Vitamin E TPGS P-glycoprotein inhibition mechanism: influence on conformational flexibility, intracellular ATP levels, and role of time and site of access.

Previous work conducted in our laboratories established the notion that TPGS 1000 (d-alpha-tocopheryl polyethylene glycol 1000 succinate), a nonionic surfactant, modulates P-glycoprotein (P-gp) efflux transport via P-gp ATPase inhibition. The current in vitro research using Caco-2 cells was conducted to further explore the P-gp ATPase inhibition mechanism. Using a monoclonal CD243 P-gp antibody shift assay (UIC2), we probed P-gp conformational changes induced via TPGS 1000. In the presence of TPGS 1000, UIC2 binding was slightly decreased. TPGS 1000 does not appear to be a P-gp substrate, nor does it function as a competitive inhibitor in P-gp substrate efflux transport. The reduction in UIC2 binding with TPGS 1000 was markedly weaker than with orthovanadate, data ruling out trapping P-gp in a transition state by direct interaction with one or both of the P-gp ATP nucleotide binding domains. An intracellular ATP depletion mechanism could be ruled out in the UIC2 assay, and by monitoring intracellular ATP levels in the presence of TPGS 1000. Indicating slow distribution of TPGS 1000 into the membrane, and in agreement with an intramembranal or intracellular side of action, Caco-2 cell monolayer experiments preincubated with TPGS 1000 produce stronger substrate inhibitory activity than those conducted by direct substrate and surfactant coapplication.

Validation of a differential in situ perfusion method with mesenteric blood sampling in rats for intestinal drug interaction profiling.

The present study explored the feasibility of a differential setup for the in situ perfusion technique with mesenteric cannulation in rats to assess drug interactions at the level of intestinal absorption. In contrast to the classic, parallel in situ perfusion setup, the differential approach aims to identify intestinal drug interactions in individual animals by exposing the perfused segment to a sequence of multiple conditions. First, the setup was validated by assessing the interaction between the P-glycoprotein (P-gp) inhibitor verapamil and the transport probes atenolol (paracellular transport), propranolol (transcellular) and talinolol (P-gp mediated efflux). While transport of atenolol and propranolol remained constant for the total perfusion time (2 h), a verapamil-induced increase in talinolol transport was observed within individual rats (between 3.2- and 5.2-fold). In comparison with the parallel setup, the differential in situ perfusion approach enhances the power to detect drug interactions with compounds that exhibit strong subject-dependent permeability. This was demonstrated by identifying an interaction between amprenavir and ketoconazole (P-gp and CYP3A inhibitor) in five out of seven rats (permeability increase between 1.9- and 4.2-fold), despite high inter-individual differences in intrinsic permeability for amprenavir. In combination with an increased throughput (up to 300%) and a reduced animal use (up to 50%), the enhanced power of the differential approach improves the utility of the biorelevant in situ perfusion technique with mesenteric blood sampling to elucidate the intestinal interaction profile of drugs and drug candidates.

Effect of pluronic F68 block copolymer on P-glycoprotein transport and CYP3A4 metabolism.

The aim of this work was to investigate the effects of pluronic F68 block copolymer on the P-gp-mediated transport of celiprolol (CEL) and CYP3A4-mediated formation of midazolam (MDZ) metabolite 1'-hydroxymidazolam. Over a range from 0.03 to 0.3%, pluronic F68 increased apical-to-basolateral permeability (AP-BL) and decreased basolateral-to-apical permeability (BL-AP) of the P-gp substrate CEL in Caco-2 cell monolayer with the efflux ratio values of 2.8+/-0.3 (0.03%), 2.6+/-0.3 (0.1%), 2.3+/-0.2 (0.3%), respectively. CEL transport across the intestinal mucosa in the everted gut sac model was also enhanced by the P-gp inhibitor verapamil and the pharmaceutical excipient pluronic F68. Furthermore, CYP3A4-catalyzed formation of 1'-hydroxymidazolam was inhibited by pluronic F68 with IC(50) and K(i) values of 0.11 and 0.16 mg/ml, respectively. The results indicate that pluronic F68 is a potent in vitro inhibitor of both P-gp and CYP3A4, suggesting a potential for pluronic F68 to modify the pharmacokinetics of orally administered drugs that are P-gp and/or CYP3A4 substrates in vivo.

