R123 Efflux Research Articles

Inhibition of P-Glycoprotein Mediated Efflux in Caco-2 Cells by Phytic Acid.

Phytic acid (IP6) is a natural phosphorylated inositol, which is abundantly present in most cereal grains and seeds. This study investigated the effects of IP6 regulation on P-glycoprotein (P-gp) and its potential mechanisms using in situ and in vitro models. The effective permeability of the typical P-gp substrate rhodamine 123 (R123) in colon was significantly increased from (1.69 ± 0.22) × 10-5 cm/s in the control group to (3.39 ± 0.417) × 10-5 cm/s (p < 0.01) in the 3.5 mM IP6 group. Additionally, IP6 can concentration-dependently decrease the R123 efflux ratio in both Caco-2 and MDCK II-MDR1 cell monolayers and increase intracellular R123 accumulation in Caco-2 cells. Furthermore, IP6 noncompetitively inhibited P-gp by impacting R123 efflux kinetics. The noncompetitive inhibition of P-gp by IP6 was likely due to decreases in P-gp ATPase activity and P-gp molecular conformational changes induced by IP6. In summary, IP6 is a promising P-gp inhibitor candidate.

Increased expression of ATP binding cassette transporter genes following exposure of Haemonchus contortus larvae to a high concentration of monepantel in vitro.

BackgroundThere is some evidence that ATP binding cassette (ABC) transporters play a role in resistance to anthelmintics, particularly against macrocyclic lactones. Some anthelmintics, including ivermectin (IVM), have been shown to induce transcription of multiple ABC transporters in nematodes; however, the effects of monepantel (MPL) on transcription of these transporter genes has not been studied.MethodsLarvae of two MPL-susceptible isolates of Haemonchus contortus were exposed to MPL at two concentrations (2.5 and 250 μg/ml) for periods of 3, 6 and 24 h. Transcription levels of sixteen ABC transporter genes were measured at the end of the incubation periods. The consequences of MPL exposure were examined by measuring rhodamine-123 efflux from the larvae, and their sensitivity to subsequent treatment with IVM or levamisole.ResultsMultiple ABC transporter genes showed significantly higher transcription in both worm isolates following exposure to MPL at 250 μg/ml for 3, 6 or 24 h, particularly the P-glycoprotein (P-gp) genes pgp-11, pgp-12 and pgp-14. Of these, only pgp-11 maintained the elevated levels 24 h after the end of the drug exposure period. In contrast, there was only a single instance of low-level upregulation as a result of exposure to MPL at 2.5 μg/ml. Larvae exposed to MPL at 250 μg/ml showed an increased efflux of rhodamine-123 and a proportion of the larval population showed an ability to subsequently tolerate higher concentrations of IVM in migration assays. There was no increased tolerance to IVM following pre-exposure to MPL at 2.5 μg/ml.ConclusionsExposure of H. contortus larvae to 250 μg/ml MPL results in increased transcription of multiple transporter genes and increased R-123 efflux. The subsequent ability of a proportion of the larvae to tolerate IVM suggests a protective role of ABC transporters across different chemical entities. However, these observations were only made at a concentration of MPL well above that experienced by parasitic life stages in vivo, and hence their significance remains unclear.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1806-9) contains supplementary material, which is available to authorized users.

Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

Interactions of human P-glycoprotein transport substrates and inhibitors at the drug binding domain: Functional and molecular docking analyses

Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis–cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean±SD) ranging from −11.8±0.54 (valspodar) to −3.98±0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of −6.9±0.30kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, 3H-1EPI, 3H-2EPI, 14C-1EPI, 14C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both 3H-2EPI and 14C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123.

Acetaminophen Inhibits Intestinal P-Glycoprotein Transport Activity

Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 μM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.

