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Department of Pharmaceutical Sciences (X.-f.Z., X.Y., Q.W., M.E.M.) and Department of Chemistry (R.A.C.), University at Buffalo, State University of New York, Amherst, New York
(Received January 4, 2005; Accepted May 16, 2005)
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Abstract |
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). It is a 655-amino acid "half-transporter" and belongs to the G subclass of the ATP-binding cassette (ABC) superfamily (Doyle and Ross, 2003). Cells that overexpress BCRP demonstrate resistance to mitoxantrone and topoisomerase I inhibitors (Maliepaard et al., 1999; Miyake et al., 1999; Kawabata et al., 2001). Increased BCRP mRNA expression in patients' mononuclear bone marrow cells has been recently reported in relapsed or refractory hematological malignancies and is associated with poor response to chemotherapy (Steinbach et al., 2002; van den Heuvel-Eibrink et al., 2002; van der Kolk et al., 2002). It is therefore important to identify clinically applicable chemotherapy modulators in treatment of malignancies such as acute leukemia, where multiple multidrug resistance transporters are expressed (Ross, 2000).
1,4-Dihydropyridine calcium channel blockers have been long viewed as P-glycoprotein modulators. The clinical application of calcium channel blockers as P-glycoprotein inhibitors has been limited due to their potent vasodilator effect. The structure of dihydropyridines has been modified in our laboratory to generate a series of analogs that are potent P-glycoprotein inhibitors and demonstrate negligible calcium channel binding (Zhou et al., 2005). Because of the substantial overlap of substrates and inhibitors for BCRP and P-glycoprotein (Borst et al., 2000; Litman et al., 2000), it is reasonable to speculate that dihydropyridines may also possess BCRP modulation effects. In the present study, these synthesized dihydropyridines and pyridines, as well as four commercially available dihydropyridines (niguldipine, nicardipine, nifedipine, and nitrendipine) were evaluated for their effects on BCRP-mediated efflux and on the cytotoxicity of the BCRP substrate and chemotherapeutic agent mitoxantrone. DHP-014 (compound Im), a new dihydropyridine compound shown to be a potent BCRP and P-glycoprotein inhibitor in vitro, was selected to investigate its effect on topotecan pharmacokinetics in Sprague-Dawley rats. Topotecan is a good BCRP substrate (Jonker et al., 2000), as well as a moderate to poor P-glycoprotein substrate (Chen et al., 1991). Pronounced changes of topotecan bioavailability due to BCRP inhibition have been reported in mice (Jonker et al., 2000), humans (Kruijtzer et al., 2002), and rats (Zhang et al., 2005). Topotecan undergoes very limited metabolism in rats (Platzer et al., 1998) and humans (Rosing et al., 1997), making it a suitable model BCRP substrate for the investigation of BCRP-mediated drug-drug interactions in vivo, since interactions between inhibitors and metabolizing enzymes will not confound the results.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The chemical structures of these test compounds are shown in Fig. 1. Series I comprises 1,4-dihydropyridines and Series II comprises corresponding aromatized pyridines. Human breast cancer MCF-7-sensitive and MCF-7/MX100 human large cell lung carcinoma NCI-H460 and NCI-H460/MX20, and fumitremorgin C were kind gifts from Dr. Susan E. Bates (National Cancer Institute, Bethesda, MD). Topotecan was purchased from ChemPacific (Baltimore, MD). Injectable anesthetics and suture materials were obtained from Henry Schein Inc. (Melville, NY). Acridine was purchased from Sigma. Cell Culture. Parental human breast cancer cell MCF/s and human non-small cell lung cancer cell H460/s were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen). Resistant MCF-7/MX100 cells and H460/MX20 were cultured in the above-mentioned medium with addition of 100 nM and 20 nM mitoxantrone, respectively. All cells were incubated at 37°C in 5% CO2/95% air.
