are influx transporters,
which bind molecules in the intestinal fluid on the apical side
and transport them to the basolateral side of enterocytes; for
example, H+/oligopeptide cotransporter (PEPT1) functions as
an uptake transporter of peptidomimetic drugs, including
angiotensin-converting enzyme inhibitors, β-lactam anti-
biotics, and renin inhibitors [19-21]. Some other transporters
have the opposite function and transport compounds from the
cell cytoplasm to the intestinal lumen and, therefore, reduce
177
 Caco-2 cell permeability assays to measure drug absorption
the absorption of the compounds. Transporters in the multi-
drug resistance (MDR) and multidrug resistance-associated
protein (MRP) families are efflux transporters that hinder the
absorption of certain molecules [22]. In particular, the efflux
transporter P-glycoprotein has been shown to prevent the net
absorption of many drugs [23]. Efflux and transporter proteins
are expressed by Caco-2 cell monolayers (Figure 1).
1.3 Role of intestinal drug metabolism in
bioavailability
As xenobiotic metabolism is usually considered a function of
the liver, most metabolic studies have focused on liver
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12
enzymes. However, enzymes of the cytochrome P450
(CYP)3A subfamily, which are the most abundant Phase I
drug-metabolising enzymes in humans, have been found to be
expressed at high levels in the mature villus tip enterocytes of
the small intestine [24-26] (Figure 1). Furthermore, immuno-
histochemical studies, protein measurements and enzyme
activity determinations have shown that some CYP3A iso-
zymes have higher concentrations in the small intestine than in
the liver [27,28]. Other enzymes, such as CYP1A and Phase II
enzymes, are also found in the small intestine. These enzymes
might have significant impact on the bioavailability of some
orally administered drugs. As summarised in Table 1 and dis-
For personal use only.
cussed below, only the biological models of intestinal permea-
bility may be used to account for intestinal metabolism as a
factor in the absorption of orally administered drugs.
1.4 Models of intestinal absorption
Lipophilicity (log P/log D) had been a commonly used
physiochemical property in the prediction of membrane
permeability [29]. Lipophilicity can be measured by deter-
mining the logarithm of the partition coefficient between
octanol and water. However, it has been recognised that
there is a substantial difference between octanol/water and
membrane/water partition coefficients [30]. As a result, the
use of this single property of lipophilicity is an oversimplifi-
cation that results in unreliable predictions. Therefore,
more sophisticated physiochemical methods have been
developed, including immobilised artificial membrane
chromatography and the parallel artificial membrane
permeability assay (PAMPA).
Immobilised artificial membrane chromatography is
essentially reverse-phase liquid chromatography in which lip-
ids emulating those of cell membranes are used to replace the
usual hydrocarbon phase on the solid support [5]. Using this
technique compounds with longer retention (k′) are pre-
dicted to have good permeability across lipid bilayers based
on the assumption that the major factor regulating drug
intestinal transport is the ability to diffuse through cell mem-
branes. In support of this assumption, reasonable correlation
has been demonstrated between the log k′ and permeability
across Caco-2 cell monolayers [30,31]. A higher throughput
type of artificial membrane assay for drug intestinal permea-
bility is the PAMPA. The system uses 96-well microtitre
178 Expert Opin. Drug Metab. Toxicol. (2005) 1(2)
plates coupled with a spectrophotometric plate reader.
Recent studies have demonstrated good correlation between
the flux in this system and the extent of absorption in
humans for some well-characterised drugs [6,30]. However,
artificial membranes are different from the biological
membranes in that they lack paracellular pores, influx and
efflux transporters, and metabolic enzymes, all of which
influence the extent of intestinal absorption of drugs. There-
fore, although artificial membranes may be useful for the
prediction permeability of passively transported compounds,
they have serious limitations for the prediction of intestinal
permeability of compounds that are small, hydrophilic, or
transported by carrier proteins [7]. Except for deficiencies
with respect to CYP isozyme expression, the Caco-2 cell assay
overcomes these limitations.
The prediction of drug absorption using computational
(in silico) methods is an area of ongoing investigation. In prin-
ciple, in silico methods might be faster and less expensive sub-
stitutes to in vitro or in vivo assays. Although existing in silico
methods range in complexity from relatively simple quantita-
tive models to complex pharmacokinetic and/or pharmaco-
dynamic models, they are still too inaccurate to replace
in vitro and in vivo approaches in determining human drug
intestinal absorption [7].
A variety of animal intestinal tissues are in use as models of
human intestinal permeability of drugs, and include the
everted gut sac, intestinal mucosa in the Ussing chamber, and
isolated membrane vesicles. In the everted sac method, an
intestinal segment is turned inside out, filled with oxygenated
buffer, tied at both ends, and the rate at which a drug crosses
from the incubation medium to the inside is measured [7,13].
In the Ussing chamber method, intestinal tissues are isolated
and cut into strips of appropriate size to fit in the opening of
diffusion chamber. The drug transport rate can then be meas-
ured based on the rate of appearance of the drug on the sero-
sal side. The Ussing chamber method provides accurate
measurements of intestinal permeability [32], but suffers from
some of the same limitations as the everted gut sacs approach.
These problems include rapid loss of viability due to a lack of
active blood and nerve supplies, the potential for morpho-
logical damage during preparation and the need for fresh
tissue with each assay [7].
Isolated membrane vesicles, prepared from either intestinal
scrapings or isolated enterocytes, have been used in suspen-
sion to model the intestinal absorption of drugs. The isolated
membrane vesicles require smaller amounts of test com-
pounds than the everted sac or Ussing chamber methods and
may be cryopreserved for use as needed. Disadvantages
include problems with transporter proteins and enzymes that
are often damaged during vesicle preparation and lack of api-
cal and basolateral polarity which characterises intestinal
mucosa cells in vivo. Due to the many disadvantages of the
everted sac, Ussing chamber and isolated membrane vesicles,
their use is much less common than the cell culture models
such as the Caco-2 cell monolayer model [13].
 van Breemen & Li