Mechanistic Studies of the Effect of Bile Salts on Rhodamine 123 Uptake into RBE4 Cells

To examine the ability of bile salts (BS) to act as permeation enhancers at the blood brain barrier, the effect of four BS (cholate, deoxycholate, monoketocholate and taurocholate) on accumulation of rhodamine 123 (R123) in rat brain endothelial (RBE4) cells was investigated. Experiments were performed using BS concentrations shown to be noncytotoxic to RBE4 cells. Uptake and efflux of R123 in the absence and presence of BS were studied by fluorescence spectroscopy and confocal microscopy. Changes in RBE4 cell membrane fluidity in the presence of BS were evaluated using fluorescence anisotropy. The direct interaction between BS and R123 (ion pairing) and the effect of BS on distribution of R123 into liposomes were studied by capillary electrophoresis. All BS influenced R123 uptake in a concentration-dependent manner and increased cell membrane fluidity. Monoketocholate produced the greatest increase in uptake and also significantly reduced R123 efflux probably by inhibition of P-glycoprotein (P-gp). Direct interaction of BS and R123 was weak, but distribution of R123 into liposomes was increased by BS. The results suggest that BS increase R123 uptake by increasing cell membrane fluidity and, in the case of MKC, by inhibiting P-gp.

N,N-Bis(cyclohexanol)amine aryl esters inhibit P-glycoprotein as transport substrates

P-Glycoprotein (Pgp) inhibition by three sets of four isomers of N,N-bis(cyclohexanol)amine aryl esters was assessed on rhodamine 123 (R123) efflux in human MDR1-gene transfected mouse T-lymphoma L5178 cells and on Sf9 ATPase activity. The most active compounds inhibited Pgp with IC50 values much lower than those of either cyclosporin A (CSA) or GF120918. As to R123 efflux inhibition, the role of the bond present in the second aryl moiety appeared important since the triple bond derivatives (3a–d) were the most powerful as compared to the double bond (2a–d) and the single bond (1a–d) counterparts. Concentration–inhibition curves of 2c and 3d exhibited a biphasic behaviour suggesting the existence of two binding sites in the recognition domain of Pgp. Persistence of inhibition by these compounds resulted to be intermediate between that caused by CSA and GF120918. R123 exhibited positive interaction with CSA, 1d, 1c, 2d, 2c and 3c, the concentration–inhibition curves being shifted leftward when R123 concentration was increased, while it exhibited negative interaction with 3d and no effect with GF120918. Sf9 ATPase activity was stimulated in an increasing order of potency by 2c, 3c, 2d, CSA, epirubicin and 3d. In a decreasing order of potency 3d, 2c, GF120918, CSA, 2d and 3c inhibited at sub-nanomolar concentrations epirubicin-stimulated ATPase activity. In conclusion, isomeric geometry and restriction of molecular flexibility of N,N-bis(cyclohexanol)amine aryl esters were crucial for their presentation to and inhibition of Pgp as transport substrates, R123 and epirubicin cooperating with them to this inhibition.

Anthelmintics Are Substrates and Activators of Nematode P Glycoprotein

P glycoproteins (Pgp), members of the ABC transporter superfamily, play a major role in chemoresistance. In nematodes, Pgp are responsible for resistance to anthelmintics, suggesting that they are Pgp substrates, as they are in mammalian cells. However, their binding to nematode Pgp and the functional consequences of this interaction have not been investigated. Our study showed that levamisole and most of the macrocyclic lactones (MLs) are Pgp substrates in nematodes. Ivermectin, although a very good substrate in mammalian cells, is poorly transported. In contrast to their inhibitory effect on mammalian Pgp, these drugs had a stimulatory effect on the transport activity of the reference Pgp substrate rhodamine 123 (R123) in the nematode. This may be due to a specific sequence of nematode Pgp, which shares only 44% identity with mammalian Pgp. Other factors, such as the affinity of anthelmintics for Pgp and their concentration in the Pgp microenvironment, could also differ in nematodes, as suggested by the specific relationship observed between the octanol-water partition coefficient (log P) of MLs and R123 efflux. Nevertheless, some similarities were also observed in the functional activities of the mammalian and nematode Pgp. As in mammalian cells, substrates known to bind the H site (Hoechst 33342 and colchicine) activated the R site, resulting in an increased R123 efflux. Our findings thus show that ML anthelmintics, which inhibit Pgp-mediated efflux in mammals, activate transport activity in nematodes and suggest that several substituents in the ML structure are involved in modulating the stimulatory effect.

