MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb 3 ) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb 3 , a water-soluble Gb 3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb 3 , but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb 3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb 3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb 3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb 3 endocytosis also upregulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb 3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs. Multidrug resistance (MDR)3 is one of the major obstacles for successful chemotherapy in patients with cancer. The MDR phenotype has been associated with overexpression of P-glycoprotein in cancer cells (1). MDR1 is a 170-kDa plasma membrane glycoprotein that uses ATP to pump hydrophobic molecules out of the cell (2, 3). Its efflux function decreases drug concentration in tumor cells that results in chemotherapy failure. MDR1 is expressed in relatively high levels in the apical membranes of epithelial cells of intestine, kidney, liver, and blood-brain/testes barriers (4). Thus, the presence of MDR1 within the endothelial cells' epithelium can affect the bioavailability and the oral availability of therapeutic drugs (5).Various attempts have been made to reverse this resistance using MDR1 inhibitors that interact with MDR1 and block drug efflux. First generation modulators such as calcium channel blockers, calmodulin inhibitors, and cyclosporin were developed for pharmacological uses other than reversal of MDR and were relatively nonspecific and weak inhibitors that were also substrates of MDR1, and their deleterious toxicities with their use at required concentrations to inhibit MDR1 ...

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The objectives of this study were to investigate raltegravir transport across several blood-tissue barrier models and the potential interactions with drug efflux transporters. Raltegravir uptake, accumulation, and permeability were evaluated in vitro in (i) Pglycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 1 (MRP1), or MRP4-overexpressing MDA-MDR1 (P-gp), HEK-ABCG2, HeLa-MRP1, or HEK-MRP4 cells, respectively; (ii) cell culture systems of the human blood-brain (hCMEC/D3), mouse blood-testicular (TM4), and human blood-intestinal (Caco-2) barriers; and (iii) rat jejunum and ileum segments using an in situ single-pass intestinal perfusion model. Our data suggest that raltegravir is a substrate but not an inhibitor of the drug efflux transporters P-gp and BCRP. These transporters might play a role in the restriction of raltegravir permeability across the blood-brain, bloodtesticular, and blood-intestinal barriers, potentially contributing to its low tissue concentrations and/or low oral bioavailability observed in the clinic setting.