INHIBITION OF P-GLYCOPROTEIN-MEDIATED VINBLASTINE TRANSPORT ACROSS HCT-8 INTESTINAL CARCINOMA MONOLAYERS BY VERAPAMIL, CYCLOSPORINE-A AND SDZ PSC-833 IN DEPENDENCE ON EXTRACELLULAR PH

The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transe pithelial flux of tritiated vinblastine was investigated using tight a nd highly resistant (R >1,400 Omega cm(2)) monolayer cultures of intes tinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-cul ture inserts. Apical addition of these chemosensitisers inhibited drug flux (137 pmol h(-1) cm(-2); range, 133-142 pmol h(-1) cm(-2)) in the basal to apical secretory direction at clinically relevant concentrat ions, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM). Acidification of the mod ulator-containing apical compartment to an extracellular pH (pHo) of 6 .8 had no influence on MDR reversal by CsA at 1 mu g/ml (0.9 mu M; flu x inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 mu M; flux inhibit ion, 60%), in contrast to R,S- and R-VPL, which showed decreased inhib ition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5). P-gp-mediated rhodamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison wi th pHo 7.5 in standard medium, but at low pHo the inhibitory activity of R-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished s ignificantly, again without a reduction in the effect of PSC 833 (rhod amine 123 flux inhibition, 75%). In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epitheli a and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for si ngle-cell suspensions, resulting in increased (2.2- to 3.7-fold) intra cellular drug accumulation. Functional apical P-gp expression, the abs ence of paracellular leakage and modulator-sensitive rhodamine 123 eff lux in single HCT-8 cells indicate a P-gp-mediated transcellular efflu x in HCT-8 monolayers. In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracel lular conditions, which reduce the VPL-induced drug retention signific antly. As far as MDR contributes to the overall cellular drug resistan ce of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.