Induction of intestinal P‐glycoprotein (Pgp) expression and activity by sub‐toxic doses of acetaminophen (AP) in rat intestine and human LS174T cells

AP induced liver Pgp expression and activity in rats.Aimto study the effect of sub‐toxic doses of AP on intestinal Pgp expression in rats and in a human intestinal cell line.M&amp;MMale Wistar rats were injected i.p. for 3 days with 0.2; 0.3; and 0.6 g/kg b.w. of AP (AP group) or vehicle (C group) (n=4). One day later Pgp expression and activity were evaluated by western‐blotting and rhodamine 123 (R123, 15 uM) serosal to mucosal transport in ileum isolated sacs with or without verapamil (V, 100 uM). Intestinal human LS174T cells were exposed to a sub‐toxic dose of AP (5 mM) for 48 hs, then Pgp expression was evaluated by QPCR and R123 efflux was determined by flow citometry with or without V.ResultsIleum Pgp expression increased in AP vs C (160%, P&lt;0.05). Cumulative R123 secretion was 44% higher in AP group (P&lt;0.05). V inhibited R123 transport in both AP and C. Cells exposed to AP expressed 60% more protein and presented 80% more activity than control cells (P&lt;0.05).ConclusionSub‐toxic doses of AP induced intestinal Pgp expression and activity in rats and in a human intestinal cell line. This suggests potential drug‐drug interactions when AP is co‐administered with other Pgp substrates. Consistently, preliminary studies have shown induction of Pgp in a human intestinal cell line exposed to sub‐toxic concentrations of APAP.

Effects of Curcuma spp. on P-glycoprotein function

The effects of Curcuma longa (khamin chan) and Curcuma sp. "khamin-oi" (khamin-oi), as well as isolated major curcuminoids on intestinal P-gp functions were evaluated in vitro. The accumulation of R123 in Caco-2 cells was increased and the R123 efflux ratios were significantly decreased by both Curcuma longa and Curcuma sp. "khamin-oi" extracts, indicating their roles on efflux transporters. The a-b transport of daunorubicin was increased by curcumin, demethoxycurcumin and bisdemethoxycurcumin while the b-a transport was significantly decreased by curcumin and demethoxycurcumin. However, calcein-AM uptake into the human P-gp overexpression cell line, LLC-GA5-COL300, was increased by curcumin and demethoxycurcumin in a concentration-dependent manner but not affected by bisdemethoxycurcumin. These results show that curcumin and demethoxycurcumin could inhibit P-gp but bisdemethoxycurcumin may modulate the function of other efflux transporters such as MRP. Taken together, the information may indicate the impact of Curcuma longa and Curcuma sp. "khamin-oi" on pharmacokinetics of orally administered drugs that are P-gp substrates.

Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P-glycoprotein substrate rhodamine 123

Reducing intracellular xenobiotic concentration is an important defence strategy used by cells challenged with foreign chemicals. One mechanism used to achieve this goal is via the use of P-glycoproteins (P-gps), ATP-dependent transporters that mediate the removal of hydrophobic compounds from cells. The energetic costs of this mechanism are unknown, therefore, the activity and respiratory costs associated with the P-gp-mediated efflux of rhodamine 123 (R123) was measured in isolated rainbow trout hepatocytes. The accumulation of R123 was rapid and concentration-dependent. Initial accumulation rates were 1.79 ± 0.41, 7.29 ± 1.06 and 15.30 ± 1.74 ng R123/min/10 6 cells when exposed to 1, 5 and 10 μM R123, respectively. Efflux was measured in cells ‘pre-loaded’ with R123 at each concentration, resulting in initial efflux rates of 0.77 ± 0.12, 2.02 ± 0.35 and 3.51 ± 0.84 ng R123/min/10 6 cells, respectively. The baseline oxygen consumption rate of hepatocytes was 33.21 ± 1.09 ng O 2/min/10 6 cells. Respiration rates were significantly higher in cells exposed to 5 and 10 μM R123 (39.08 ± 0.80 and 41.72 ± 0.61 ng O 2/min/10 6 cells), representing increases over basal rates of 18.5 and 25.7%, respectively. Measurements of isolated mitochondrial respiration established that changes in hepatocyte oxygen consumption were not through the direct effects of R123 on mitochondria. The P-gp inhibitor, XR9576 significantly inhibited R123 efflux from cells with a concomitant return of respiration rates to baseline values. This study demonstrates that increased P-gp transport of xenobiotics can significantly raise cellular respiration rates and may result in higher energy costs for organisms living in P-gp-substrate contaminated environments.