Solute

Solute

AP-to-BL permeability BL-to-AP permeability

Figure 2. Caco-2 intestinal epithelial cell assay for intestinal absorption. A solution of a test compound or mixture of compounds
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12

is placed on the apical side of a Caco-2 cell monolayer, and the rates of appearance of the test compounds on the basolateral side of the
cells are measured to assess the permeability of the monolayer for each compound. Alternatively, the test compound or compounds may
be added to the basolateral side in order to test for the presence of active transport or efflux in one direction or the other across the
Caco-2 cell monolayer. Reprinted from LI Y, SHIN YG, YU C et al.: Increasing the throughput and productivity of Caco-2 cell permeability
assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism. Comb. Chem. High
Throughput Screen. (2003) 6:757-767 [10] with permission.
AP: Apical; BL: Basolateral.

Many cell monolayer models have been developed to emu-
late the human intestinal epithelium and are gaining in popu-
larity. These models use immortalised cells that grow rapidly
into confluent monolayers and undergo spontaneous differen-
tiation. Therefore, these cell monolayer models provide an
ideal system for the study of drug absorption by the intestine.
For personal use only.
and for mechanistic studies of drug transport [33]. The Caco-2
cell monolayer permeability assay is reliable, easy to carry out,
and requires only small quantities of compounds. Originally
isolated from a human colon adenocarcinoma, Caco-2 cells
undergo spontaneous enterocytic differentiation in culture to
resemble epithelial cells of the small intestine [34]. When

