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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 from a patient with a colon adenocarcinoma, exhibit features in culture demonstrating key human physiology found in the small A key aspect for developing a successful drug involves designing the intestine, including expression of the major efflux transporters on the drug so it can be effectively delivered to the intended site of action. apical membrane and robust efflux activity when used in the 21-day The absorption of a drug is a key component to achieving good bidirectional transwell assay. bioavailability and ensuring the drug is able to reach the systemic circulation. For oral drugs, the majority of drug absorption occurs in Current cell-based transporter assays, like the Caco-2 assay, require the small intestine where the presence of villi and microvilli greatly the use of transporter-specific compounds as substrates or inhibitors increase the surface area for optimal absorption. Drug absorption in in order to identify potential drug-transporter interactions. However, the small intestine is greatly influenced by multiple interacting factors, substrates are often recognized by multiple transporters at different including drug properties (solubility, formulation, concentration, affinities, and the specificity of inhibitors is often unknown or poor, etc.), gastrointestinal properties (pH, food intake, region of the small leading to ambiguous interpretations of drug-transporter interactions. intestine, etc.), metabolism, permeability, and active transport across MK571 for example, is highly regarded as a selective inhibitor for the the intestinal epithelial membrane. efflux transporter MRP2. However, at concentrations that are typically used by researchers, 25 - 50 µM, MK571 effectively inhibits the other Efflux Transporters in the Intestine major efflux transporters BCRP and MDR1. Membrane drug transporter proteins have been identified as a determinant of drug disposition in the body, potentially affecting Caco-2 Efflux Transporter Knockout Cells absorption, pharmacokinetics, drug-drug interactions, and safety Utilizing CompoZr® Zinc Finger Nucleases (ZFNs), MDR , BCRP, and profiles. The major efflux transporters of the ABC family include MDR1 MRP2 efflux transporter genes were targeted for ZFN-mediated (P-glycoprotein, P-gp, ABCB1), BCRP (ABCG2), and MRP2 (ABCC2). They knockout in the C2BBe1 (Caco-2) cell line. The resultant panel of are localized to barrier tissues of the body such as intestine, liver, kidney, single and double knockout (KO) cells demonstrated disruption of blood-brain barrier, and placenta, where they efflux a wide range of gene sequence as well as complete loss of transporter function in xenobiotics such as statins, macrolide antibiotics, angiotensin blockers, bidirectional transport assays out to at least 40 passages post-ZFN and chemotherapeutic agents, affecting exposure and clearance in vivo. genomic modification. In the intestine, drug-transporter interactions involving the efflux MDR1 transporters often result in poor absorption and low oral bioavailability BCRP KOParental (Wild type) as the drug is readily effluxed back into the intestinal lumen and MRP2 Wild Type C2BBe1 Caco-2 cells 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 drug’s Single KO cell lines potential in vivo absorption properties. MDR1 KO BCRP KO MRP2 KO

In Vitro Transporter Models While there are many in vitro models to test drugs for both intestinal Double KO MDR1/BCRP MDR1/MRP2 BCRP/MDR2 cell lines permeability and efflux liabilities, the Caco-2 assay has been used KO KO KO for over two decades and demonstrates good correlation to human permeability and active efflux for most drugs. Caco-2 cells, derived 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 drug- These novel intestinal efflux transporter knockout cell lines have been transporter interactions by comparison of drug transport between the fully characterized and are proving to be powerful tools for elucidating wild-type (WT) and the knockout cell lines. Sample data are shown drug-transporter interactions without dependence on chemical for several model compounds known to be substrates for these efflux inhibitors and for clarifying the potential impact of specific efflux transporters – digoxin and erythromycin (MDR1), estrone 3-sulfate and transporters in drug absorption and disposition. These cell lines are nitrofurantoin (BCRP), and CDCF (MRP2). In each case, the efflux ratio currently available for academic and pharmaceutical researchers, and of the compound decreases to unity in the appropriate KO cell line contract research organizations (CROs) in multiple formats including versus the WT cell line (Figures 1, 2, and 3). In addition, cimetidine was licensing and a recently launched assay-ready plate format. identified as a dual substrate for both MDR1 and BCRP transporters using the single and double KO cell lines compared to WT (Figure 4).

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5.0 2.0 2.0 0.0 0.0 Wild Type MDR1 KO MDR1/BCRP KO MDR1/MRP2 KO Wild Type MRP2 KO MRP2/MDR1 KO MRP2/BCRP KO

Digoxin Erythromycin CDCF

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

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30.0 8 25.0 B) B) A- A- A/ 20.0 A/ 6 B- B- atio ( atio (

R 15.0 R 4 E ux

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2 5.0 2.0 0.0 0 Wild Type BCRP KO BCRP/MDR1 KO BCRP/MRP2 KO Wild Type MDR1 KO BCRP KO MRP2 KO MDR1/BCRP MDR1/MRP2 MRP2/BCRP Estrone sulfate Nitrofurantoin KO KO KO

Figure 2. Efflux of BCRP Substrates in wild-type (WT) and BCRP knockout (KO) cell lines 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 References sigma.com/transporterko 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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