This document summarizes the main transporters expressed in the intestinal tract and techniques used to evaluate interactions between drugs and these transporters. It discusses the two major families of transporters - SLC and ABC transporters. Specific transporters discussed include P-gp, MRP2, BCRP, OCTs, OCTNs, OATPs, and PEPT1. The roles and substrates of these transporters are described. Techniques to evaluate drug-transporter interactions that are discussed include brush border membrane vesicles, everted intestinal sacs, Caco-2 cell monolayers, and in vivo methods using gene knockout animals.

1. Review
The transporters of intestinal tract and techniques applied to
evaluate interactions between drugs and transporters
Zhihao Liu, Kexin Liu*
College of Pharmacy, Dalian Medical University, Dalian, China
a r t i c l e i n f o
Article history:
Received 19 March 2013
Received in revised form
22 April 2013
Accepted 30 April 2013
Keywords:
Intestinal tract
Transporter
SLC
ABC
Technique
a b s t r a c t
Most drug products on the global pharmaceutical market are administered orally. The
absorption of oral drug in the intestine is an important factor to determine the drug
bioavailability. There are many intestinal transporters expressed on the small intestine
and the transporters can be classified into two major families, SLC family and ABC family.
They mediate drug absorption, distribution, excretion and drugedrug interaction. Under-
standing the transport mechanism can improve the effectivity and safety of drug and guide
clinical rational use of drugs. The roles of drug transporters can be assessed in vitro and
in vivo, using techniques spanning from cellular expression systems to gene knockout
animals. The purposes of this article were to introduce the main transporters in the in-
testinal tract, to explain the transport mechanism and to compare the limitations and
applications of techniques used to evaluate interactions of drugs and transporters.
ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All
rights reserved.
1. Introduction
In 1980s, studies of membrane vesicles and cultured epithelial
cell lines were introduced into the research field of drug
transport and the biochemical characterization of trans-
porters advanced remarkably. Then many drug transporters
have been recognized [1e3]. The transporters play important
roles in drug disposition, drug targeting, drugedrug in-
teractions (DDI), and drug-induced toxicity. Uptake (SLC
family) and efflux (ABC family) transporters interact with the
pharmacokinetics and pharmacodynamics of drugs. There are
many transporters expressed on the intestine (Fig. 1). They are
responsible for the absorption of drugs. The roles of drug
transporters can be assessed in vitro and in vivo, using tech-
niques spanning from cellular expression systems to gene
knockout animals. In this article, we provide an overview of
the basic characteristics of major drug transporters of the
intestine. And this review also provides a broad overview of
the current in vitro and in vivo models that are used to evaluate
the interaction between a drug candidate and transporters.
* Corresponding author. Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, 9 West Section, Lvshun
South Road, Lvshunkou District, Dalian 116044, China. Tel. /fax: þ86 411 86110407.
E-mail address: kexinliu@dlmedu.edu.cn (K. Liu).
Peer review under responsibility of Shenyang Pharmaceutical University
Production and hosting by Elsevier
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a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 1 5 1 e1 5 8
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http://dx.doi.org/10.1016/j.ajps.2013.07.020

2. 2. Main transporters expressed on the
intestine
2.1. ABC (ATP-binding cassette) transporters
2.1.1. P-gp (P-glycoprotein, MDR1, ABCB1)
P-gp belongs to the ABC transporter family and involved in
the pharmacokinetics of a wide range of drugs and xenobi-
otics. It was first isolated from cancer cells where it extrudes
drugs out of the cell thereby leading to multidrug resistance.
The amino acid sequence of P-gp has been deduced from
full-length or partial cDNA sequences of human, mouse,
or Chinese hamster P-gp genes. The human MDR1 gene en-
codes a protein of 1280 amino acids which consists of two
highly homologous halves. The molecular weight of P-gp is
170e180 kDa. This molecule contains 12 transmembrane do-
mains (TMD) and two putative ATP binding sites. P-gp is
encoded by two genes in humans (MDR1 and MDR3), and three
in rodents (MDR1a, MDR1b and MDR2) [1]. It is expressed at
brush border membranes of enterocytes, where it functions as
the efflux pump for xenobiotics before they can access the
portal circulation. It is a frequent of cause of treatment failure
in cancer patients.