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol.

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

Nanotherapeutics with suitable properties for advanced anticancer therapy based on HPMA copolymer-bound ritonavir via pH-sensitive spacers.

Ritonavir (RIT) is a widely used antiviral drug that acts as an HIV protease inhibitor with emerging potential in anticancer therapies. RIT causes inhibition of P-glycoprotein, which plays an important role in multidrug resistance (MDR) in cancer cells when overexpressed. Moreover, RIT causes mitochondrial dysfunction, leading to decreased ATP production and reduction of caveolin I expression, which can affect cell migration and tumor progression. To increase its direct antitumor activity, decrease severe side effects induced by the use of free RIT and improve its pharmacokinetics, ritonavir 5-methyl-4-oxohexanoate (RTV) was synthesized and conjugated to a tumor-targeted polymer carrier based on a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Here we demonstrated that polymer-bound RTV enhanced the internalization of polymer-RTV conjugates, differing in RTV content from 4 to 15 wt%, in HeLa cancer cells compared with polymer without RTV. The most efficient influx and internalization properties were determined for the polymer conjugate bearing 11 wt% of RTV. This conjugate was internalized by cells using both caveolin- and clathrin-dependent endocytic pathways in contrast to the RTV-free polymer, which was preferentially internalized only by clathrin-mediated endocytosis. Moreover, we found the co-localization of the RTV-conjugate with mitochondria and a significant decrease of ATP production in treated cells. Thus, the impact on mitochondrial mechanism can influence the function of ATP-dependent P-glycoprotein and also the cell viability of MDR cancer cells. Overall, this study demonstrated that the polymer-RTV conjugate is a promising polymer-based nanotherapeutic, suitable for antitumor combination therapy with other anticancer drugs and a potential mitochondrial drug delivery system.

Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoprotein mediated efflux of rhodamine 123 partially due to modifying P-glycoprotein protein expression within Caco-2 cells.

PURPOSE: The objective of this study was to determine the influence of two lipid excipients, Peceol(c) and Gelucire(c) 44/14 on P-glycoprotein (Pgp) activity and protein expression in human colon adenocarcinoma cells (Caco-2). Lipid excipients are increasingly used as drug delivery systems for hydrophobic drugs to increase their bioavailability by overcoming the barrier of low absorption. This study will probe a novel mechanism by which lipid excipients reduce Pgp-mediated efflux and thereby increase bioavailability of orally administered therapeutics. METHODS: Non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 were determined for 24-hour treatments of Caco-2 cells using integrity of the cell membranes and mitochondrial respiration as markers. Pgp activity after treatment with non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 was measured with a fluorescent Pgp substrate, rhodamine 123 (Rh123). The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123 using the Transwell(c) semi-permeable cell culture support system. To assess the effect of Peceol(c) and Gelucire(c) 44/14 on Pgp protein expression, Western blotting with a specific Pgp antibody was performed. RESULTS. The two assays for cytotoxicity were in agreement and showed that concentrations of less than 0.5% (v/v) Peceol(c) and less than 0.02% (w/v) Gelucire(c) 44/14 were not toxic to Caco-2 cells. Rh123 accumulation was increased up to 3-fold in cells treated with sub-toxic concentrations of the excipients. The flux of Rh123 across the cell monolayer was unaffected by treatment in the absorptive (apical to basolateral) direction but the efflux transport was reduced after treatment with Peceol(c), Gelucire(c) 44/14 or the positive control , 100microM verapamil. Some of the reduction in Pgp efflux activity can be explained by the reduction in protein expression after treatment with the lipid excipients; treatment with 0.25% (v/v) and 0.5% (v/v) Peceol(c) reduced Pgp protein levels to 62.4% and 68.4% of the control respectively while Gelucire(c) 44/14 treatments of 0.01% (w/v) and 0.02% (w/v) reduced Pgp to 64.5% and 51.8% respectively. CONCLUSION: In this study we utilized established methodologies to assess the inhibitory effect of the excipients on the Pgp-mediated efflux of the probe, Rh123 and tested the hypothesis that long-term treatment of Caco-2 cells with the lipid excipients, Peceol(c) and Gelucire(c) 44/14, decreased Pgp protein expression. The results suggest a new mechanism which may contribute to the improved bioavailability seen for drugs formulated with lipid-based excipients.