Evidence for modulation of P-glycoprotein-mediated efflux by methoxypolyethylene glycol-block-Polycaprolactone amphiphilic diblock copolymers.

The purpose of this study was to investigate the pathways involved in rhodamine 123 (R-123) accumulation enhancement in Caco-2 cells with a low molecular-weight methoxypolyethylene glycol-block-polycaprolactone (MePEG-b-PCL) diblock copolymer. R-123 accumulation by Caco-2 cells with MePEG17-b-PCL5 was measured in the presence of endocytosis inhibitors or under ATP depletion conditions. Directional flux studies were conducted with cell monolayers on Transwell plates. Endocytosis inhibitors had no effect on reducing R-123 accumulation with MePEG17-b-PCL5. The apical to basolateral (AP-->BL) flux of R-123 with MePEG17-b-PCL5 or verapamil was similar to R-123 alone. However the BL-->AP flux was significantly decreased with MePEG17-b-PCL5 and verapamil. The efflux ratio for R-123 flux was 3.2 and was reduced to 1.06 with MePEG17-b-PCL5 confirming the inhibition of P-glycoprotein (P-gp)-mediated efflux. R-123 accumulation at the conclusion of each of the flux experiments was similar for MePEG17-b-PCL5 and verapamil in the BL-->AP direction. The AP-->BL direction demonstrated a 2-fold increase for verapamil and a 5-fold increase with MePEG17-b-PCL5. This difference in R-123 accumulation was possibly due to the diblock enhancing passive membrane diffusion of R-123. MePEG17-b-PCL5 diblock reduced R-123 efflux through inhibition of P-gp efflux, and unimers may interact with cell membranes, increasing permeability and enhancing R-123 influx through increased transmembrane diffusion.

HMG-CoA reductase inhibitors and P-glycoprotein modulation.

1. Five 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P-glycoprotein (P-gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over-expresses the multi-drug resistance protein 1a/b (mdr1a/1b). 2. P-gp modulation was studied by a fluorimetric assay and confocal microscopy by means of R123 efflux and uptake experiments, respectively. 3. Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration-dependent manner. Lovastatin acid, simvastatin acid, fluvastatin and pravastatin did not show a significant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest affinities for P-gp and results were comparable for both methods. 4. In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to fluorimetric assay is a sensitive tool to study P-gp affinity in drug screening that is especially useful for early phases of drug development.

Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts.

The substitution of gluconate for Cl- is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl- on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl- (Cl--Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl- by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl--Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl-. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport.

Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation.

Drug disposition is often altered in inflammatory disease. Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked. The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs. Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp. AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration. Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay. P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes. Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123. Diminished mRNA levels of all three MDR isoforms were seen. LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver. Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific. Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver. This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.

Effects of pluronic block copolymers on drug absorption in Caco-2 cell monolayers.

The present work characterizes the effects of Pluronic copolymers on the transport of a P-gp-dependent probe, rhodamine 123 (R123) in Caco-2 cell monolayers. The accumulation and efflux studies were performed on the confluent Caco-2 monolayers using fluorescent probes with and without Pluronic copolymers. At concentrations below the critical micelle concentration single chains ("unimers") of Pluronic P85 enhanced the accumulation and inhibited the efflux of R123 in Caco-2 monolayers. The transport of the P-gp-independent probe, rhodamine 110 was not altered under these conditions. In contrast the micelles increased R123 accumulation to a much lower extent when compared to the unimers and enhanced R123 efflux in Caco-2 monolayers. Pluronic P85 unimers increase accumulation of a P-gp-dependent drug in Caco-2 monolayers through inhibition of the P-gp efflux system. The mechanism of the micelle effect is not known, however, it is very similar to the micelle effects in BBMEC. This has been previously shown to involve vesicular transport of the micelle-incorporated drug. The study suggests that Pluronic copolymers can be useful in increasing oral absorption of select drugs.

