digoxin and verlukast

Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system. In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (K(m) of 2.2 ± 0.3 µM and V(max) of 115 ± 26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC(50) values ≤ 1.1 µM. Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2. A highly significant correlation (r(2) = 0.959) between the in vitro IC(50) values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds. This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.

Topics: Animals; ATP-Binding Cassette Transporters; Benzbromarone; Bile Canaliculi; Biliary Tract; Biological Transport; Cyclosporine; Digoxin; Drug Evaluation, Preclinical; Drug Interactions; Fluoresceins; High-Throughput Screening Assays; Metabolic Clearance Rate; Propionates; Quinolines; Rats; Taurocholic Acid; Transport Vesicles

Efflux transporters expressed in the apical membrane of intestinal enterocytes have been implicated in drug oral absorption. The current study presents a strategy and tools to quantitatively predict the impact of efflux on oral absorption for new chemical entities (NCEs) in early drug discovery. Sixty-three marketed drugs with human absorption data were evaluated in the Caco-2 bidirectional permeability assay and subjected to specific transporter inhibition. A four-zone graphical model was developed from apparent permeability and efflux ratios to quickly identify compounds whose efflux activity may distinctly influence human absorption. NCEs in "zone 4" will probably have efflux as a barrier for oral absorption and further mechanistic studies are required. To interpret mechanistic results, we introduced a new quantitative substrate classification parameter, transporter substrate index (TSI). TSI allowed more flexibility and considered both in vitro and in vivo outcomes. Its application ranged from addressing the challenge of overlapping substrate specificity to projecting the role of transporter(s) on exposure or potential drug-drug interaction risk. The potential impact of efflux transporters associated with physicochemical properties on drug absorption is discussed in the context of TSI and also the previously reported absorption quotient. In this way, the chemistry strategy may be differentially focused on passive permeability or efflux activity or both.

Topics: Adenosine; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Sub-Family B Member 4; ATP-Binding Cassette Transporters; Biological Transport; Caco-2 Cells; Chromatography, Liquid; Dibenzocycloheptenes; Diketopiperazines; Drug Discovery; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Absorption; Mass Spectrometry; Models, Biological; Neoplasm Proteins; Pharmaceutical Preparations; Predictive Value of Tests; Propionates; Quinolines; Substrate Specificity

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

The objective was to directly compare the four different "calculation" methods of assessing P-gp inhibition potential using experimental data obtained from approximately 60 structurally diverse internal research and marketed compounds. Bidirectional studies for digoxin (probe for P-gp substrate) were performed with and without test compounds (at 10 microM). Four different calculation methods were applied to the same dataset (raw bidirectional permeability values) to obtain the "percent inhibition of P-gp" for these compounds using the different methods. Significantly different inhibition potential was obtained with the "exact" same experimental dataset depending on the calculation method used. Subsequently, entirely different conclusions regarding the "inhibition potential" of test compound was reached due to the different calculation methods. Based on the direct comparison of these methods, method no. 3 (i.e., inhibition of B to A permeability of digoxin) is recommended as the calculation method ideal during screening stages due to its high throughput amenability. The methodology is capable of rapidly screening compounds with adequate reliability for early stage drug discovery. Method no. 3 provides an abridged version of a bidirectional study that is fully capable of identifying all non-inhibitors (0-20%), moderate inhibitors (20-60%), and potent inhibitors (>60%) and demonstrates high correlation with method no. 1 (inhibition based on both A to B and B to A permeability of digoxin). Nevertheless, method no. 1 might be appropriate for more detailed mechanistic studies required in late stage discovery and development.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Caco-2 Cells; Cell Line, Tumor; Cell Membrane Permeability; Digoxin; Drug Evaluation, Preclinical; HIV Protease Inhibitors; Humans; Indinavir; Kinetics; Propionates; Quinolines; Saquinavir; Verapamil

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 extracts from the roots of Salvia miltiorrhiza Bunge (Danshen) are widely and traditionally used in the treatment of angina pectoris, acute myocardial infarct, hyperlipidemia and stroke in China and other Asian countries. In this study, we have investigated the role of P-glycoprotein (P-gp) in the intestinal absorption of tanshinone IIA (TSA), a major active constituent of Danshen, using several in vitro and in vivo models. The oral bioavailability of TSA was about 2.9-3.4% in rats, with non-linear pharmacokinetics when its dosage increased. In a single pass rat intestinal perfusion model, the permeability coefficients (P(app)) based on TSA disappearance from the luminal perfusates (P(lumen)) were 6.2- to 7.2-fold higher (P < 0.01) than those based on drug appearance in mesenteric venous blood (P(blood)). The P(blood), but not P(lumen), was significantly increased when co-perfused with verapamil, or quinidine (both P-gp inhibitors). The uptake and efflux of TSA in confluent Caco-2 cells were significantly altered in the presence of verapamil, quinidine, MK-571, or probenecid. The transport of TSA across Caco-2 monolayers was pH-, temperature- and ATP-dependent. Furthermore, the transport from the apical (AP) to basolateral (BL) side of the Caco-2 monolayers was 3.3- to 8.5-fold lower than that from the BL to AP side, but such a polarized transport was attenuated by co-incubated verapamil or quinidine. A polarized transport was also observed in the control MDCKII cells and more apparent in MDR1-MDCKII monolayers, with the P(app) values of TSA in the BL-AP direction being 7- to 9-fold higher in MDR1-MDCKII monolayers than those in the control MDCKII cells. Moreover, TSA significantly inhibited P-gp-mediated transport of digoxin in P-gp-overexpressing membrane vesicles with an IC(50) of 2.6 microM, but stimulated vanadate-sensitive P-gp ATPase activity with estimated K(m) and V(max) values of 10.70 +/- 0.69 microM and 67.65 +/- 1.31 nmol/min/mg protein, respectively. TSA was extensively metabolized to tanshinone IIB (TSB), and two other oxidative metabolites in rat liver microsomes, but the formation rate of TSB in rat intestinal microsomes was only about 1/10 of that in liver microsomes. These findings indicate that TSA is a substrate and reversing agent for P-gp; and P-gp-mediated efflux of TSA into the gut lumen and the first-pass metabolism contribute to the low oral bioavailability. Further studies are needed to explore the role of other drug transp