Bioavailability and tissular distribution of docetaxel, a P-glycoprotein substrate, are modified by interferon-alpha in rats.

Interferon-alpha (IFN-alpha) inhibits intestinal P-glycoprotein (P-gp) expression in rats. In the present study, the effects of repeated pre-treatment with recombinant human INF-alpha (rhIFN-alpha) on oral and intravenous pharmacokinetics of a P-gp substrate, docetaxel (DTX; Taxotere) were investigated in a rat model. The bioavailability and distribution in different organs were also studied. Sprague-Dawley rats were subcutaneously pre-treated with either rhIFN-alpha for 8 days (4MIU kg(-1), once daily) or with pegylated-IFN-alpha (ViraferonPeg; 60 microg kg(-1), Days 1, 4 and 7). The rats were then distributed into sub-groups (n = 5-6) according to the pre-treatment type, and received one dose of [(14)C]DTX (20 mgkg(-1)) either orally or intravenously. Pharmacokinetics studies were then performed over 240 min, at the end of which tissues (intestine, liver, kidneys, lung, heart and brain) were immediately removed for radioactivity quantitation. Non-pegylated and pegylated IFN-alpha both increased DTX oral bioavailability parameters: C(max) (17.0+/-4.0 microg L(-1) (P < 0.02) and 18+/-5.5 microg L(-1) (P < 0.05), respectively, vs 7.4+/-2.5 microg L(-1) for the control) and AUC (0.036+/-0.010 microg h mL(-1) (P < 0.01) and 0.033+/-0.009 microg h mL(-1) (P < 0.01), respectively, versus 0.012+/-0.004 microg h mL(-1) for the control). IFN-alpha also delayed DTX absorption from 60 min in controls to about 95 min and 80 min in non-pegylated and pegylated treated animals, respectively. However, IFN-alpha did not affect intravenous DTX pharmacokinetics and it had a limited effect on tissue distribution at 240 min. [(14)C]DTX was decreased in intestine and enhanced in brain in both pre-treated groups. rhIFN-alpha modified the P-gp-dependent pharmacokinetics of DTX, limited its intestinal efflux and markedly enhanced its oral bioavailability.

The functional purification of P-glycoprotein is dependent on maintenance of a lipid-protein interface.

P-Glycoprotein (P-gp) is a 180-kDa membrane-bound transporter which can confer the multi-drug resistance phenotype on tumor cells. We have examined the factors required to preserve activity of P-gp during its purification. The starting material for purification was plasma membranes from Chinese hamster ovary (CHrB30) cells, overexpressing P-glycoprotein. These membranes displayed drug stimulated ATPase activity (Vm = 897 +/- 55 nmol min(-1) mg(-1); Km = 1.8 +/- 0.4 mM) and high affinity binding of [3H]vinblastine (Kd = 36 +/- 5 nM; Bm = 161 +/- 11 pmol/mg). Several non-ionic detergents which readily solubilized P-glycoprotein significantly inhibited ATPase activity and drug binding at concentrations well below their respective CMC values. This inactivation was prevented by excess crude lipid mixtures, with the greatest protection afforded against dodecyl-maltoside. Furthermore, the significantly reduced binding affinity and capacity of solubilized P-gp was partly reversed by the addition of lipids. A combination of anion-exchange and hydroxyapatite chromatography were used to purify P-gp with high yield to greater than 90%. The purified, reconstituted P-gp displayed high ATPase activity (Vm = 2137 +/- 309; Km = 2.9 +/- 0.9 mM) which was stimulated by verapamil (EC50 = 3.8 +/- 0.6 microM) and inhibited by orthovanadate (3.1 +/- 0.8 microM). Pure P-gp also displayed high affinity vinblastine binding (Kd = 64 +/- 9 nM) with a capacity of 2320 +/- 192 pmol/mg. This purification scheme yields the highest P-gp activity reported to date, and indicates a dependence of function on maintaining a lipid-protein interface.

