estrone-sulfate and verlukast

The objective of this study was to determine an appropriate culture period to assess whether a compound of interest is transported by efflux transporters such as human multidrug resistance 1 (hMDR1), human multidrug resistance-associated protein 2 (hMRP2), and human breast cancer resistance protein (hBCRP) in Caco-2 cells. Caco-2 cells were cultured on a Transwell for 1 to 6 weeks. The expression of these transporters in the mRNA and protein levels was examined using a real-time polymerase chain reaction and Western blotting, respectively. Transcellular transport activities using digoxin, ochratoxin A, olmesartan, and estrone-3-sulfate were also examined. Except for digoxin, the permeability coefficient (P(app)) ratio of the three compounds at 2 weeks was the highest in the periods tested. The P(app) ratio of digoxin at 2 weeks was higher than that at 3 weeks. The temporal expression profile of each transporter in the mRNA level was similar to that in the protein level, and the functions of hMRP2 and hBCRP were roughly correlated with the expression in the mRNA and protein levels, but that of hMDR1 was not. These data suggest that among all the culture periods evaluated a 2-week culture is the best culture period for transport studies to identify whether a compound is a substrate for hMDR1, hMRP2, and hBCRP.

Topics: Angiotensin II Type 1 Receptor Blockers; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Caco-2 Cells; Calcium Channel Blockers; Digoxin; Electric Conductivity; Estrone; Humans; Imidazoles; Leukotriene Antagonists; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Ochratoxins; Propionates; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazoles; Tight Junctions; Time Factors; Verapamil

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.

Topics: Animals; Biological Transport; Choroid Plexus; Colforsin; Cyclic AMP-Dependent Protein Kinases; Estrone; Female; In Vitro Techniques; Kinetics; Leukotriene C4; Male; Meglumine; Methotrexate; Models, Biological; Organic Anion Transporters; p-Aminohippuric Acid; Potassium; Propionates; Protein Kinase C; Quinolines; Rats; Rats, Sprague-Dawley; Sodium Cyanide; Squalus acanthias; Taurocholic Acid; Tetradecanoylphorbol Acetate; Xanthenes

The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug-transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER=17) and MDR-MDCK (ER=120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER=15 and 88, respectively) but not in MDR-MDCK cells (ER=1.1 and 1.3, respectively). CsA at 10 microM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug-drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug-drug interactions.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Caco-2 Cells; Cell Line; Cyclosporine; Digoxin; Dogs; Drug Interactions; Estrone; Humans; Indoles; Kidney; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Permeability; Propionates; Quinolines; Sulfasalazine

The purpose of this study was to determine the mechanisms responsible for transport of raloxifene and its hydrophilic conjugates. Human intestinal Caco-2 cell culture model and Caco-2 cell lysate were used for the studies. The results indicated that absorptive permeability (PAB) of raloxifene was lower than its secretory permeability (PAB). As the concentration increased, the efflux ratio (PBA/PAB) decreased, but PBA increased. PAB was also increased in the presence of verapamil and cyclosporine A, two P-glycoprotein inhibitors. Raloxifene was extensively metabolized into sulfated and glucuronidated conjugates. The extent of metabolism or clearance was decreased as the concentration increased from 3.4 (96%) to 30 (22%) microM. Multidrug resistance-related protein inhibitors MK-571 (C26H26ClN2O3S2) and leukotriene C4 significantly decreased (maximal 80%) apical efflux of both conjugates. They also significantly decreased (maximal 85%) basolateral efflux of glucuronides but not sulfates. On the other hand, organic anion transporter (OAT) inhibitor estrone sulfate and estrone glucuronide significantly decreased (maximal 50%) the efflux of sulfate from both sides but had variable effects on glucuronide efflux. Inhibition of conjugate efflux with the OAT inhibitor estrone sulfate was concentration dependent, which resulted in increased transport of intact raloxifene (maximal 90%). This increase in raloxifene transport was also observed in the presence of another OAT inhibitor estrone glucuronide (70%). In conclusion, this is the first report that inhibition of an efflux transporter responsible for the transport of metabolites can result in increase in the transport of the intact compound. It also provides additional explanation why raloxifene has low bioavailability but a long half-life.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Availability; Biological Transport; Caco-2 Cells; Cyclosporine; Drug Interactions; Estrogen Antagonists; Estrone; Humans; Intestines; Multidrug Resistance-Associated Proteins; Organic Anion Transporters; Propionates; Quinolines; Raloxifene Hydrochloride; Time Factors