Several cell lines are in use for modelling human absorption
including Madin Darby canine kidney (MDCK), TC-7,
HT29-MTX, 2/4/A1, and the most popular, Caco-2 cells.
In situ perfusion of intestinal segments from rats or rabbits
has been used for the study of intestinal drug absorption. In
the in situ perfusion experiment, drug solution in physio-
logical buffer is perfused through the isolated cannulated
intestinal segments. Drug absorption is estimated based on
disappearance of drug from the intestinal lumen. Because the
blood supply, nerve and clearance capabilities of the intest-
inal segments remain intact, this method avoids many of the
problems associated with the everted gut sac model and the
Ussing chamber model. This method appears to accurately
predict in vivo intestinal permeability of passively trans-
ported compounds, but needs a scaling factor for accurate
prediction of the permeability of carrier-mediated com-
pounds [8]. In addition, in situ perfusion requires relatively
large numbers of animals for statistically significant data and
larger quantities of test compounds to perform the experi-
ment than the Caco-2 cell assays [7]. Therefore, Caco-2 cell
monolayer assays are more practical for drug discovery and
early drug development studies.
2. Caco-2 cell monolayers as models of
intestinal absorption and metabolism
2.1 Caco-2 cells and intestinal absorption
Human epithelial Caco-2 cell monolayers were used for the
first time to model human intestinal absorption in the late
1980s [3,4]. Since then, this model has become a standard tool
for the prediction of intestinal drug absorption in humans
grown to confluence, cell polarity and tight junctions are
established in the Caco-2 cell monolayers, and several active
transport systems are expressed as in the walls of the human
small intestine. These active transport systems include trans-
porters for bile acids, amino acids, and sugars [35]. P-glyco-
protein, which is the product of the MDR gene, and the
MRPs are also expressed in the cell membrane of Caco-2 cells
and induce a basolateral-to-apical efflux of specific xenobiotic
compounds [36,37]. The apparent permeability coefficients
measured for reference compounds across Caco-2 cell mono-
layers have shown good correlation with in vivo absorption
[38,39]. As a result of these favourable properties, the Caco-2
cell monolayer assay has become a standard in vitro model for
assessing the intestinal permeability and transport of drug
candidates and lead compounds.
Caco-2 cells are cultured on semipermeable polycarbonate
surfaces on inserts that fit into an assay chamber establishing
apical and basolateral chambers. These two chambers are con-
nected only through the monolayer of cells and their semi-
permeable substrate (Figure 2). The apical and basolateral
chambers represent the luminal and blood/mesenteric lymph
sides of the gastrointestinal tract, respectively. Caco-2 cell
monolayer assay culture dishes usually contain 6, 12 or
24 wells. Typically, Caco-2 cells are cultured for ∼ 21 days to
reach confluence and to differentiate into enterocytes exhibit-
ing transporter proteins and tight junctions. In preparation
for the Caco-2 cell monolayer assays, the cell culture medium
is removed from both the apical and basolateral chambers and
replaced with HBSS containing 25 mM HEPES at pH 7.4
and 37 °C. To evaluate the integrity of the monolayers, the
transepithelial electrical resistance (TEER) is usually

Expert Opin. Drug Metab. Toxicol. (2005) 1(2) 179

 Caco-2 cell permeability assays to measure drug absorption

A. B.
MS response at m/z 260

MS response at m/z 181

1800
3.6 1200 6.6 Mannitol
1400 Internal
standard 800
1000
9.2 Propranolol
600 400
200

4 6 8 10 4 6 8 10
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Retention time (min) Retention time (min)

Figure 3. Representative LC-MS chromatograms of (A) the high permeability standard propranolol, and (B) the low
permeability standard mannitol. For the analysis of propranolol, selected ion monitoring of the protonated molecule of m/z 260 and
that of the internal standard acebutolol of m/z 337 was carried out. A reversed-phase C18 column (2.1 × 100 mm, 3.5 µm) was used for
the HPLC separation. The mobile phase for the propranolol analysis consisted of a linear gradient from 0.5% acetic acid to methanol at a
flow rate of 0.2 ml/min. For the analysis of mannitol, the deprotonated molecule of m/z 181 was monitored, and HPLC separation was
carried out using an aminopropyl column (2.0 × 150 mm, 5 µM) with a mobile phase consisting of a linear gradient from water to
acetonitrile at a flow rate of 0.5 ml/min.
HPLC: High performance liquid chromatography; LC-MS: Liquid chromatography-mass spectrometry; MS: Mass spectrometry.
For personal use only.

