ADME/Tox Assays

CompoZr Transporter Knockout Cell Lines

Sigma® Life Science has utilized CompoZr Zinc Finger Nuclease (ZFN) genome editing technology to create genetically modified cell lines for research within the field of ADME/Tox. Engineered cell lines include hepatocytes that enable the study of metabolism, CYP induction and uptake and excretion of xenobiotics. To study renal function primary renal cells were engineered that display normal epithelial morphology and exhibits uptake/efflux transporter activity along with a highly sensitive response to renal toxicants. To model the intestine C2BBe1 cells, a Caco-2 subclone, have efflux transporters of interest functionally knocked out. All CompoZr ADME/Tox Cell Lines are available in multiple formats that include cryopreserved vials, assay ready plates, Petaka plates or as services through select CRO providers.

An example of the utility of CompoZr ADME/Tox Cell Lines is the functional knockout of membrane transporters which enables unambiguous interpretation of transporter/substrate interactions as potentially non-specific chemical inhibitors are not required. The non-specific chemical inhibitor, MK571, is used to study MRP2/substrate interactions in Caco-2 cells. Published data demonstrates the promiscuity of MK571 leads to ambiguous results, data with the chemotherapeutic vinblastine being a prime example. While previously generated data indicates vinblastine may be a substrate for both MDR1 and MRP2, when tested in Caco-2 Transporter Knockout cell lines the data clearly indicates vinblastine is specifically an MDR1 substrate.

Click here for additional technical and ordering information on each respective CompoZr ADME/Tox Cell Line or click on a link below to order directly from the website.

   
BSEP Knockout
Transporter
*Coming Soon


MDR1 Knockout
 Transporter
*Coming Soon

CAR Knockout
 CYP Induction
*Coming Soon
  RPTEC Cell Line
*Coming Soon
  Wild Type Caco-2
Assay Ready Plates

MDR1 Knockout
Transporter

BCRP Knockout
Transporter

MRP2 Knockout
Transporter

MRP4 Knockout
Transporter
*Coming Soon

 Efflux of Vinblastine in Caco-2 Transporter Knockout Cells

An example of the utility of Transporter Knockout Cells can be seen with Vinblastine which 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 by 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 1. 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.