Animals. Female Sprague-Dawley rats (220–260 g in body weight) were obtained from Harlan (Indianapolis, IN) and housed according to institutional guidelines. The rats were kept in a temperature-controlled environment with a 12-h light/dark cycle and given a standard diet with water ad libitum. Rats were fasted overnight before oral administration of drug. The animal protocol was approved by the Institutional Animal Use and Care Committee at the University at Buffalo.
Western Blotting Assay. Cells grown in 100 x 15 mm culture dishes were washed with phosphate-buffered saline and harvested using a cell scraper. Total cell lysates were prepared by adding a lysis buffer (20 mM Tris, pH 7.5, 120 mM sodium chloride, 100 mM sodium fluoride, 1% octylphenoxy polyethoxy ethanol, 200 µM sodium orthovanadate, 50 mM ß-glycerolphosphate, 10 mM sodium pyrophosphate, 4 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) to the harvested cells and keeping on an ice bath for 30 min. Soluble extracts were obtained by centrifuging cell lysates at 13,000g for 20 min. The protein concentration of the supernatant was determined by the Bradford method (Bradford, 1976). Lysates were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Invitrogen). Membranes were then blocked overnight at 4°C in Tris-buffered saline containing 0.2% (v/v) Tween 20 and 5% (w/v) fat-free dry milk (Bio-Rad, Hercules, CA) with a 1:750 dilution of the BXP-21 monoclonal antibody against BCRP (Maliepaard et al., 2001a). The blots were then incubated at room temperature for 1 h with a horseradish peroxidase-conjugated sheep anti-mouse IgG secondary antibody (Amersham Biosciences, Inc., Piscataway, NJ), followed by enhanced chemiluminescence (ECL) detection (Amersham Biosciences, Inc.).
Mitoxantrone Cellular Accumulation Studies. Mitoxantrone cellular accumulation studies with analysis by flow cytometry followed the protocols described by Minderman et al. (2002) and Zhang et al. (2004). Briefly, cells were incubated at a density of 1 x 106 cells/ml for 30 min at 37°C in RPMI 1640 medium with 3 µM mitoxantrone. At the end of the 30-min incubation period, cells were washed once with ice-cold phosphate-buffered saline and then resuspended in ice-cold phosphate-buffered saline. An aliquot of cells was kept on ice until flow cytometry analysis for drug retention. Samples were analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA) equipped with a standard argon laser for 488-nm excitation and with 530/30-nm band pass (FL1), 585/42 band pass (FL2), and 670 nm long pass (FL3) filters for emission collection. All flow-cytometric data were analyzed with the WinList software program (Verity Software House, Topsham, ME).
Mitoxantrone Cytotoxicity Assay. One hundred microliters of MCF-7/MX100 cells were seeded in 96-well plates at a density of 4000 cells per well. The cells were allowed to attach for 24 h at 37°C, following which, an additional 100 µl of medium was added to each well containing the desired final concentration of mitoxantrone (0.1 µM to 1000 µM) with or without the modulator. After a 6-h exposure to the drug, the cells were washed twice with sterile 1x phosphate-buffered saline, and fresh medium was added to each well. The cells were allowed to grow for 4 more days. After 4 days, the total protein was measured by a sulforhodamine B staining assay (Skehan et al., 1990). Briefly, 10% trichloroacetic acid was added to the cells for an hour on ice to fix cellular protein to the wells, rinsed five times with water, and allowed to air dry. Sulforhodamine B (0.4% w/v in 1% glacial acetic acid) was added to each well for 15 min and washed four times with 1% acetic acid. After drying the plates, protein-bound dye was solubilized in 10 mM Tris base and quantitated by measuring the absorbance at 570 nm.