measured before and after an experiment as an indication of
the tightness of the cellular junctions [40]. Typically, TEER
values > 300 Ω cm-2 indicate adequate monolayer integrity.
During Caco-2 cell assays, the compounds to be evalu-
ated are added to either the apical or the basolateral side of
each cell monolayer to simulate the influx or efflux of com-
pounds across the intestinal epithelium. The concentrations
of these compounds in the Caco-2 permeability assays are
usually in the range of 10 – 500 µM. By carrying out multi-
ple assays with different initial concentrations of test com-
pounds, the participation of saturable receptors or
transporter pathways may be detected. At various time
points (i.e., 10, 20, 30 and 40 min), the concentration of
the test compounds in the receiving chambers are assayed.
The transport rate for a particular concentration of a test
compound is typically expressed as the apparent permeabil-
ity coefficient (Papp), which is calculated using the following
equation:
(1)
P app = V r × ( dC ) ⁄ ( dt ) × 1 ⁄ AC o
where Vr is the volume of the recipient compartment, dC/dt
is the slope of the cumulative concentration of the compound
in the recipient chamber over time, A is the membrane surface
area, and C0 is the compound initial concentration in the
donor chamber [38].
As the Papp is not a constant, it varies according to the
assay conditions and batch-to-batch characteristics of the
Caco-2 cell monolayers. To compare the Papp values for
compounds determined on different days or even in differ-
ent laboratories (interassay variation), it is convenient to
measure some reference compounds with each assay.
Ideally, reference compounds representing high (i.e., pro-
pranolol) and low cellular permeability (i.e., mannitol)
should be measured along with the test compounds [41].
Typical Papp values for mannitol and propranolol from the
apical to the basolateral side of the Caco-2 cell monolayer
are ∼ 0.3 × 10-6 cm/sec and ∼ 5 × 10-5 cm/sec, respectively.
Examples of the liquid chromatography-mass spectrometry
(LC-MS) measurement of mannitol and propranolol in
aliquots from the basolateral side of a Caco-2 cell mono-
layer are shown in Figure 3. Additional details concerning
the measurement of the concentrations of test compounds
in Caco-2 cell experiments are discussed below.
If the Papp values in the apical-to-basolateral direction are
not equal to those in the basolateral-to-apical direction, or if
the Papp values decrease with increasing test compound con-
centration, this suggests the involvement of transporter path-
ways. Additional incubations may then be carried out to
probe for the involvement of specific transporter or efflux sys-
tems. For example, the measurement of Papp values for a test
compound may be repeated in the presence of verapamil
(100 µM) [42] or MK-571 (50 µM) [37], which are inhibitors
of P-glycoprotein and MRP, respectively [43]. Changes in the
Papp value in the presence of such an inhibitor would confirm
its participation in the transport or efflux of the compound.
2.2Caco-2 cells and intestinal drug metabolism
As in the liver, certain CYP isozymes are abundant in the
human small intestine. In particular, CYP3A4 accounts for

180 Expert Opin. Drug Metab. Toxicol. (2005) 1(2)

 van Breemen & Li

Resveratrol-3-sulfate

Absorbance
100
300 nm

(mAU)
Resveratrol-3-glucuronide Resveratrol
0
2500 m/z 227
Resveratrol

0
Mass spectrometer response

600
Resveratrol-3-sulfate m/z 307
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12

Resveratrol-3-glucuronide m/z 403

150
0

2500 Total ion chromatogram

0
0 5 10 15 20 25
Retention time (min)
For personal use only.

Figure 4. Liquid chromatography-mass spectrometry chromatograms of resveratrol and its metabolites after incubation of
resveratrol with Caco-2 cells. Reprinted from LI Y, SHIN YG, YU C et al.: Increasing the throughput and productivity of Caco-2 cell
permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism. Comb. Chem.
High Throughput Screen. (2003) 6:757-767 [10] with permission.