The substrates of P-gp include anticancer drugs (such
as vincristine, vinblastine, doxorubicin, daunorubicin, etopo-
side, and paclitaxel), immunosuppressants (such as cyclo-
sporin A), verapamil, digoxin, and steroids (such as
aldosterone and cortisole) [4]. What’s more, P-gp can be
interacted by some other drugs. The AUC of talinolol was
significantly decreased when coadministered with P-gp
inducer rifampicin [5]. At some times, P-gp and CYP3A4 were
suggested to cooperate in the intestinal absorption of
drugs. Van Waterschoot RA et al. reported that ritonavir
increased lopinavir oral bioavailability by inhibiting CYP3A,
and MDR1 [6].
2.1.2. MRP2 (multidrug resistance-associated protein 2,
ABCC2)
MRP family is one of the main families of ABC transporter. In
the same manner, MRP2 is the main transporter of MRP family.
MRP2 encodes a 190e200 kDa polytopic transmembrane
protein comprising 1545 amino acids and belongs to ABC
transporter subfamily C [7]. MRP2 was first functionally char-
acterized as a canalicular multispecific organic anion trans-
porter in canalicular membranes of hepatocytes. MRP2 has 2
hydrophobic TMDs (TMD1 & TMD2) and 2 cytoplasmic NBDs.
The NBDs are responsible for the ATP binding/hydrolysis that
drives drug transport, and their structures are conserved
independently of the degree of primary-sequence homology
[8]. The amino acids of MRP2 have 49% identity with MRP1 [9].
The location of MRP2 is unique, as it is present on the apical
plasma membranes of the intestine [10,11], while other MRPs
are all located on basolateral membrane of polarized cells.
MRP2 could play a major role in cancer chemotherapy and
in the pharmacokinetics of substrate drugs [12]. It is respon-
sible for the efflux of drugs and endogenous compounds. In
mice lacking Mrp2, drug level fosinopril was reduced in the
intestine by 1.5-fold [13]. The substrates of Mrp2 include
cisplatin, BQ-123, etoposide, vinca alkaloids, anthracyclines,
camptothecins, MTX, lopinavir and olmesartan [14]. The in-
hibitors of Mrp2 include MK571 and probenecid.
2.1.3. BCRP (breast cancer resistance protein, ABCG2)
Human BCRP encodes a 655 amino acid, containing a single N-
terminal ATP binding cassette, followed by 6 putative TMDs
[15]. BCRP are thought to have a protective role in the intes-
tinal tract by preventing toxic substances absorption. It is a
pharmacologically important ABC drug efflux transporters
located at the apical part of the plasma membrane in polar-
ized cells such as epithelia, and they influence the pharma-
cokinetics and tissue distribution of drugs [15].
BCRP mediates concurrent resistance to chemotherapeutic
agents, presumably by pumping drugs out of the cell, thereby
resulting in concentrations lower than cytotoxic levels. The
substrates of BCRP transporter include mitoxantrone, etopo-
side doxorubicin and methotrexate [16].
2.2. Organic ion transporters
2.2.1. OCTs (organic cation transporter, SLC22A)
In 1994, the first member of the SLC22 family, OCT1, was first
identified from a rat kidney cDNA library by expression clon-
ing [17]. Then OCT2 was identified from rat in 1996 [18]. OCTs
contain 12 TMDs and they mediate intracellular uptake of a
broad range of structurally diverse organic cations with mo-
lecular masses generally lower than 400 Da [19].
Substrates of OCT1 include endogenous compounds, such
as choline, creatinine, and monoamine neurotransmitters;
clinically used drugs, such as metformin, oxaliplatin and
cimetidine; and a variety of xenobiotics, such as tetraethy-
lammonium (TEA, model cation), N-methylquinine, and 1-
methyl-4-phenylpyridinium (MPPþ) [20,21].
2.2.2. OCTNs (novel organic cation transporter, SLC22A)
OCTN1-3 are the new members of OCT family. The amino acids
of OCTNs have 30% identity with OCTs. OCTNs encode a 551
Fig. 1 e Drug transporters in the intestinal epithelial cells
(Tomohiro Terada, Ken-ichi Inui, Impact of Drug Transport
Proteins).
