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. The major efflux transporters of the ABC family, including P-glycoprotein (P-gp, ABCB1, MDR1 gene), BCRP (ABCG2), and MRP2 (ABCC2), 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 and chemicals such as statins, macrolide antibiotics, angiotensin blockers, and chemotherapeutic agents, affecting exposure and clearance in vivo.
Recently, the FDA and EMA have issued recommendations and guidelines for drug transporter studies in the pharmaceutical and biotech industries. New chemical entities are now routinely screened for potential transporter recognition and impact; interaction at clinical concentrations may indicate additional clinical trials during drug development.
Current cell-based transporter studies may involve cell lines such as Caco-2 intestinal enterocytes that express multiple transporters, requiring the use of transporter-specific substrates or inhibitors. However, substrates are often recognized by multiple transporters at different affinities, and the specificity of inhibitors is often uncertain or unknown, leading to mistaken interpretations of transporter interaction.
Utilizing CompoZr® zinc finger nucleases (ZFNs) from Sigma® Life Science, MDR1, BCRP, and MRP2 efflux transporter genes were targeted for ZFN-mediated knockout in a Caco-2 cell line. The resultant panel of single and double knockout cells show disruption of gene sequence as well as complete loss of transporter function in bidirectional transport assays to at least 40 passages post ZFN genomic-modification.
In this article, we describe the functional characterization of these cells using a set of industry-standard compounds targeting single transporters, as well as compounds displaying overlapping transporter specificity. These novel cells are powerful tools to elucidate transporter interactions without dependence on chemical inhibitors and clarify the potential impact of specific efflux transporters in drug disposition.