|
||||||||||||||||||||||||||||||
|
|
Membrane drug transporters have been identified as a determinant of drug disposition in the body, potentially affecting absorption, pharmacokinetics, drug-drug interactions and safety profiles. A key challenge in drug transporter studies is that substrates are often recognized by multiple transporters and the specificity of chemical inhibitors can be uncertain. To improve upon existing assays Sigma® Life Science has applied its exclusive CompoZr Zinc Finger Nuclease (ZFN) technology to create functional knockouts of the intestinal efflux transporters MDR1, BCRP and MRP2 in C2BBe1 cells, a Caco-2 subclone. The use of transporter knockout cell lines allows unambiguous interpretation of transporter/substrate interactions as potentially nonspecific chemical inhibitors are not required. C2BBe1 transporter knockout cells are ideal in that they express multiple transporters, are human derived and grow in a homogenous monolayer that forms tight junctions. Transporter Knockout Cell Lines are available in three different formats: 24 and 96-well Transwell Assay Ready Plates, licensing at for-profit institutions and analysis of test compounds by a service provider. To learn more about these options click below:
|
|||||||||||||||||||||||||||||
MDR1 Single and Double Knockout Cell Lines
|
Figure 1. MDR1 Single and Double Knockout Cell Lines can be used, in conjunction with wild type C2BBe1 cells, to identify interactions between the MDR1 efflux transporter and test substrates. Digoxin and Erythromycin are examples of MDR1 substrates with efflux ratios ≤ 2 when tested in a transwell assay with the MDR1 Single and Double Knockout Cell Lines, no chemical inhibitors were utilized to generate these data. Values are the mean +/- standard deviation, n ≥ 3 assays of triplicates. |
|
BCRP Single and Double Knockout Cell Lines
|
Figure 2. BCRP Single and Double Knockout Cell Lines can be used, in conjunction with wild type C2BBe1 cells, to identify interactions between the BCRP efflux transporter and test substrates. Estrone Sulfate and Nitrofurantoin are examples of BCRP substrates with efflux ratios ≤ 2 when tested in a transwell assay with BCRP Single and Double Knockout Cell Lines, no chemical inhibitors were utilized to generate these data. Values are the mean +/- standard deviation, n ≥ 3 assays of triplicates. |
|
MRP2 Single and Double Knockout Cell Lines
|
Figure 3. The non-fluorescent 5(6)-carboxy-2’,7’-dichlorofluorescein-diacetate (CDCFDA) is taken up by passive diffusion into wild type C2BBe1 cells. It undergoes hydrolysis to form the fluorescent product CDCF; CDCF is then effluxed by active transport. When tested in MRP2 Single and Double Transporter Knockout Cell Lines the efflux ratio for CDCF is reduced to ≤ 2 which indicates CDCF is no longer undergoes active efflux, no chemical inhibitors were utilized to generate these data. Values are the mean +/- standard deviation, n ≥ 3 assays of triplicates. |
|
Analysis of Cimetidine in Double Transporter Knockout Cell Lines
|
Figure 4. Cimetidine is a crossover substrate that has an efflux ratio ≥ 2 in all Single Knockout Cell Lines. When analyzed with the MDR1/BCRP Double Knockout Cell Line cimetidine has an efflux ratio < 2. This loss of active transport indicates cimetidine is a substrate for both MDR1 and BCRP while active efflux still occurs in all other knockout cell lines. Values are the mean +/- standard deviation, n ≥ 3 assays of triplicates. |
Efflux of Vinblastine with MDR1 and MRP2
Vinblastine is a prescription drug that’s used to treat multiple types of cancer which include Hodgkin’s disease, Kaposi’s sarcoma, non-Hodgkin’s lymphoma, breast cancer, and testicular cancer. Previous data indicates that Vinblastine is a substrate of both MDR1 and MRP2 (see link). A recent publication, Mease K, et al. J Pharm Sci., 2012 May; 101(5):1888-97, conducted a series of experiments to further elucidate Vinblastine as a substrate for MDR1 and MRP2 in Caco-2 cells. In this study the chemical inhibitors zosuquidar (LY335979) and MK571 where used to inhibit MDR1 and MRP2 respectively. Data demonstrated MK571 is non-specific, inhibiting multiple transporters including MDR1, BCRP and MRP2 which are all expressed in Caco-2 cells. Specifically with Vinblastine, the reduction of efflux with MDR1 is primarily due to the inhibition of MDR1 by MK571. The promiscuity of MK571 leads to the inability to conclusively determine if Vinblastine is a substrate for MDR1, MRP2 or is a crossover substrate for both transporters.
Efflux of Vinblastine in MDR1 and MRP2 Transporter Knockout Cells
|
Figure 5. To conclusively determine if Vinblastine is a substrate for MDR1 or MRP2 the drug was tested in both the MDR1 and MRP2 Transporter Knockout Cell Lines. Wild type Caco-2 cells demonstrated active efflux of Vinblastine, ER ≥ 2. In the MRP2 Knockout Cell Line, where MRP2 is completely absent and MDR1 is present, active efflux of Vinblastine also occurs. In the MDR1 Knockout Cell Line, where MDR1 is completely absent and MRP2 is present, active efflux of Vinblastine is eliminated. Since efflux was eliminated in the MDR1 knockout cell line but not the MRP2 knockout cell line this data indicates that Vinblastine is an MDR1 substrate and not an MRP2 substrate. This result was confirmed in the MDR1/MRP2 Double Knockout Cell Line. The data represents an average and standard deviation of 12 replicates for each cell line. No chemical inhibitors were used to generate these data. |
| Learning Center | Related Topics | |