DHP-014 (Compound Im)-Topotecan Interaction Study in Rats. Topotecan was freshly prepared in 5% D-glucose for oral dosing. Three groups of four animals each were given 2 mg/kg topotecan orally, alone or coadministered with DHP-014 (10 mg/kg or 20 mg/kg) by intraperitoneal injection. Blood samples (150 µl) taken from the jugular vein cannula were collected in heparinized tubes prior to drug administration and at 2, 7, 15, 30, 60, 120, 240, 480, and 720 min after dosing. Plasma samples were obtained by centrifugation at 1000g for 10 min and analyzed by HPLC (Chen and Balthasar, 2002; Zhang et al., 2005). The HPLC assay utilized a mobile phase of methanol/10 mM potassium phosphate (KH2PO4) (25:75, v/v) containing 2% triethylamine with a pH of 3.72 and a flow rate of 1.0 ml/min. For sample preparation, 40 µl of plasma was mixed with 4 µl of acridine (1.5 µg/ml as the internal standard), 120 µl of methanol, and 40 µl of 100 mM phosphoric acid. The mixture was then centrifuged at 2000g for 8 min. A clear supernatant was collected, and 100 µl of the sample solution was injected into the HPLC system. Topotecan was detected with excitation and emission wavelengths set at 361 and 527 nm, respectively. The lower limit of quantitation of this method was 0.02 ng. Standard curves are linear over the concentration range of 1 to 500 ng/ml. The intraday and interday variabilities were 9.48% and 14.2%, respectively.
Data Analysis. For mitoxantrone accumulation studies, mitoxantrone fluorescence in modulator-treated cells and vehicle (0.1% DMSO)-treated cells were compared. Statistical significance was determined using a one-way analysis of variance followed by Dunnett's post hoc test. Differences were considered to be significant when p < 0.05. In the concentration-dependent study, the EC50 values of compounds for increasing mitoxantrone accumulation in MCF-7/MX100 and H460/MX20 cells were obtained by fitting the fraction of maximal increase (F) by eq. 1 using the computer program Win-Nonlin (Pharsight, Mountain View, CA).
 | (1) |
represents the curve fitting coefficient. F was calculated as the ratio of the net increase of mitoxantrone accumulation in the presence of the test compound (A - A0) to the maximal net increase, in this study, represented by the net increase of mitoxantrone accumulation in the presence of 40 µM nicardipine (Anicardipine - A0). A0, A, and Anicardipine are the mitoxantrone accumulation in the presence of the vehicle control (0.1% DMSO), the test compound, and 40 µM nicardipine, respectively.
In mitoxantrone cytotoxicity studies, the IC50 values were obtained by fitting the growth inhibition data to an inhibitory sigmoidal model of a Hill Equation (eq. 2 using WinNonlin (Pharsight, Mountain View, CA).
 | (2) |
represents the curve-fitting coefficient, or Hill coefficient. The pharmacokinetic parameters of topotecan were obtained by noncompartmental analysis using WinNonlin version 2.1 (Pharsight). The area under the plasma concentration-time curves (AUC) was calculated using the trapezoidal method. Analysis of variance was applied to assess the statistical significance of pharmacokinetic parameters of topotecan among different dosing regimens. Differences were considered to be statistically significant when p < 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mitoxantrone Retention Studies. Figures 3 and 4 show the effect of the modulators on mitoxantrone retention in BCRP-overexpressing H460/MX20 and MCF-7/MX100 cells, respectively. To exclude the possibility that the dihydropyridine/pyridine compounds may contribute to the fluorescence intensity, H460/MX20 and MCF-7/MX100 cells were incubated in medium with a 10 µM concentration of each test compound. No test compound-associated fluorescence was detected with the FACScan instrument settings used to detect mitoxantrone (data not shown). The 30-min accumulation of 3 µM mitoxantrone in MCF-7/adr cells was measured in the presence of 2.5 µM synthesized dihydropyridines and pyridines. Fumitremorgin C (10 µM) served as the positive control in these experiments. As shown in Fig. 3, all synthesized dihydropyridine compounds demonstrated a significant increase of intracellular mitoxantrone concentration at p < 0.001 or p < 0.05 except for compound Ih (no significant difference versus control). All of the corresponding pyridine analogs also significantly increased the intracellular mitoxantrone concentrations in the drug-resistant breast cancer cells.