∼ 50% of all CYP isozymes in the human intestine [44]. Expres-
sion of CYP3A4 has also been reported in Caco-2 cell mono-
layers, too. For example, Gan et al. [45] observed a hydroxylated
metabolite of cyclosporin A in Caco-2 cell incubations, which
was a product of CYP3A4. However, other investigators have
reported neither immunological nor functional evidence of
Phase I enzymes in Caco-2 cells [46].
As Caco-2 cells typically underexpress CYP isozymes com-
pared with the human small intestine or even human jejunal
microsomes, this is a limitation in the use of Caco-2 cells as a
model for intestinal Phase I metabolism of orally administered
compounds. To try and overcome this limitation, Schmiedlin-
Ren et al. [47] determined that treatment of Caco-2 cell mono-
layers at confluence with 1α,25-dihydroxyvitamin-D3
resulted in a dose-dependent increase in CYP3A4 mRNA and
protein expression. This treatment also increased levels of
NADP CYP reductase and P-glycoprotein expression in
Caco-2 cells. Subsequently, Fisher et al. [11] used 1α,25-dihy-
droxyvitamin-D3 treated Caco-2 cell monolayers for in vitro
modelling of first-pass intestinal metabolic kinetics. When
applying this system to the metabolic kinetics of midazolam,
which is a substrate for CYP3A4, the results were similar to
those observed in vivo.
Although CYP isozymes are poorly expressed in Caco-2
cells, many other drug-metabolising enzymes are expressed at
levels that are useful for studies of intestinal drug metabolism
[48,49]. For example, hydrolases resembling those at the intesti-
nal brush border are expressed in Caco-2 cells. In addition,
carboxylesterases, uridine diphosphoglucuronosyl transferases,
glutathione-S-transferases, sulfotransferases [50,51] and cyate-
chol-O-methyltransferase are present and functional in
Caco-2 cells. Among these enzymes, the Phase II sulfotrans-
ferases and glucuronyltransferases are particularly significant
in the determination of the bioavailability of orally adminis-
tered compounds, because conjugation of pharmacologically
active compounds usually results in reduction or elimination
of their activity.
For example, Caco-2 cells have been shown to produce sul-
fate and glucuronide conjugates of resveratrol [10,52] (Figure 4).
In another example, sulfation by Caco-2 cells was the primary
route of intestinal metabolism of epicatechin, which is an
antioxidant flavonoid in tea [53]. In another investigation of a
flavonoid natural product, Hu et al. [54] reported that api-
genin was conjugated by Caco-2 cells to form sulfate and glu-
curonide Phase II metabolites and that the sulfate conjugate
was a substrate for an organic anion transporter.
2.3 Higher throughput Caco-2 cell monolayer assays
Caco-2 cells are usually cultured for ∼ 21 days to allow time
for the formation of confluent, differentiated monolayers.

