Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918.

We sought to develop an assay for measuring the inhibition of P-glycoprotein (Pgp) function in whole blood as an indicator of in vivo drug activity. Since the CD56(+) subset of peripheral blood lymphocytes (PBLs) has been shown to express functional Pgp, the changes in rhodamine 123 (R123) uptake by CD56(+) PBLs from GG918-treated and untreated whole blood were used as the basis for these studies. In an ex vivo study, heparin-treated whole blood was obtained from normal volunteers, and GG918 and R123 were added at various concentrations for time course analyses of dye loading. GG918 concentrations from 2.5 to 800 nm were tested in incubations ranging from 15 min to 3 h prior to R123 addition. R123 loading times ranged from 0 to 80 min. Flow cytometric analyses of the CD56(+) PBLs indicated that the resolution of Pgp inhibition was dependent on inhibitor concentration and time of R123 loading and independent of the R123 concentrations tested. In this ex vivo assay model, a dose-dependent response was seen for GG918 with a 2-fold increase in cellular R123 intensity being produced at a drug concentration of 80 nm. When this assay method was applied to blood samples from volunteers dosed p.o. with GG918, similar shifts in R123 fluorescence of the CD56(+) PBLs were observed with significant increases in R123 intensity occurring at serum concentrations as low as 40 nm. In contrast to assays in which target cell populations are enriched prior to testing, the addition of the substrate (R123) directly to the blood sample combined with the segregation of the target cells by specific immunofluorescence provides the investigator an indication of in situ activity of circulating drug. Thus, CD56(+) PBLs may prove useful as a surrogate target for monitoring multidrug resistance inhibitor activity in situ.