Pumping of Drugs by P-Glycoprotein: A Two-Step Process?

Abstract

The apparent inhibition constant, K app , for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, K app was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows that K app for inhibition should increase linearly with the amount of pumps present in the membrane for a reverser that inhibits pumping from the cytoplasmic face. In contrast, if the reverser acts by blocking transport from the outer face, i.e., preemptively, K app should be independent of the number of pumps present. The experimental data suggest that verapamil blocks pumping at the extracellular face of the membrane, whereas the other three blockers act on pumping from the cytoplasmic phase. The maximum degree of inhibition was the same for all four blockers; thus, they do not act in parallel but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin at the cytoplasmic face and transfers it to the verapamil-binding site, from where daunorubicin is emptied at the extracellular surface.Multidrug resistance (MDR) constitutes a major barrier to effective chemotherapy of cancer (Bates et al., 1996). Although alternate resistance mechanisms exist (Borst et al., 2000;Litman et al., 2001;Sparreboom et al., 2003), the MDR phenotype is most often characterized by a reduced intracellular drug level and overexpression of the ATP-binding cassette transporter P-glycoprotein (P-gp) that is capable of transporting a wide range of xenobiotic compounds out of tumor cells (Chabner and Fojo, 1998;Seelig and Landwojtowicz, 2000). The transport activity of P-gp can be blocked and multidrug resistance reversed by the addition of substrate analogs, or so-called chemosensitizers or modulators (Skovsgaard et al., 1984;Krishna and Mayer, 2001).In the literature, variable values for IC 50 (the drug concentration that gives half-maximal inhibition) for P-glycoprotein reversal are reported for the same drug in different models.For example, for the blocker cyclosporin A, the IC 50 for inhibition of daunorubicin pumping by P-gp ranges from 0.04 M (P388/ADR cells) to ca. 4 M (CEM/ vinblastine1000 cells) (Hu et al., 1990), with a range of intermediate values (Cardarelli et al., 1995;Lan et al., 1996;Litman et al., 1997). For verapamil, the IC 50 for reversal of daunorubicin extrusion by P-gp ranges from 0.4 M (SW2780AD cells) (Javaheri et al., 1983) to 10 M (CEM/ vinblastine1000 cells) (Hu et al., 1990),...

PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism

“…Therefore, we prepared PMs from DOX40 cells to further characterize PK11195 binding in Pgp-expressing cells and documented that PM fractions contained Pgp but were depleted of mitochondrial inner and outer mitochondrial membrane markers ( Figure 6A). DOX40 PM fractions showed specific 3 H-CSA binding, consistent with documented Pgp binding by CSA, 31,32,[43][44][45][46][47][48][49][50][51] and specific 3 H-PK11195 binding was also reproducibly measured in DOX40 PMs. Specific PK11195 binding that was measured in 8226 PM assays (data not shown) suggests that pBR can be PM-localized, consistent with other reports.…”

“…CSA and many other efflux modulators bind transporters directly. 31,32,[43][44][45][46][47][48][49][50][51] First, we asked whether PK11195 binds Pgp, first by comparing PK11195 binding in 9 primary AML samples for which additional aliquots were available. CSA and PK11195 had increased dye retention in 5 of these samples (Pgp-positive AMLs), whereas 4 samples showed no efflux capacity (Pgp-negative AMLs).…”

Natural Products Modulate the Multifactorial Multidrug Resistance of Cancer

“…Since the substrates appear to be freely diffused into the cells, they can be found by looking for MDR1 in the plasma membrane, as it appears from in vitro testing that MDR1 recognizes its substrates before they enter the cytoplasm [55]. Studies find that many substrates of MDR1 are also substrates of drug-metabolizing enzymes, such as CYP3A4.…”

If the KI Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC50 for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?

“…They speculated on reasons to explain this, including the possibility that there was a higher P-gp surface density in the blood-brain barrier, following the analysis in Litman et al (2003). Kurnik et al (2008) found in humans that tariquidar could fully inhibit P-gp in lymphocytes but not in the blood-brain barrier.…”