Potentiation by alpha-tocopheryl succinate of the etoposide response in multidrug resistance protein 1-expressing glioblastoma cells.

Multidrug resistance protein 1 (MRP1) is one of the representative members of the ATP-binding cassette superfamily of transporters that is involved in resistance to chemotherapeutic agents in cancer patients. MRP1 functions as an efflux pump of drugs, primarily those conjugated to glutathione (GSH). Decreases in the intracellular concentration of GSH have been shown to enhance the response of MRP1-overexpressing cells to MRP1-substrate drugs by limiting the available drug-GSH conjugates. We report here that alpha-tocopheryl succinate (TOS), a vitamin E analogue, decreased intracellular GSH concentration and blocked MRP1 function in glioblastoma cells. Functional blockade by TOS of MRP1 was confirmed by the enhanced accumulation of etoposide (VP-16), an MRP1-substrate drug. As a result, co-treatment of TOS with VP-16 or treatment with liposomes containing both TOS and VP-16 greatly enhanced the response of MRP1-expressing glioblastoma cells to VP-16. TOS may be a promising adjuvant for enhancing the therapeutic efficacy of VP-16 in patients with MRP1-expressing glioblastomas.

[The multidrug resistance phenotype: some morphological and biological characteristics except efflux pump]. / Le phénotype de résistance multidrogue: quelques caractéristiques morphologiques et biologiques autres que la pompe à efflux.

Recent data from the literature together with personal results strongly suggest that multidrug resistance phenotype is overwhelming the sole expression of P170 glycoprotein efflux pump. Morphological alterations have been put in evidence in MDR cells after transmission and scanning electron microscopy. They include presence of osmiophilic vesicles and modifications of nuclear and nucleolar chromatin. Biological characteristics include the hypersecretory pattern of lysosomal enzymes from MDR cells. Such a fact could be more or less related to the increased occurrence of mdr1 RNA in metastasis, especially in breast cancers, compared to primary tumors. If the P170-mediated efflux is one of the key mechanism of MDR, a decreased influx of anticancer drugs cannot be excluded. Liposomes, for instance made of cardiolipin, are thus able to increase the intracellular drug uptake of vinblastine without any action upon efflux mechanism.

Commonly used surfactant, Tween 80, improves absorption of P-glycoprotein substrate, digoxin, in rats.

Tween 80 (Polysorbate 80) is a hydrophilic nonionic surfactant commonly used as an ingredient in dosing vehicles for pre-clinical in vivo studies (e.g., pharmacokinetic studies, etc.). Tween 80 increased apical to basolateral permeability of digoxin in Caco-2 cells suggesting that Tween 80 is an in vitro inhibitor of P-gp. The overall objective of the present study was to investigate whether an inhibition of P-gp by Tween 80 can potentially influence in vivo absorption of P-gp substrates by evaluating the effect of Tween 80 on the disposition of digoxin (a model P-gp substrate with minimum metabolism) after oral administration in rats. Rats were dosed orally with digoxin (0.2 mg/kg) formulated in ethanol (40%, v/v) and saline mixture with and without Tween 80 (1 or 10%, v/v). Digoxin oral AUC increased 30 and 61% when dosed in 1% and 10% Tween 80, respectively, compared to control (P < 0.05). To further examine whether the increase in digoxin AUC after oral administration of Tween 80 is due, in part, to a systemic inhibition of digoxin excretion in addition to an inhibition of P-gp in the GI tract, a separate group of rats received digoxin intravenously (0.2 mg/kg) and Tween 80 (10% v/v) orally. No significant changes in digoxin IV AUC was noted when Tween 80 was administered orally. In conclusion, Tween 80 significantly increased digoxin AUC and Cmax after oral administration, and the increased AUC is likely to be due to an inhibition of P-gp in the gut (i.e., improved absorption). Therefore, Tween 80 is likely to improve systemic exposure of P-gp substrates after oral administration. Comparing AUC after oral administration with and without Tween 80 may be a viable strategy in evaluating whether oral absorption of P-gp substrates is potentially limited by P-gp in the gut.