Expert Opin. Drug Metab. Toxicol. (2005) 1(2) 181

 Caco-2 cell permeability assays to measure drug absorption
Modified 3- to 7-day Caco-2 cell culture systems have been
developed in order to expedite the preparation process [55,56].
Although the rank ordering of compounds with respect to
their passive permeability across the Caco-2 cell monolayer
has been demonstrated to be similar whether using the stand-
ard 21-day culture or the 3- to 7-day culture, the expression of
active transporter and efflux proteins, such as P-glycoprotein,
was significantly lower in the accelerated Caco-2 cell cultures.
In addition, as indicated by TEER measurements and micro-
scopy, the monolayer of the accelerated culture is less conflu-
ent as well as less differentiated. Baranchzyk-Kuzma et al. [50]
observed that the specific activity of the sulfotransferases in
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12
Caco-2 cells increases with the confluence and age of the cell
monolayer from 7 to 21 days in culture. Chikhale and Borc-
hardt [57] also found that Caco-2 cells that had been cultured
for 25 days showed greater drug-metabolising activity than
cells cultured for only 11 days. Therefore, the 21-day Caco-2
cell culture period still remains the standard for studies that
require proteins such as P-glycoprotein that are expressed
primarily in fully differentiated Caco-2 cells.
Other approaches have been pursued with greater success to
increase the throughput of Caco-2 cell assays. For example, cell
culture plates with semipermeable polycarbonate inserts are
available from commercial sources for the parallel culture and
For personal use only.
testing of Caco-2 cell monolayers. These plates are available in
6-, 12- and 24-well formats with insert diameters of 24, 12
and 6.5 mm, respectively. The use of these multiwell plates has
enhanced the convenience as well as throughput of Caco-2 cell
monolayer assays.
In its original form, measurement of apparent permeability
coefficients using the Caco-2 cell monolayer assay required
radiolabelling and liquid scintillation counting for the quanti-
tative analysis of the concentration of a test agent in the desti-
nation well [39,43]. However, radiolabelled compounds are
expensive to synthesise, their use requires special safety pre-
cautions, and disposal of radioactive waste is expensive.
Another disadvantage of the use of radiolabelled compounds
in the Caco-2 cell assay is that only one compound can be
assayed per well, which limits the throughput of the assay.
Liquid chromatography coupled with ultraviolet (UV) or
fluorescence detection has also been used for quantification
[58], but this approach requires that the analytes have a strong
UV or fluorescent chromophore for adequate sensitivity.
Many compounds, such as mannitol, which is used as a low-
permeability standard in Caco-2 cell assays to test the inte-
grity of the monolayers, lack either type of chromophore.
Another severe limitation of the use of radioisotope, UV or
fluorescence detection is the inability to identify metabolites
formed from the test compound by Caco-2 cells. Typically,
the radiolabelling method overestimates the apparent permea-
bility coefficient of compounds by failing to distinguish
between the test compounds and their metabolites. UV and
fluorescence usually lack sufficient sensitivity to detect meta-
bolites and, in any case, cannot provide structural information
for their identification.
182 Expert Opin. Drug Metab. Toxicol. (2005) 1(2)
Recently, compound quantification in the Caco-2 permea-
bility assay has been improved considerably by the application
of LC-MS [59] and LC-tandem mass spectrometry (LC-MS-
MS) [60,61]. Compared with the radiolabelling approach and
high performance liquid chromatography (HPLC)-UV meth-
ods, LC-MS and LC-MS-MS assays are more sensitive and
more selective. Another advantage of these mass spectro-
metry-based detection methods is the ability to distinguish
the test compounds from their metabolites. Furthermore,
MS-MS facilitates the identification of these metabolites;
thereby adding another dimension to the information that
may be obtained using Caco-2 cell monolayers [10].
As mass spectrometry is an inherently fast detection
method, efforts have been made to increase the throughput of
the chromatography step used for Caco-2 cell assays. For
example, fast chromatographic separations have been deve-
loped for LC-MS-MS analyses of samples from Caco-2 cell
incubations that have a throughput of ∼ 2 min/sample [62].
These fast chromatographic methods are able to sacrifice chro-
matographic efficiency to save time by taking advantage of the
extremely high selectivity of tandem mass spectrometric detec-
tion. As a result, improvement in LC-MS-MS throughput of
> 10-fold may be achieved compared with routine reversed-
phase HPLC separations. Another approach to enhancing the
throughput of the HPLC separation while taking advantage of
the speed of the mass spectrometric detection is to interface
multiple HPLC columns to a single electrospray mass spec-
trometer. For example, Fung et al. [63] reported the simultane-
ous use of four multiplexed HPLC columns with a single mass
spectrometer for Caco-2 cell applications. By using a combina-
tion of fast HPLC separations and a multiple sprayer LC-MS-
MS system, a throughput of 100 compounds/week was
achieved for Caco-2 cell monolayer assays [63].
Finally, another advantage of the selectivity of LC-MS-
MS for quantitative analyses in support of Caco-2 cell intes-
tinal permeability assays is the ability to analyse multiple
compounds simultaneously. Instead of being limited to the
measurement of one compound per well in a Caco-2 cell
experiment, HPLC separation and mass spectrometric
detection facilitate the quantitative analysis of multiple com-
pounds in a single well. This approach has been called N-in-
one analysis, cassette dosing, cocktail analysis and sample
pooling; for example, Bu et al. [64] reported the analysis of
pools of up to five drugs, and Laitinen et al. [65] and Tannergren
et al. [66] reported the analysis of pools of up to 10 compounds
during Caco-2 cell monolayer permeability assays.
3. Conclusions
The Caco-2 cell monolayer system has become a standard
model for human intestinal absorption of xenobiotic
compounds that is more convenient and less expensive than
in vivo models, more accurate than physical models such as
artificial membranes, more convenient and higher throughput
than in vitro models such as everted gut sacs, and more
 van Breemen & Li

