Advanced in Vitro Tools for Addressing Complex Transporter Issues

Tami Dvash, R&D, Sigma Aldrich, Rehovot, Israel

Membrane transporters P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP2), and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic.  Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter.  Our aim was to evaluate potential substrates that have generated ambiguous results in other models using conventional chemical inhibitors.   The studies described were carried out using Sigma's novel double knock out (KO) cell lines in C2BBe1 cells. The three double KO cell lines were generated using zinc finger nuclease technology to specifically introduce a stop codon in the protein and render that particular transporter(s) non- functional.  The lines used to generate the data presented are MDR1/BCRP KO, MDR1/MRP2 KO, and BCRP/MRP2 KO.   The wild-type and KO cell lines were tested for transporter function in transwell bidirectional assays using potential substrates.  Three case studies will be presented of substrates that had produced ambiguous results in wild-type Caco-2 cells, MDCKII over-expressing cell lines, and membrane vesicle studies (respectively):

1. Our double KO cell line, MDR1/MRP2 KO, conclusively demonstrated no interaction with BCRP with a compound previously suggested to be a substrate for both MDR1 and BCRP.
2. Using both MDR1/MRP2 KO and BCRP/MRP2 KO lines a compound was determined to be an MDR1 only substrate while it was previously known to interact with BCRP.
3. Using both MDR1/MRP2 KO and BCRP/MRP2 KO lines a compound was determined to be a dual MDR1 and BCRP substrate.

These cell lines have proven useful in clarifying complex drug interactions when more than one transporter is involved without some of the current chemical inhibition selectivity issues and genetic knock down approaches.