Reversion of multidrug resistance by co-encapsulation of doxorubicin and curcumin in chitosan/poly(butyl cyanoacrylate) nanoparticles.

Co-encapsulated doxorubicin (DOX) and curcumin (CUR) in poly(butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared with emulsion polymerization and interfacial polymerization. The mean particle size and mean zeta potential of CUR-DOX-PBCA-NPs were 133 ± 5.34 nm in diameter and +32.23 ± 4.56 mV, respectively. The entrapment efficiencies of doxorubicin and curcumin were 49.98 ± 3.32% and 94.52 ± 3.14%, respectively. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs+CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX+CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of doxorubicin and curcumin achieved the highest reversal efficacy and down-regulation of P-glycoprotein in MCF-7/ADR cell lines, an MCF-7 breast carcer cell line resistant to adriamycin. Multidrug resistance can be enhanced by combination delivery of encapsulated cytotoxic drugs and reversal agents.

Self-microemulsifying drug delivery system for improved oral bioavailability of dipyridamole: preparation and evaluation.

Dipyridamole shows poor and variable bioavailability after oral administration due to pHdependent solubility, low biomembrane permeability as well as being a substrate of P-glycoprotein. In order to improve the oral absorption of dipyridamole, a self-microemulsifying drug delivery system (SMEDDS) for dipyridamole was prepared and evaluated in vitro and in vivo. The optimum formulation was 18% oleic acid, 12% Labrafac lipophile WL 1349, 42% Solutol HS 15 and 28% isopropyl alcohol. It was found that the performance of self-microemulsification with the combination of oleic acid and Labrafac lipophile WL 1349 increased compared with just one oil. The results obtained from an in vitro dissolution assay indicated that dipyridamole in SMEDDS dissolved rapidly and completely in pH 6.8 aqueous media, while the commercial drug tablet was less soluble. An oral bioavailability study in rats showed that dipyridamole in the SMEDDS formulation had a 2.06-fold increased absorption compared with the simple drug suspension. It was evident that SMEDDS may be an effective approach to improve the oral absorption for drugs having pH-dependent solubility.

Inhibition of P-glycoprotein pumps by PEO-PPO amphiphiles: branched versus linear derivatives.

UNLABELLED: Inhibition of the activity of efflux transporters may relevantly improve the chemotherapy of cancer and infectious diseases. AIM: To explore the ability of poloxamines (Tetronic(®), X-shaped structure with a central ethylendiamine group and four branches of poly[ethylene oxide]-poly[propylene oxide] [PEO-PPO]) to inhibit the activity of P-glycoprotein (P-gp) on Caco-2 cell monolayers and to elucidate the incidence of the molecular architecture of PEO-PPO block copolymers on the intracellular accumulation of a relevant substrate, doxorubicin, by comparison with poloxamers (Pluronic(®), linear triblock copolymers), well-known inhibitors of this efflux transporter. METHODS: Both pristine and N-methylated poloxamines displaying a wide range of molecular weights and EO/PO ratios were tested regarding cytocompatibility and accumulation of doxorubicin in Caco-2 monolayers. Verapamil was used as a control. RESULTS: The most active anti-P-gp poloxamines (which enhanced two- to three-fold doxorubicin accumulation compared with verapamil) resulted to be pristine medium-to-high hydrophobic T304, T904, T1301, T901 and T150R1. A notable dependence of the anti-P-gp activity on the copolymer concentration was found. A joint diagram of the inhibitory activity of poloxamers and poloxamines as a function of the effective length of the PPO block is proposed. CONCLUSION: The anti-P-gp activity is maxima for block copolymers possessing a low-to-medium hydrophilic-lipophilic balance and an 'effective number' of PO units ranging from 30 to 50.

