In vitro-to-in vivo prediction of p-glycoprotein-based drug interactions at the human and rodent blood- brain barrier

In vitro inhibition of P-glycoprotein (P-gp) expressed in cells is routinely used to predict the potential of in vivo P-gp drug interactions at the human blood-brain barrier (BBB). The accuracy of such predictions has not been confirmed because methods to quantify in vivo P-gp drug interactions at the human BBB have not been available. With the development of a noninvasive positron emission topography (PET) imaging method by our laboratory to determine P-gp-based drug interactions at the human BBB, an in vitro-in vivo comparison is now possible. Therefore, we developed a high throughput cell-based assay to determine the potential of putative P-gp inhibitors [including cyclosporine A (CsA)] to inhibit (EC50) the efflux of verapamil-bodipy, a model P-gp substrate. LLCPK1-MDR1 cells, expressing recombinant human P-gp, or control cells lacking P-gp (LLCPK1) were used in our assay. Using this assay, quinine, quinidine, CsA, and amprenavir were predicted to be the most potent P-gp inhibitors in vivo at their respective therapeutic maximal unbound plasma concentrations. The in vitro EC50 of CsA (0.6 mu M) for P-gp inhibition was virtually the same as our previously determined in vivo unbound EC50 at the rat BBB (0.5 mu M). Moreover, at 2.8 mu M CsA (total blood concentration), our in vitro data predicted an increase of 129% in [C-11] verapamil distribution into the human brain, a value similar to that observed by us (79%) using PET. These data suggest that our high throughput cell assay has the potential to accurately predict P-gp drug interactions at the human BBB.