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Figure 5 represents the relationship between mitoxantrone accumulation in the two BCRP-overexpressing cell lines after treatment with modulators. A statistically significant correlation for the inhibition of mitoxantrone efflux by the modulators in MCF-7/MX100 and H460/MX20 cells was observed (r2 = 0.71, p < 0.01).
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Mitoxantrone Cytotoxicity Studies. Nicardipine, niguldipine, nitrendipine, compound Im and compound IIb restored the sensitivity of resistant MCF-7/MX100 cells to mitoxantrone cytotoxicity in a concentration-dependent manner. As shown in Table 2, the IC50 values of mitoxantrone in MCF-7/MX100 were significantly reduced by treatment with compounds Im and IIb at concentrations ranging from 1 to 40 µM. Nicardipine demonstrated the most potent effect on mitoxantrone cytotoxicity compared with the other commercially available dihydropyridines. At 2.5 µM, nicardipine potentiated mitoxantrone cytotoxicity by nearly 2-fold compared with the vehicle control, an effect comparable to that of the positive control fumitremorgin C at10 µM. However, nifedipine showed no significant effect on mitoxantrone cytotoxicty in BCRP-overexpressing MCF-7/MX100 cells.
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Coadministration of Topotecan with DHP-014 (Compound Im) in Rats. Topotecan was administered to rats (2 mg/kg orally) either alone or with DHP-014 (10 or 20 mg/kg i.p.). DHP-014 was selected for further study based on the following criteria: good inhibitory activity for BCRP, negligible calcium channel binding activity, and predicted low systemic clearance based on quantitative structure-pharmacokinetic relationship analysis (Zhou et al., 2003, 2005). The plasma concentration-time profiles of topotecan are given in Fig. 7 and the pharmacokinetic parameters are summarized in Table 3. The area under the curve (AUC) and peak concentration (Cmax) of topotecan were significantly increased (p < 0.05) by coadministration with DHP-014 at a dose of 20 mg/kg, whereas the oral clearance (CL/F) of topotecan was markedly decreased (p < 0.05) compared to topotecan given alone. No statistically significant differences of topotecan terminal half-life (t1/2) were observed among the three treatments.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
BCRP is a recently identified ABC transporter and is highly expressed in the placenta (Maliepaard et al., 2001a), with lesser expression in brain, prostate, small intestine, testis, ovary, colon, and liver (Doyle et al., 1998). Several substances have been reported as BCRP inhibitors: GF120918 (de Bruin et al., 1999), the mycotoxin fumitremorgin C (Rabindran et al., 2000), its derivatives demethoxyfumitremorgin C (van Loevezijn et al., 2001), Ko132, Ko134, and Ko143 (Allen et al., 2002), the HER tyrosine kinase inhibitor CI1033 (Erlichman et al., 2001), experimental camptothecin analogs (Maliepaard et al., 2001b; Perego et al., 2001), and estrogens such as estrone and 17ß-estradiol (Imai et al., 2002).