Euphoportlandols A and B, tetracylic diterpene polyesters from Euphorbia portlandica and their anti-MDR effects in cancer cells.

Two new tetracyclic diterpene polyesters, euphoportlandols A (1) and B (2), have been isolated along with 12 known tetracyclic triterpenes from an acetone extract of Euphorbia portlandica. Their structures were established as 5alpha,11alpha,14alpha,17-tetraacetoxy-3beta-benzoyloxy-6beta,15beta-dihydroxy-9-oxoseget-8(12)-ene (1) and 5alpha,11alpha,14alpha,17-tetraacetoxy-3beta-benzoyloxy-6beta,15beta-dihydroxy-9-oxosegetane (2), respectively, by spectroscopic data interpretation. Compounds 1 and 2 were evaluated for their ability to inhibit multidrug resistance in cancer cells. Both compounds were found to be inhibitors of P-glycoprotein activity.

Membrane fluidization by ether, other anesthetics, and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells.

The anesthetics benzyl alcohol and the nonaromatic chloroform and diethyl ether, abolish P-glycoprotein (Pgp) ATPase activity in a mode that does not fit classical competitive, noncompetitive, or uncompetitive inhibition. At concentrations similar to those required for inhibition of ATPase activity, these anesthetics fluidize membranes leading to twofold acceleration of doxorubicin flip-flop across lipid membranes and prevent photoaffinity labeling of Pgp with [125I]-iodoarylazidoprazosin. Similar concentrations of ether proved nontoxic and modulated efflux from Pgp-overexpressing cells. A similar twofold acceleration of doxorubicin flip-flop rate across membranes was observed with neutral mild detergents, including Tween 20, Nonidet P-40 and Triton X-100, and certain Pgp modulators, such as verapamil and progesterone. Concentrations of these agents, similar to those required for membrane fluidization, inhibited Pgp ATPase activity in a mode similar to that observed with the anesthetics. The mode of inhibition, i.e. lack of evidence for classical enzyme inhibition and the correlation of Pgp ATPase inhibition with membrane fluidization over a wide range of concentrations and structures of drugs favors the direct inhibition of Pgp ATPase activity by membrane fluidization. The unusual sensitivity of Pgp to membrane fluidization, as opposed to acceleration of ATPase activity of ion transporters, could fit the proposed function of Pgp as a 'flippase', which is in close contact with the membrane core.

Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport.

The human multidrug transporter MDR1 P-glycoprotein and the multidrug resistance proteins MRP1 and MRP2 transport a range of cytotoxic drugs, resulting in multidrug resistance in tumour cells. To overcome this form of drug resistance in patients, several inhibitors (reversal agents) of these transporters have been isolated. Using polarized cell lines stably expressing human MDR1, MRP1 or MRP2cDNA, and 2008 ovarian carcinoma cells stably expressing MRP1 cDNA, we have investigated in this study the specificity of the reversal agents V-104 (a pipecolinate derivative), GF120918 (an acridone carboxamide derivative also known as GG918), and Pluronic L61 (a (poly)oxypropethylene and (poly)oxypropylene block copolymer). Transport experiments with cytotoxic drugs with polarized cell lines indicate that all three compounds efficiently inhibit MDR1 Pgp. Furthermore, V-104 partially inhibits daunorubicin transport by MRP1 but not vinblastine transport by MRP2. V-104 reverses etoposide resistance of 2008/MRP1 cells, whereas GF120918 does not reverse resistance due to MRP1. V-104 partially inhibits the export of the organic anion dinitrophenyl S-glutathione by MDCKII-MRP1 but not by MDCKII-MRP2 cells. Unexpectedly, export of the organic anion calcein by MDCKII-MRP1 and MDCKII-MRP2 cells is stimulated by Pluronic L61, probably because it relieves the block on entry of calcein AM into the cell by endogenous MDR1 Pgp.