Inhibition of drug transport by genistein in multidrug-resistant cells expressing P-glycoprotein

It has been claimed that the flavonoid genistein could be used to distinguish multidrug-resistant tumors expressing the multidrug resistance-associated protein (MRP) from those expressing P-glycoprotein (Pgp). Genistein would block drug transport by MRP without affecting Pgp-mediated drug transport. However, we found that exposure to 200 μM genistein elicited an elevation in intracellular accumulation of rhodamine 123 (R123) and daunorubicin (DNR) in Pgp-expressing cell lines. Genistein inhibited R123 efflux in a rapidly reversible manner ( ca. 2 min). The flavonoid also decreased photoaffinity labeling of Pgp by [ 3H]azidopine, a Pgp substrate. The present results show that genistein interacts with Pgp and inhibits Pgp-mediated drug transport. Hence, genistein cannot be used in simple assays to distinguish MRP- and Pgp-expressing cells.

Modulation of multidrug resistance by BIBW22BS in blasts of de novo or relapsed or persistent acute myeloid leukemia ex vivo.

The phenylpteridine derivative BIBW22BS (BIBW22) is a potent modulator of multidrug resistance (MDR). We investigated BIBW22 in comparison to dexniguldipine and verapamil as modifier of MDR in blasts of de novo, relapsed or persistent acute myeloid leukemia (AML) in vitro. All patients with relapsed or persistent AML had been pretreated with idarubicin and cytosine arabinoside. The degree of MDR was determined by efflux kinetics of rhodamine 123 (R123), daunorubicin, and idarubicin measured by flow cytometry (FACS). A total of 51 patients with AML, 25 de novo and 26 relapsed or persistent, were investigated. While only 6 out of 25 de novo AML blast populations showed moderate efflux of R123 and daunorubicin, 17 out of 26 blast populations of relapsed or persistent AML had an efflux between 20% and 44% within 15 min ex vivo. This efflux could be significantly inhibited by 1 microM BIBW22, 1 microM dexniguldipine, or 10 microM verapamil. For idarubicin we found an effusion of 40+/-9% within 15 min in all blast populations that could not be inhibited by the modulators. Clinically achievable drug concentrations causing only moderate side-effects are in the range of 0.5 microM dexniguldipine and 3 microM verapamil. Up to now, BIBW22 has not been investigated clinically. Thus the potential toxicity of concentrations of 0.5-1 microM BIBW22, sufficient for an optimal efflux inhibition ex vivo, is not known yet. We conclude from our ex vivo investigations in blast populations of de novo, relapsed or persistent AML that BIBW22 is a potent modulator of MDR.

Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane.

P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ("vacuum cleaner" hypothesis). A possible mechanism for this model is that the Pgp is a flippase--i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains low at short times after external addition). The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors. The unidirectional influx of R123 was the same in sensitive and resistant cells--i.e., independent of the level of Pgp expression and insensitive to inhibitors of R123 efflux. From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.

Relationships between rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein.

The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl- channel activated by cell swelling. The partial reversal of multidrug resistance by Cl- transport blockers suggests a possible role for Cl- in Pgp-mediated drug transport. We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl- (and also Na+ and HCO3-/CO2) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123). In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a approximately 60-min removal of Cl- (or by exposure to Na(+)-free, or nominally HCO3-/CO2-free medium); short term (2-3 min) ion substitutions were also ineffective. In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl- current but had no effect on the Pgp-mediated unidirectional efflux of R123. Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl- current by hyposmotic solution. The lack of detectable effect of removal of Cl-, Na+, or HCO3- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp. The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl- current does not hinder the Pgp pump function. The lack of effect of R123 on swelling-activated Cl- current denotes that Pgp-mediated transport of organic substrates and Pgp-associated Cl- currents can occur at the same time in a single cell. These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl- transport.