RANKING OF P-GLYCOPROTEIN SUBSTRATES AND INHIBITORS BY A CALCEIN-AM FLUOROMETRY SCREENING ASSAY

In order to compare the capacities of a variety of compounds to interf ere with P-glycoprotein (Pgp) function, a novel assay was set up to wo rk on a large screening scale. The model assay measures the capacity o f parental sensitive (Par) and multidrug-resistant (MDR) cells to effl ux a small fixed amount of acetoxymethyl calcein (calcein-AM) after th eir pretreatment with concentration ranges of known Pgp modulators. Th is microplate cytometry-based assay was performed with two different p airs of cell lines, the human lymphocytic leukemia CEM cells and the m urine monocytic leukemia P388 cells. For a given Pgp-expressing MDR ce ll line, a Pgp modulator EC(50) was defined as the concentration requi red to restore half of the calcein retention shown by similarly treate d Par cells. With both MDR-P388 and MDR-CEM cells, EC(50) comparisons ranked five reference Pgp modulators as follows: SDZ 280-446 > SDZ PSC 833 > cyclosporin A > verapamil > vinblastine. Further use of the MDR -CEM cells could rank 15 Pgp modulators for their capacity to interfer e with calcein-AM efflux as follows: SDZ 280-446 1.9 x > SDZ PSC 833 8 .3 x > cyclosporin A 3.8 x > amiodarone 1.1 x > quinacrine 1.6 x > ver apamil 1.4 x > quinidine 1.1 x > vinblastine 11 x > vincristine 2 x > chloroquine > beta-lumicolchicine greater than or equal to gamma-lumic olchicine 2 colchicine > etoposide greater than or equal to doxorubici n. This calcein-AM assay should open the way for ranking large numbers of novel structures for their potential Pgp modulator properties, par ticularly for an efficient screening of Pgp function antagonists, but it does not allow defining whether their inhibition may be competitive or not.