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Volume 8 | No. 8

The Role of Intestinal Efflux Transporters

In Drug Absorption
Michael D. Mitchell, Product Manager, Sigma Life Science
David C. Thompson, Ph.D, R&D Manager, Sigma Life Science
Drug Absorption in the Intestine
A key aspect for developing a successful drug involves designing the
drug so it can be effectively delivered to the intended site of action.
The absorption of a drug is a key component to achieving good
bioavailability and ensuring the drug is able to reach the systemic
circulation. For oral drugs, the majority of drug absorption occurs in
the small intestine where the presence of villi and microvilli greatly
increase the surface area for optimal absorption. Drug absorption in
the small intestine is greatly influenced by multiple interacting factors,
including drug properties (solubility, formulation, concentration,
etc.), gastrointestinal properties (pH, food intake, region of the small
intestine, etc.), metabolism, permeability, and active transport across
the intestinal epithelial membrane.

Efflux Transporters in the Intestine

Membrane drug transporter proteins have been identified as a
determinant of drug disposition in the body, potentially affecting
absorption, pharmacokinetics, drug-drug interactions, and safety
profiles. The major efflux transporters of the ABC family include MDR1
(P-glycoprotein, P-gp, ABCB1), BCRP (ABCG2), and MRP2 (ABCC2). They
are localized to barrier tissues of the body such as intestine, liver, kidney,
blood-brain barrier, and placenta, where they efflux a wide range of
xenobiotics such as statins, macrolide antibiotics, angiotensin blockers,
and chemotherapeutic agents, affecting exposure and clearance in vivo.
In the intestine, drug-transporter interactions involving the efflux
transporters often result in poor absorption and low oral bioavailability
as the drug is readily effluxed back into the intestinal lumen and
excreted out of the body. Given that the vast majority of drugs are
developed for oral delivery, utilization of in vitro permeability and
transporter assays have become critical tools for assessing a drugs
potential in vivo absorption properties.

from a patient with a colon adenocarcinoma, exhibit features in

culture demonstrating key human physiology found in the small
intestine, including expression of the major efflux transporters on the
apical membrane and robust efflux activity when used in the 21-day
bidirectional transwell assay.
Current cell-based transporter assays, like the Caco-2 assay, require
the use of transporter-specific compounds as substrates or inhibitors
in order to identify potential drug-transporter interactions. However,
substrates are often recognized by multiple transporters at different
affinities, and the specificity of inhibitors is often unknown or poor,
leading to ambiguous interpretations of drug-transporter interactions.
MK571 for example, is highly regarded as a selective inhibitor for the
efflux transporter MRP2. However, at concentrations that are typically
used by researchers, 25 - 50 M, MK571 effectively inhibits the other
major efflux transporters BCRP and MDR1.

Caco-2 Efflux Transporter Knockout Cells

Utilizing CompoZr Zinc Finger Nucleases (ZFNs), MDR , BCRP, and
MRP2 efflux transporter genes were targeted for ZFN-mediated
knockout in the C2BBe1 (Caco-2) cell line. The resultant panel of
single and double knockout (KO) cells demonstrated disruption of
gene sequence as well as complete loss of transporter function in
bidirectional transport assays out to at least 40 passages post-ZFN
genomic modification.
MDR1
BCRP
MRP2

Wild Type

MDR1 KO

BCRP KO

MDR1/BCRP
KO

MDR1/MRP2
KO

KOParental (Wild type)

C2BBe1 Caco-2 cells

MRP2 KO

Single KO
cell lines

In Vitro Transporter Models

While there are many in vitro models to test drugs for both intestinal
permeability and efflux liabilities, the Caco-2 assay has been used
for over two decades and demonstrates good correlation to human
permeability and active efflux for most drugs. Caco-2 cells, derived

Double KO
BCRP/MDR2 cell lines
KO

Your Biology Resource

The Role of Intestinal Efflux Transporters In Drug Absorption | Volume 8 No. 8

These novel knockout cell lines can be used to identify specific drugtransporter interactions by comparison of drug transport between the
wild-type (WT) and the knockout cell lines. Sample data are shown
for several model compounds known to be substrates for these efflux
transporters digoxin and erythromycin (MDR1), estrone 3-sulfate and
nitrofurantoin (BCRP), and CDCF (MRP2). In each case, the efflux ratio
of the compound decreases to unity in the appropriate KO cell line
versus the WT cell line (Figures 1, 2, and 3). In addition, cimetidine was
identified as a dual substrate for both MDR1 and BCRP transporters
using the single and double KO cell lines compared to WT (Figure 4).

These novel intestinal efflux transporter knockout cell lines have been
fully characterized and are proving to be powerful tools for elucidating
drug-transporter interactions without dependence on chemical
inhibitors and for clarifying the potential impact of specific efflux
transporters in drug absorption and disposition. These cell lines are
currently available for academic and pharmaceutical researchers, and
contract research organizations (CROs) in multiple formats including
licensing and a recently launched assay-ready plate format.

40.0

20.0

35.0
30.0
Efflux Ratio (B-A/A-B)

Efflux Ratio (B-A/A-B)

15.0

10.0

25.0
20.0
15.0
10.0

5.0

5.0

2.0

2.0
0.0

0.0
Wild Type

Digoxin

MDR1 KO

MDR1/BCRP KO

Wild Type

MDR1/MRP2 KO

MRP2 KO

MRP2/MDR1 KO

MRP2/BCRP KO

CDCF

Erythromycin

Figure 1. Efflux of P-gp Substrates in wild-type (WT) and MDR1 knockout (KO) cell lines

Figure 3. Efflux of the MRP2 Substrate CDCF in wild-type (WT) and MRP2 knockout (KO)
cell lines

10

35.0
30.0

Efflux Ratio (B-A/A-B)

Efflux Ratio (B-A/A-B)

8
25.0
20.0
15.0
10.0

5.0
2.0
0.0
Wild Type

Estrone sulfate

BCRP KO

BCRP/MDR1 KO

BCRP/MRP2 KO

0
Wild Type

Nitrofurantoin

Figure 2. Efflux of BCRP Substrates in wild-type (WT) and BCRP knockout (KO) cell lines

MDR1 KO

BCRP KO

MRP2 KO

MDR1/BCRP
KO

MDR1/MRP2
KO

MRP2/BCRP
KO

Figure 4. Efflux of Cimetidine in wild-type (WT) and P-gp, BCRP, and MRP2 knockout (KO)
cell lines

For more information or to place an order, visit

sigma.com/transporterko

References
1. P
 ratt, J. et al., Use of Zinc Finger Nuclease Technology to Knock Out Efflux Transporters in
C2BBe1 Cells. Curr. Protoc. Toxicol. 52:2. 23.2.1-23.2.22 (2012).
2. T he International Transporter Consortium, Membrane transporters in drug development. Nat.
Rev. Drug Discov, 9, 215-236 (2010).
3. H
 ighlights from the International Transporter Consortium Second Workshop. Clin. Pharmacol.
her, 92. 553-556 (2012).
4. M
 atsson et al. Identification of Novel Specific and General Inhibitors of the Three Major Human
ATP-Binding Cassette Transporters P-gp, BCRP and MRP2 Among Registered Drugs. Pharm. Res.
Vol. 26, No. 8, (2009).

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