We have previously reported that our new series of dihydropyridines and pyridines were synthesized based on the structural modification of dexniguldipine (Zhou et al., 2005). Most compounds from this series have been shown to be potent P-glycoprotein inhibitors, with little or no calcium channel blocking activity (Zhou et al., 2005). Moderate effects on MRP1-mediated drug efflux were also observed (unpublished data). In the present study, we have demonstrated that these compounds can effectively increase the intracellular accumulation of mitoxantrone in BCRP-overexpressing mitoxantrone-resistant cell lines. To the best of our knowledge, effects of dihydropyridines on BCRP have not been previously reported. Four commercially available dihydropyridines, nicardipine, niguldipine, nifedipine, and nitrendipine, were also evaluated for their effects on BCRP-mediated drug transport. All compounds except nifedipine demonstrated a BCRP modulation effect by enhancing intracellular mitoxantrone accumulation and sensitizing resistant cells to mitoxantrone cytotoxicity in a concentration-dependent manner. In the mitoxantrone accumulation studies in H460/MX20 cells, compound IIb exhibited more than a 4-fold increase of drug accumulation in BCRP-expressing cells at the concentration of 2.5 µM. Compound Im also showed a nearly 4-fold enhancement of mitoxantrone accumulation at the same concentration. These increases are larger than that observed with the positive control, 10 µM fumitremorgin C. Compounds Im and IIb were selected for the mitoxantrone cytotoxicity study in MCF-7/MX100 cells, and they were able to substantially reduce the IC50 values of mitoxantrone in a concentration-dependent manner. Since H460/MX20 and MCF-7/MX100 cells do not express P-glycoprotein or MRP1, and the dihydropyridines/pyridines do not affect transport of mitoxantrone in the parental cell line, our results suggest that the effects of the dihydropyridines/pyridines are mediated through BCRP.
Our in vivo studies with topotecan confirmed the results of the in vitro studies. Topotecan is a BCRP substrate that also demonstrates moderate to weak affinity for P-glycoprotein. Moderate to large interpatient variability was noted in topotecan pharmacokinetics, and 30% to 44% oral bioavailability was reported in clinical studies (Schellens et al., 1996; Zamboni et al., 1999). The absolute bioavailability of topotecan in rats was approximately 29.7% (Zhang et al., 2005). In humans, renal clearance accounts for approximately 40% of the clearance of topotecan (Herben et al., 1996). GF120918 is a potent inhibitor both for P-glycoprotein (den Ouden et al., 1996) and BCRP in humans and mice (Allen et al., 1999; de Bruin et al., 1999; Maliepaard et al., 2001b). The preclinical study by Jonker et al. (2000) demonstrated that treatment with GF120918 increased the systemic exposure of oral topotecan approximately 7-fold in mdr1a/1b(-/-) P-glycoprotein knockout mice and 10-fold in wild-type mice (Jonker et al., 2000), suggesting the importance of BCRP in topotecan disposition. Treatment with oral GF120918 in combination with intravenous topotecan in mdr1a/1b(-/-) mice decreased the plasma clearance and hepatobiliary excretion of topotecan (Jonker et al., 2000). Recently, Zhang et al. (2005) demonstrated for the first time that GF120918 could significantly increase the AUC and bioavailability of topotecan in Sprague-Dawley rats by more than 4-fold after oral coadministration. In the study reported here, coadministration of 20 mg/kg DHP-014 (compound Im) produced a 2-fold increase of the systemic exposure and significantly enhanced the peak concentration of oral topotecan in female Sprague-Dawley rats. Tanaka et al. (2004) demonstrated that BCRP mRNA in rats is predominantly expressed in kidney and intestine. We speculate that the observed increase of the systemic exposure levels of topotecan is due to inhibition of BCRP in rat small intestine, increasing the amount of topotecan entering the circulation. Inhibition of topotecan metabolism by DHP-014 is unlikely to contribute significantly to the increased systemic availability of topotecan because topotecan metabolism in rat liver is low (Platzer et al., 1998).
P-glycoprotein inhibition by DHP-014 will also contribute to the enhanced topotecan systemic exposure observed in this investigation. P-glycoprotein is expressed in the apical membrane of intestinal epithelial cells and is associated with a decreased bioavailability for several drugs (Lin and Yamazaki, 2003). DHP-014 is a potent P-glycoprotein inhibitor, capable of substantially enhancing the intracellular accumulation of vinblastine in P-glycoprotein-overexpressing human breast cancer MCF-7/adr cells (Zhou et al., 2005). However, topotecan is a moderate to weak P-glycoprotein substrate (Chen et al., 1991); therefore, the effect of P-glycoprotein inhibition on the bioavailability of topotecan is likely to be less important than that of BCRP inhibition.