accurate than most other in vitro models such as isolated
membrane vesicles. Rates of absorption (determined as appar-
ent permeability coefficients) may be determined, and the
involvement of transporter proteins, such as P-glycoprotein,
and the MRP may be identified. In addition, the involvement
of Phase II conjugation reactions, such as glucuronidation and
sulfation, may be assessed using the Caco-2 model. However,
the role of intestinal CYP isozymes in the metabolism of
compounds during absorption can only be investigated in
specially treated Caco-2 cell monolayers, as these cells
typically do not express these isozymes.
Recently, the use of LC-MS and LC-MS-MS in place of
Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Guelph on 09/09/12
may be used to predict whether intestinal absorption and
metabolism might reduce bioavailability. In addition, the use
of the Caco-2 cell assay facilitates the identification of specific
mechanisms at the level of intestinal absorption that causes
poor or good oral bioavailability.
The use of radiolabelled compounds is no longer required
for the measurement of intestinal permeability using the
Caco-2 cell monolayer assay. Instead, LC-MS and LC-MS-
MS have emerged as the fastest and most selective techniques
to measure the concentrations of test compounds and stand-
ards such as mannitol and propranolol on either side of the
cell monolayer. Higher throughput Caco-2 cell assays of mix-

radiolabelled test compounds has helped increase the
throughput of Caco-2 cell assays [67,68]. In addition, mass
spectrometric detection now provides investigators with the
opportunity to investigate the Phase II intestinal metabolism
of test compounds using Caco-2 cells [10]. Mass spectrometry
has the advantages of high sensitivity, high selectivity and high
speed, which makes it an ideal detector for target analytes in
trace quantities.
During drug discovery and development, early predictions of
drug metabolism and pharmacokinetic properties help stream-
line the selection of lead compounds that are most likely to
become useful drugs. In vitro models, such as the human intes-
For personal use only.
tures of test and control compounds have also been developed
that take advantage of the ability of LC-MS and LC-MS-MS
to measure the concentrations of multiple species in a single
analysis. Finally, LC-MS and LC-MS-MS provide the analyst
with the ability to detect and even quantify metabolites of test
compounds that were formed during incubation with Caco-2
cells. Together, Caco-2 cell incubations in combination with
LC-MS and LC-MS-MS facilitate the rapid determination of
intestinal permeability, absorption and even metabolism of
test compounds in support of preclinical studies of
metabolism and bioavailability.
During the next several years, we should anticipate the

tinal epithelial Caco-2 cell monolayer assay for drug permeabil-
ity, have become standard during the early stages of drug
discovery and development [3,10]. The application of the Caco-2
cell monolayer assay not only facilitates the determination of
intestinal permeability, but also enables the identification of
specific transporter or efflux proteins and intestinal Phase II
drug-metabolising enzymes that might be responsible for
enhancing or preventing intestinal absorption. Therefore, this
cell-based assay provides valuable information for drug develop-
ment in an efficient and reproducible manner that is not
available from nonbiological models and not even available
from many other biological models.
4. Expert opinion
Compared with whole animal assays or clinical trials, the
Caco-2 cell assay is less expensive and provides information
regarding the specific roles of intestinal permeability, trans-
port and efflux. During drug discovery, the Caco-2 cell assay
development of higher throughput innovations in the Caco-2
cell assays. Improvements in the efficiency of these assays
should help reduce their cost and would enable even greater
applicability of this assay to early drug discovery and develop-
ment. One of the deficiencies of the Caco-2 cell monolayer
assay at present is the lack of expression of the normal levels of
intestinal CYP isozymes in these cells. Correction of this
enzyme expression deficiency through genetic engineering or
perhaps through novel approaches to cell culture would add
considerable value to these assays as more complete models of
human intestinal absorption and metabolism. Another limita-
tion of the Caco-2 cell monolayer assay is the inability to
differentiate between compounds of intermediate permeabil-
ity. Although the assay is reliable for distinguishing between
compounds of high, middle and low permeabilities, the
sigmoidal nature of the measurements do not provide quanti-
tative distinction between compounds in the intermediate
range. Hopefully, improvements in the assay will help address
these current limitations.

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Affiliation
Richard B van Breemen† PhD &
Yongmei Li PhD
†Author for correspondence
University of Illinois College of Pharmacy,
Department of Medicinal Chemistry and
Pharmacognosy, 833 S. Wood Street, Chicago,
IL 60612, USA
Tel: +1 312 996 9353; Fax: +1 312 996 7107;
E-mail: Breemen@uic.edu
185