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Volume 8 | No. 8

The Role of Intestinal Efflux TransportersIn Drug AbsorptionMichael D. Mitchell, Product Manager, SigmaLife 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 IntestineMembrane 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 andexcreted 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

potentialin vivoabsorption properties.

In VitroTransporter 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

from a patient with a colon adenocarcinoma, exhibit features in

culture demonstrating key human physiology found in the smallintestine, 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

KOParental (Wild type)

C2BBe1 Caco-2 cells

Double KOcell lines

Single KOcell lines

MDR1 KO BCRP KO MRP2 KO

MDR1/BCRPKO

MDR1/MRP2KO

BCRP/MDR2KO

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These novel knockout cell lines can be used to identify specific drug-

transporter 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 ratioof 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).

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

Digoxin Eryth romyci n

0.0

5.0

2.0

10.0

15.0

20.0

Wild Type MDR1 KO MDR1/BCRP KO MDR1/MRP2 KO

Effl

uxRatio(B-A/A-B)

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

Estrone sulfate Nitrofurantoin

0.0

5.0

2.0

10.0

15.0

20.0

25.0

30.0

35.0

Wild Type BCRP KO BCRP/MDR1 KO BCRP/MRP2 KO

EffluxRatio(B-A/A-B)

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

CDCF

0.0

5.0

2.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

Wild Type MRP2 KO MRP2/MDR1 KO MRP2/BCRP KO

Effl

uxRatio(B-A/A-B)

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

0

2

4

6

8

10

Wild Type MDR1 KO BCRP KO MRP2 KO MDR1/BCRP

KO

MDR1/MRP2

KO

MRP2/BCRP

KO

EffluxRatio(B-A/A-B)

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

References

1. Pratt, 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. The International Transporter Consortium, Membrane transporters in drug development. Nat.

Rev. Drug Discov,9, 215-236 (2010).

3. Highlights from the International Transporter Consortium Second Workshop. Clin. Pharmacol.

her, 92. 553-556 (2012).

4. Matsson 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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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, andcontract research organizations (CROs) in multiple formats including

licensing and a recently launched assay-ready plate format.

For more information or to place an order, visitsigma.com/transporterko