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3. amino acid, containing 12 TMDs. OCTN2 is highly expressed in
the human intestine from the jejunum to colon [22].
OCTN2 is clinically important because deficiency of the
OCTN2 gene in humans causes systemic carnitine defi-
ciency (SCD) [23]. OCTN2, is involved in the distribution of
endogenous and exogenous substrates, including drugs
such as quinidine, verapamil, and some b-lactam antibiotics
[24,25].
2.2.3. OATPs (organic anion-transporting polypeptide, SLCO)
OATPs families were first isolated from rats [26]. So far 36
OATPs have been identified in humans, rat and mouse [27]. All
members contain between 643 and 722 amino acids. And they
contain 12 TMDs [28,29]. Although some OATPs are selectively
involved in the hepatic clearance of albumin-bound com-
pounds from portal blood plasma, most OATPs are expressed
in multiple tissues including the bloodebrain barrier (BBB),
choroid plexus, lung, heart, intestine, kidney, placenta and
testis [26,30]. OATP-B is widely expressed on the apical side of
the intestinal epithelial cells, mediating intestinal absorption
of drugs by pH-dependent [31].
OATPs family mediates the Naþ-independent transport of
a wide range of amphipathic organic compounds, including
bile salts, organic dyes, steroid conjugates, thyroid hormones,
anionic oligopeptides, numerous drugs, and other xenobiotic
substances [29,32]. Grapefruit juice was also reported to
inhibit OATP transporter thus reducing the blood levels of
OATP substrates [33].
2.3. PEPT1 (Hþ
/peptide cotransporter, SLC15A1)
The cDNA of PEPT1 was firstly identified by expression cloning
using a rabbit small intestinal cDNA library [34]. Homologous
cDNAs were then found in human, rat, mouse, cow and
chicken [35]. PEPT1 consists of 707e710 amino acid residues,
with several putative glycosylation and phosphorylation sites.
It contains 12 TMDs, with both the C- and N-terminal localized
inside the cell [35]. PEPT1 is a low-affinity Hþ/peptide
cotransporter that is primarily expressed in the brush border
membrane of the small intestine [36]. Currently, PEPT1 can be
divided into two types; i.e., the one localized at the brush-
border membranes of epithelial cells, and the other one at
the basolateral membranes. The former one plays a major role
on the transport of the substrates [37].
Studies have demonstrated that many peptide-like drugs
are absorbed by the Hþ-coupled peptide transporter, PEPT1.
For example, b-lactam antibiotics [38], the anti-cancer agent
Bestatin [39], renin inhibitors [40], and several angiotensin
converting enzyme (ACE) inhibitors [41] were all reported to be
recognized by PEPT1.
3. Techniques used to evaluate the
interaction of drugs and transporter
3.1. In vitro methods
3.1.1. Brush border membrane vesicles (BBMV)
Under ether anesthesia, rapidly removed the jejunum of rats
and flushed with ice-cold saline, then the mucosa was scra-
ped. BBMV were prepared by the calcium precipitation
method. Prepared BBMV were suspended in 100 mM D-
mannitol, 100 mM KCl and 10 mM Hepes/Tris (pH 7.5) and
stored at À80
C. Purity of BBMV was evaluated by comparison
of the activities of alkaline phosphatase (a marker enzyme of
the brush border membrane) and Naþ
/Kþ
ATPase (a marker
enzyme of the basolateral membrane) with those of the initial
homogenate [42,43]. The model of BBMV is shown in Fig. 2. Our
previous study indicated that the anti-hepatitis drug JBP485
can be transported by Pept1 by using rat BBMV technique [44].
BBMV can only measure the top side of the intestinal mem-
brane transport and has low survival rate. All these short-
comings limit the use of BBMV in the experiment.