P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes.

Recently, sandwich-cultured (SC) rat hepatocytes have been used as an in vitro model to assess biliary excretion of drugs and xenobiotics. The purpose of the present study was to validate the use of SC rat hepatocytes for the in vitro assessment of P-glycoprotein (P-gp)-mediated biliary drug excretion. The specific and fluorescent P-gp substrate rhodamine 123 (Rh123) and the P-gp substrate digoxin were selected as model compounds. Rh123 and digoxin accumulation and Rh123 efflux under standard and Ca(2+)-free conditions were quantified in SC rat hepatocytes to determine substrate secretion into canalicular networks in vitro. The major role of P-gp in the biliary excretion of these compounds was confirmed by inhibition experiments with the potent P-gp inhibitor GF120918. Hepatocyte culture conditions, including media type and time in culture, significantly affected Rh123 biliary excretion. P-gp expression, as assessed by Western blot, was increased with culture time. Dexamethasone (an in vivo inducer of P-gp) concentrations ranging from 0.01 to 1 microM in the cell culture medium did not influence P-gp expression or Rh123 biliary excretion. Rh123 and digoxin biliary clearance values, predicted from SC rat hepatocyte data, were consistent with values reported in vivo and in isolated perfused rat liver studies. In conclusion, the results of this study demonstrate the utility of SC rat hepatocytes as an in vitro model to study and predict the biliary excretion of P-gp substrates.

In vitro cellular accumulation and cytotoxicity of liposomal and conventional formulations of daunorubicin and doxorubicin in resistant K562 cells.

Previous investigations have indicated the possibility to circumvent multidrug resistance (MDR) by incorporation of an anthracycline into liposomes. We examined the in vitro cytotoxicity and cellular drug accumulation of the anthracyclines daunorubicin and doxorubicin compared with the commercially available liposomal formulations DaunoXome and Caelyx in human myelogenous leukemia K562 cells. The drug-sensitive parental K562/K line was compared with the P-glykoprotein (P-gp)-expressing cell lines K562/Dnr and K562/Vcr. Two cell lines with reduced levels of topoisomerase II (K562/Nov and K562/Ida) were also included. The cytotoxicity was determined by fluorometric microculture cytotoxicity assay and the cellular drug levels were determined by high performance liquid chromatograghy. There was a strong inverse correlation between P-gp levels and cellular drug accumulation (rho = -0.83, p = 0.04) and cytotoxicity (rho = -0.95, p = 0.01) of daunorubicin. Also the cytotoxicity of DaunoXome and doxorubicin was related to P-gp levels (rho = -0.96, p = 0.01 and rho = -0.90, p = 0.07, respectively). Caelyx did not show any cytotoxic effect due to impaired cellular uptake of the pegylated liposome. Regardless of the P-gp levels of the treated cells, DaunoXome showed the same cytotoxic effect despite lower intracellular accumulation (range 22-47%), compared with conventional daunorubicin.

Role of formulation vehicles in taxane pharmacology.

The non-ionic surfactants Cremophor EL (CrEL) and Tween 80, both used as formulation vehicles of many (anticancer) agents including paclitaxel and docetaxel, are not physiological inert compounds. We describe their biological properties, especially the toxic side effects, and their pharmacological properties, such as modulation of P-glycoprotein activity. In detail, we discuss their influence on the disposition of the solubilized drugs, with focus on CrEL and paclitaxel, and of concomitantly administered drugs. The ability of the surfactants to form micelles in aqueous solution as well as biological fluids (e.g. plasma) appears to be of great importance with respect to the pharmacokinetic behavior of the formulated drugs. Due to drug entrapment in the micelles, plasma concentrations and clearance of free drug change significant leading to alteration in pharmacodynamic characteristics. We conclude with some perspectives related to further investigation and development of alternative methods of administration.