Topics: Abietanes; Adenosine Triphosphate; Administration, Oral; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Availability; Caco-2 Cells; Digoxin; Dogs; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Humans; Hydrogen-Ion Concentration; Intestinal Absorption; Intestinal Mucosa; Intestines; Male; Microsomes, Liver; Multidrug Resistance-Associated Proteins; Phenanthrenes; Plant Roots; Probenecid; Propionates; Quinidine; Quinolines; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Temperature; Transfection; Verapamil

The potential multiple carrier-mediated mechanisms involved in the transport of digoxin in rat intestine were investigated by the rapid filtration method in rat intestinal brush-border vesicles (BBMV) and in vitro Ussing chambers. The uptake of digoxin showed a typical overshoot phenomenon in the presence of an inward proton gradient and an outward bicarbonate gradient, or an outward glutathione gradient in BBMV. Good fitting to an equation consisting of both saturable and linear terms was obtained using non-linear regression analysis. GF120918, a specific P-gp inhibitor, significantly increased the absorptive permeability of digoxin in rat ileum (7.02 x 10(-7) cm/s versus 2.11 x 10(-6) cm/s with GF120918) but the addition of DIDS (0.5 mM), an anionic transporter inhibitor, or bromosulfophthalein (0.1 mM), an Oatp inhibitor, in the presence of GF120918 decreased the absorptive permeability compared with GF120918 alone (2.11 x 10(-6) cm/s versus 1.46 x 10(-6) cm/s, p<0.01 and 2.11 x 10(-6) cm/s versus 1.60 x 10(-6) cm/s, p<0.05, respectively). The above results suggest the involvement of carrier-mediated uptake mechanism, possibly Oatp, in digoxin absorption. Interestingly, GF120918 (1 microM) did not abolish the polarized transport of digoxin in rat jejunum and ileum, and DIDS (0.5 mM), not a P-gp inhibitor, and MK571 (50 microM), an MRP-selective inhibitor, can also significantly decrease the exsorptive permeability of digoxin. This result indicates the involvement of non-P-gp efflux transporter in digoxin secretion and this transporter is DIDS and MK571-sensitive. Contrary to conventional concept, our studies show that intestinal absorption of digoxin may involve both active uptake and efflux transporters. Our study may have clinical implications in drug-drug or drug-food interactions involving transporters.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acridines; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Digoxin; Ileum; In Vitro Techniques; Intestinal Absorption; Jejunum; Male; Microvilli; Organic Anion Transporters; Propionates; Quinidine; Quinolines; Rats; Rats, Sprague-Dawley; Sulfobromophthalein; Tetrahydroisoquinolines; Verapamil

One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.

Topics: Animals; Biological Transport; Bronchodilator Agents; Choroid Plexus; Digoxin; Enzyme Inhibitors; Epithelial Cells; Fluoresceins; Male; Methotrexate; Microscopy, Confocal; Organic Anion Transporters; Propionates; Quinolines; Rats; Rats, Sprague-Dawley

Caco-2 epithelial layers were used as a model to re-evaluate the mechanism(s) by which intestinal digoxin absorption is limited by its active secretion back into the lumen. It is widely recognised that intestinal secretion of digoxin is mediated by the ATP-binding cassette (ABC) transporter Multidrug Resistance 1, MDR1. In MDR1-transfected Madin-Darby canine kidney, MDCKII, cell monolayers, digoxin secretion was reduced by the MDR1 inhibitor cyclosporin A, whereas no inhibition was seen in the presence of MK-571, 3-([(3-(2-[7-chloro-2-quinolinyl]ethyl)phenyl]-[(3-dimethylamino-3-oxoprphyl)-thio)-methyl]-thio) propanoic acid, a Multidrug Related Protein (MRP) inhibitor. In contrast, digoxin secretion by Caco-2 epithelia was significantly inhibited by both cyclosporin A and MK-571, suggesting that an additional non-MDR1 component may contribute to this transport. Since digoxin secretion by MRP2-transfected MDCKII monolayers was increased by only 1.2-fold relative to controls, it is likely that the contribution of MRP2 to digoxin secretion by Caco-2 cells is negligible. An additional MK-571-sensitive secretory pathway for digoxin, together with MDR1, is likely to mediate digoxin secretion in Caco-2 epithelia.

Topics: Animals; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Caco-2 Cells; Cyclosporine; Digoxin; Humans; Kinetics; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Transfection; Vinblastine