3.1.2. Everted intestinal sac
The abdomen of rats was opened by a midline incision, and
the jejunum was removed by cutting across the upper end of
the duodenum (i.e., w2 cm distal to the ligament of Treitz) and
the lower end of the ileum and manually stripping the mes-
entery. The small intestine was washed out carefully with
cold normal oxygenated saline, using a syringe equipped with
a blunt end. Intestinal segments (10 Æ 1 cm) were everted
according to the conventional technique described by Wilson
and Wiseman (1954) with modifications. The everted intestine
was placed in glucose-saline at room temperature in a flat
dish. A thread ligature was tied around one end to facilitate
subsequent identification and to check for perforation. The
empty sac was filled with 1 ml of KrebseRinger buffer (KRB)
Fig. 2 e The model of BBMV system.
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4. (pH 7.4) containing 0.5 mM MgCl2, 4.5 mM KCl, 120 mM NaCl,
0.7 mM Na2HPO4, 1.5 mM NaH2PO4, 1.2 mM CaCl2, 15 mM
NaHCO3, 10 mM glucose, and 20 mg/L of phenolsulfonph-
thalein as a nonabsorbable marker [45,46]. The distended sac
was placed in incubation medium containing drugs. The in-
cubation medium was surrounded by a water jacket main-
tained at 37
C. A gas mixture of 95% O2 and 5% CO2 was
bubbled through the external incubation medium during the
incubation period. At the end of the incubation period, the
serosal fluid was drained through a small incision into a test
tube.
Everted intestinal sac is an excellent in vitro model to
evaluate the absorption of drugs by transporters. The advan-
tages of using this system for transport studies are (a) drug
transport across the basolateral and apical membrane can be
measured separately, (b) buffers inside and outside vesicles
can be changed easily. But there also have some limitations
such as compounds with high nonspecific binding and high
passive diffusion which may result in false negative results.
3.1.3. Caco-2 cells method
Caco-2 cells are derived from human colorectal adenocarci-
nomas and form monolayer (like human intestinal epithe-
lium) under conventional culture conditions. They have been
widely used as a potent in vitro model to predict drug ab-
sorption in humans, to explore mechanisms of drug absorp-
tion, and to identify substrates or inhibitors of transporters
[47,48]. These cells exhibit remarkable morphologic and
biochemical similarity to the small intestinal columnar
epithelium; they are extremely useful for mechanistic studies
of drug absorption and are widely used by pharmaceutical
companies in absorption screening assays for preclinical drug
selection [49].
Caco-2 cells were plated on 24-well dishes at a density of
5 Â 105
cells/well. The cell monolayers were given fresh me-
dium every other day and were used on the 15th day for up-
take experiments. Hank’s balanced salt solution (HBSS:
145 mM NaCl, 3 mM KCl, 1 mM CaCl2, and 0.5 mM MgCl2)
containing 5 mM D-glucose and 5 mM MES (for pH 6.0) was
used as the uptake medium. HBSS containing 5 mM D-glucose
and 5 mM HEPES (for pH 7.4) was used as the rinse medium.
The culture medium was removed on the day of experiment,
and the Caco-2 cell monolayers were washed twice with HBSS
(pH 7.4). After washing, the monolayers were preincubated
with HBSS (pH 7.4) for 15 min at 37
C. The medium was
removed after the preincubation period and uptake was
initiated by adding 1 ml of the preincubated drug solution. The
drug solution was aspirated at the appropriate time to termi-
nate uptake, and the cells were then quickly rinsed four times
with ice-cold rinse medium (pH 7.4). The amount of drug up-
take was determined by lysing the cells with 0.3 ml of 0.1%
Triton X-100, and the cell lysate was then transferred to a
plastic vial for quantitation.
Ogihara, T et al. indicated that the antivirus drug Oselta-
mivir is a substrate of PEPT1 by using Caco-2 cells [50]. In the
same manner, our previous study also used the Caco-2 cells to
investigate the uptake characters of JBP485 by PEPT1 [51].
Caco-2 cells system is also a useful model to estimate the
epithelial permeability of intestine [52]. Intestinal perme-
ability is a basic parameter that can be easily assessed using a
variety of experimental models and it can be linked mecha-
nistically to the pathways of drug absorption as well as to the
overall absorption rate constant for drug absorption into the
body.
