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-glycop rotein (Pgp) function in whole blood as an indicator of irt vivo drug activity, Since the CD56(+) subset of peripheral blood lymphocytes (PB Ls) has been shown to express functional Pgp, the changes in rhodamine 123 (R123) uptake by CD56(+) PBLs from GG918-treated and untreated wh ole blood were used as the basis for these studies, In an ex vivo stud y, heparin-treated whole blood was obtained from normal volunteers, an d 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 additio n, R123 loading times ranged from 0 to 80 min, Flow cytometric analyse s 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 m odel, a dose-dependent response was seen for GG918 with a 2-fold incre ase in cellular R123 intensity being produced at a drug concentration of 80 nM. When this assay method was applied to blood samples from vol unteers dosed p.o. with GG918, similar shifts in R123 fluorescence of the CD56(+) PBLs were observed with significant increases in R123 inte nsity occurring at serum concentrations as low as 40 nM. In contrast t o assays in which target cell populations are enriched prior to testin g, the addition of the substrate (R123) directly to the blood sample c ombined with the segregation of the target cells by specific immunoflu orescence provides the investigator an indication of irt situ activity of circulating drug, Thus, CD56(+) PBLs may prove useful as a surroga te target for monitoring multidrug resistance inhibitor activity in si tu.