In vitro assays that reliably correlate with observed clinical drug-drug interactions (DDIs) facilitate drug development and safety testing and reduce reliance on animal testing. One important mechanism of DDI is the interaction of drugs with transport proteins in various tissues, including intestine, kidney, liver, brain and placenta. The human intestinal cell line, Caco-2, is widely used by academic, governmental and pharmaceutical labs to study drug transporters, but interpretation of the results is hampered by the expression of multiple drug efflux transporters in these cells. Chemical inhibitors of transporters are available and are used to partially dissect transport mechanisms. Unfortunately, currently available inhibitors are not totally specific, creating ambiguities in data interpretation. Knockdown of gene expression by interfering RNA is much more specific;therefore, transport assays employing Caco-2 cells in which a given transporter has been knocked down by continuous presence of RNAi targeted to that message have the potential of yielding specific and unambiguous transport results. In Phase I we used lentiviral-mediated short hairpin RNAi constructs targeted at drug efflux transporter sequences to construct Caco-2 knockdown cell lines and appropriate vector controls. We characterized the differentiation state, knockdown phenotype stability and drug transport properties of the constructs and determined that they met the success criteria spelled out in the Phase I application. In Phase II we propose to optimize the growth media for the various cell lines, scale-up the manufacture of assay plates and determine the stability of the cell lines to frozen storage. In addition we propose to develop and validate fluorescence-based assays for characterization of cell transporter function and to develop novel assay systems for monitoring the interaction of apical and basolateral transporters present in these cell lines. Successful completion of this Phase II grant will allow us to provide pre-plated assay plates or license these human knockdowns cells to academic, governmental and industrial labs that wish to study DDI's using an assay format compliant with the recommendations of the 2006 FDA draft guidance on in vitro testing for DDIs.
It has been estimated that roughly 2.8% of all hospitalizations are a result of drug-drug interactions (DDIs). The intent of this project is to develop new in vitro research tools that provide superior prediction of DDIs mediated by drug transport proteins in humans. In Phase I of this project we successfully created human intestinal cell lines that produce more specific DDI assays by reducing the expression of unwanted human drug transport proteins. In Phase II of the project we propose optimizing and scaling up these cell-based assay systems so that they can be made available to academic, governmental and industrial research labs involved in investigating DDIs and improving drug safety.