Transepithelial transport studies were conducted using
Caco-2 cell monolayers grown for 21 days on 24-well transwell
inserts (Fig. 3). The cell monolayers were given fresh complete
medium every other day. Monolayers formed after 2 weeks of
culture. The integrity of the cell layer was evaluated by
measuring the transepithelial electrical resistance (TEER)
using Millicell-ERS equipment. Monolayers with TEER values
(250 U cm2
) were used for the experiments. The monolayer
cells were rinsed gently three times with HBSS (pH 7.4). The
TEER values of the monolayer were measured before and after
addition of an assay sample to the insert. Then the buffer
added to the apical or basal side was replaced by the test so-
lutions maintained at 37
C and the plate was incubated at
37
C for the appropriate time. The apparent permeability
values (Papp) were calculated in all experiments according to
the equations:
Papp ¼
dQ=dt
AC0
(1)
where dQ/dt is the slope of the cumulative amount trans-
ported during the time course of the period studied, A is the
area of the inserts, and C0 is the starting concentration.
Permeability direction ratios (PDR) were calculated ac-
cording to the following equation:
PDR ¼
PappðAP À BLÞ
PappðBL À APÞ
(2)
where AP-BL is the apical-to-basolateral transport and BL-AP
is the basolateral-to-apical transport. This equation will be
reversed if transporter is localized on the basolateral cell
membrane.
The transport assay is the most direct assay for evaluating
a given transporter’s function and measuring the permeability
of a test compound across cell monolayer. And the Caco-2
system has now become the gold standard for investigating
drug permeability in oral drug absorption.
3.1.4. Transfected cells
Although Caco-2 cells system has many advantages, there
also include some limitations such as the long (21 day) cell
culturing time, heterogeneity, and non-specificity due to the
Fig. 3 e Transwell system with microporous filter (Cindy Q.
Xia et al., Current Drug Metabolism, 2007, 8, 341e363).
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5. expression of multiple transporters. The heterogeneity of
Caco-2 cells can be caused by different origins, culture con-
ditions and number of cell passages [53]. Thus cells stably
expressed drug transporters were constructed to study the
transport characters. The transfected cells can ensure the
cells expressing high level of a single transporter. This
approach can be more intuitive to analyze the effects of
transporters on drugs. For generation of stable transfectants,
the cells were transfected with the plasmid using Lipofect-
AMINE 2000 reagent, and were grown in G418 in DMEM
(high glucose) for 4 weeks [54]. Ten surviving cell colonies
were randomly selected and examined by immunostaining
and western blot [55]. One cell colony that exhibited the
highest expression of plasmid was chosen for the following
experiments. Knutter, I et al. transfected PEPT1 into human
retinal pigment epithelial cells and confirmed the affinity
of 14 different vascular converting enzyme inhibitors for
PEPT1 [41].
In the same manner, double-transfected cells system is
also an effective method to identify the substrates and in-
ducers of transporters. Iwai, M. et al. assessed the interaction
of YM155 with P-glycoprotein (MDR1/ABCB1) using double-
transfected cells system: LLC-OCT1/MDR1 cell line [56]. They
get the conclusion that YM155 is a substrate of MDR1, and that
MDR1 may play an important role in the pharmacokinetics of
YM155. What’s more, Masakazu Horiuchi et al. successfully
constructed OATP1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4
three-transfected cells to study drug transport through the
transporters [57]. Because of the scarcity of human tissue
sources, transporter expressing systems will be useful for
predicting transporter-mediated drugedrug interactions. In
many cases, the endogenous transporter existing in the
transfected cells can interference test results. Appropriate
cells should be chosen in the transfection process.
3.1.5. Xenopus laevis oocytes
The xenopus laevis oocytes expression system has been used
by many scientists to probe different transporters using influx
and efflux studies [58,59]. Briefly, surgically removed oocytes
from female X. laevis (African Xenopus Facility, Knysna, South
Africa) were separated by collagenase treatment and were
injected with 30 nl of RNA solution containing 30 ng of cRNA.
The two-electrode voltage clamp technique was applied to
characterize responses in inward current (I ) to substrate
addition as described [60]. Currentevoltage (IeVm) relation-
ships were measured in the potential range from À160 to
þ80 mV. IeVm measurements were made immediately before
and 30 s after substrate application when current flow reached
steady state [61].
Knutter I et al. used xenopus laevis oocytes transfected
with PEPT1 to investigate the affinity of sartans for PEPT1.
They concluded that the sartans tested in the study displayed
high-affinity interaction with PEPTs [62].
Due to their relatively large size, they are readily accessible
by microinjection. What’s more, the system shows low ac-
tivity of endogenous transporters and high sensitivity. But the
expression system displays a distinct variability in perfor-
mance between individual oocytes. Quality and quantity of
oocytes can vary considerably between individual donor ani-
mals [63].
3.1.6. Positron emission tomography (PET)
PET is a molecular and functional imaging technique with
sensitivity, which permits repeated, non-invasive assessment
and quantification of specific biological and pharmacological
processes. Now it is playing an increasing role in both drug
discovery and development by assessing their pharmacoki-
netics and pharmacodynamics. It is noteworthy that many
PET tracers used for cancer detection are thought to be sub-
strates of specific transporters [64,65].
To evaluate the functions of P-gp and BCRP in Caco-2 cells,
Yamasaki T et al. carried out a small animal PET study using
[11C]
GF120918. PET results indicated that [11C]
GF120918 uptake
in the tumor was low, but was significantly increased by
treatment with unlabeled GF120918. The study indicated that
a PET study combining the administration of [11C]
GF120918
with unlabeled GF120918 may be a useful tool for evaluating
the functions of P-gp in tumors [66]. PET is thus a promising
approach to determine the functional change in transporters
associated with drugedrug interactions. However, due to the
higher cost of PET, it is not widely used in scientific research.
3.1.7. ATPase assays
The transport function of the ABC efflux pumps depends on
the binding and the hydrolysis of cytoplasmic ATP within
NBD. The ATPase activity of P-gp, MRP or BCRP expressing in
insect cell or mammalian cell membranes is vanadate sensi-
tive and can be stimulated or inhibited by substrates of these
transporters [67]. P-gp, exhibits a high-capacity drug-depen-
dent ATP hydrolytic activity that is a direct reflection of its
drug transport capability [68]. The drug-stimulated ATPase
activity is a useful alternative to conventional screening sys-
tems for identifying high-affinity drug substrates of the P-gp.
Using this assay system, a variety of drugs have been directly
shown to interact with the P-gp [69,70].
The membrane ATPase assay is compound-independent,
easy to perform and does not require the use of radiolabeled
compounds. Therefore it can easily be applied in a high
throughput mode [71]. However, the ATPase assay is not a
functional assay and cannot be used to distinguish between
substrates and inhibitors. Other drawbacks of this assay
include large inter-day or intra-day variations and potential
false negative results.
3.2. In vivo methods
3.2.1. In situ jejunal perfusion technique
In situ jejunal perfusion technique is a well-established
technique to investigate the intestinal absorption behavior
of drugs in which the compound of interest is monitored in
perfusate. Rats were anesthetized with an intraperitoneal
(i.p.) injection of 25% ethylurethane (0.4 ml/kg), and a heating
pad and heating lamp were used to maintain body tempera-
ture. Briefly, a laparoscopy was performed, and an inflow
annual made of silastic tubing was inserted in the jejunum
approximately 1 cm below the ligament of Treitz [38,45,72]. An
outflow annual was set up at a distance of 10 cm. The bile duct
was ligated to prevent possible enterohepatic circulation. The
jejunal segment was then flushed with saline solution (pre-
warmed to 37
C) to remove residual intestinal contents [73].
Oxygenated perfusion solution was delivered with a
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6. peristaltic pump at a flow rate of 4 ml/15 min through an inlet
tube water-jacketed at 37
C, before its entry into the jejunal
segment. The solution for jejunal perfusion was KrebseRinger
buffer (pH 7.5, KRB) containing 0.5 mM MgCl2, 4.5 mM KCl,
120 mM NaCl, 0.7 mM Na2HPO4, 1.5 mM NaH2PO4, 1.2 mM
CaCl2, 15 mM NaHCO3, 10 mM glucose, and 20 mg/L of phe-
nolsulfonphthalein as a nonabsorbable marker. Portal vein
blood was collected using a vein-detained needle after giving
drugs. Zhang et al. used the technique to ensure that JBP485
and cephalexin can compete the same sites of PEPT1 and
OATs [45].
In situ jejunal perfusion technique would maintain the
nerve intact and high activity of enzyme. However the organ
integrity and enzyme activity may become fragile and
compromised during long-term perfusions. Positive control
should be taken to examine the success of the model.
3.2.2. Genetic knockout mice
Genetic knockout mice have become important models in
understanding the physiological functions of drug trans-
porters and in evaluating the effects of transporters on the
pharmacokinetics and pharmacodynamics of drugs [67].
Schinkel et al. used transgenic technology to build the mdr1a
(similar to the human MDR1) gene-deficient mice [74].
Mdr1a(À/À) mice displays drastic alterations in the pharma-
cological handling of drugs, demonstrating an important role
for mdr1 in the intestine. These mice are overall healthy, but
they accumulate much higher levels of substrate drugs in the
body, and have markedly slower elimination of these drugs
from the circulation [75]. Compared to wild-type mice, the
mice of this type have no obvious physical defects.
The gene knockout mice can confirm the role of drug
transporters without inhibitors. It is the best in vivo mode to
study the drug substrates, inhibitors and transporter effects
on the pharmacokinetics of transporters. However, caution
needs to be taken when using this model to evaluate transport
mechanisms since deletion of one transporter can often cause
alteration in the expression of other transporters or enzymes
as well change the physiology of the knockout animal. As re-
ported by Schuetz and Schinkel’s groups [76,77], the female
Mdr1a/1b(À/À) and Mdr1a(À/À) knockout mice housed in
Amsterdam had dramatically increased level of CYP3A, 2B,
and 1A proteins and activities.
4. Conclusion
In this review, basic characteristics of major intestinal trans-
porters and techniques applied to evaluate interactions of
drugs and transporters are discussed. Then the advantages
and limitations of each model are discussed in this review
(Table 1). The study of drug transporters can be actually
applied to clinical science and drug development; that is, ap-
plications of drug delivery, the clarification of DDI, application
of personalized pharmacotherapy, and clarification of the
relationship of each transporter to particular disease(s). To
avoid adverse consequences of such transporter-mediated
drugedrug interactions, we need to be more aware of the
role played by drug transporters.
r e f e r e n c e s
[1] Kunta JR, Sinko PJ. Intestinal drug transporters: in vivo
function and clinical importance. Curr Drug Metab
2004;5:109e124.
[2] Muller F, Fromm MF. Transporter-mediated drug-drug
interactions. Pharmacogenomics 2011;12:1017e1037.
Table 1 e The comparisons of the techniques used to study the intestinal transporters.
Methods Advantages Limitations
In situ jejunal perfusion Maintain the nerve intact; high activity of
enzyme.
The organ integrity and enzyme activity may become fragile
and compromised during long-term perfusions.
Genetic knockout mice Meet the real environment of body. The model is difficult to establish; It is expensive and not
suitable for high-throughput screening.
BBMV Suitable for high-throughput screening. The survival rate is low; It can only measure the top side of
the intestinal membrane transport.
Everted intestinal sac The cells can receive sufficient oxygen. The
concentration of serosal side is high and easy
to detect. The experiment cycle is short and
can save animals.
The activity demanding of the small intestine is high.
Caco-2 cells They are similar to small intestinal epithelial
cells. The conditions are easy to control. The
experiment is economic.
The cells require long-time culture and have multiple
transporters expressed. They also lack intestinal mucus
layer and certain metabolic enzymes. The expression of
transporters is different in different culture conditions.
Transfected cells The transporters are specific and suitable for
regulating.
Transected cells have endogenous transporters. The Km or
Vmax obtained from transfected system may be different
from natural cell lines. The cells are lack of regulation way.
Xenopus laevis oocytes The activity of endogenous transporters is
low. The sensitivity is high.
The expression of protein can’t be regulated. Quality and
quantity of oocytes can vary considerably between
individual donor animals
PET High resolution; high accuracy. The instrument is complicated and expensive.
ATPase assays Easy to operate; saving time. The study is indirect and cannot provide dynamic
information.
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