The mechanism underlying the interaction of dihydropyridines and pyridines with BCRP has not been elucidated. It has been reported that the dihydropyridine derivative dexniguldipine can inhibit P-glycoprotein-mediated drug extrusion through a direct interaction with P-glycoprotein (Borchers et al., 2002). Dihydropyridines and pyridines may act on BCRP in a similar manner because these compounds have a planar, multiring structure, like mitoxantrone; thus, they may compete with mitoxantrone for binding sites on BCRP. Pascaud et al. (1998) characterized the modulation of P-glycoprotein ATPase activity by five dihydropyridines: nicardipine, nimodipine, nitrendipine, nifedipine, and azidopine. They reported that P-glycoprotein ATPase was activated by approximately 2-fold in the presence of 3 to 4 µM nicardipine. Nifedipine had no effect on P-glycoprotein ATPase. In the present study, nicardipine was the most potent BCRP inhibitor among the four commercially available dihydropyridines, whereas nifedipine showed no inhibitory effect on BCRP-mediated mitoxantrone efflux. It is possible that nicardipine and other dihydropyridines may interact with BCRP by a similar mechanism, i.e., activation of BCRP ATPase activity.
The extrapolation of drug-transporter interactions from animals to humans should be done cautiously due to potential species differences in the expression and activity of these transporters. Whereas Jonker et al. (2000) have demonstrated a substantial increase in AUC and a decreased plasma clearance in mdr1a/1b(-/-) knockout and wild-type mice when topotecan was administered intravenously in combination with oral GF120918, the effect was less pronounced in humans than in mice (Kruijtzer et al., 2002). One possible explanation for this difference is that the expression of BCRP in kidneys is higher in rats and mice than in humans (Tanaka et al., 2004), although other possibilities, such as differences in topotecan/GF120918 affinity for human versus murine BCRP, may also be important.
In summary, the present study indicates that most of the dihydropyridines and pyridines in our new series of compounds reverse resistance against mitoxantrone in MCF-7/MX100 and H460/MX20 cell lines. All these compounds were synthesized based on the structure optimization of dexniguldipine to maximize P-glycoprotein binding affinity and decrease calcium channel binding activity. Therefore, these dihydropyridines/pyridines may be promising agents for clinical application due to their potent inhibition of both BCRP and P-glycoprotein. The commercially available dihydropyridines, nicardipine, niguldipine, and nitrendipine were also demonstrated to be potent BCRP inhibitors; nifedipine, on the other hand, did not show any significant effect on BCRP function. This study represents the first report that dihydropyridines and pyridines are potent inhibitors of BCRP.
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Acknowledgments |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: BCRP, breast cancer resistance protein; ABC, ATP-binding cassette; DHP-014, 3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate; MRP1, multidrug resistance-associated protein 1; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; AUC, area under the curve; FTC, fumitremorgin C; CI1033, N-(4-(3-chloro-4-fluorophenylamino)-7-(3-morpholinopropoxy)quinazolin-6-yl)acrylamide.
Address correspondence to: Dr. Marilyn E. Morris, 517 Hochstetter Hall, University at Buffalo, State University of New York, Amherst, NY 14260-1200. E-mail: memorris{at}buffalo.edu
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) cells and MCF-7/MX100 (
) cells. The 30-min accumulation of mitoxantrone in MCF-7/MX100 cells and H460/MX20 cells in the presence of various concentrations (1–40 µM) of test compounds (nicardipine, nifedipine, niguldipine, nitrendipine, Im, and IIb) or the vehicle control (0.1% DMSO) was performed as described under Materials and Methods. FTC was used as a positive control at a concentration of 10 µM. Data are expressed as mean ± S.D. (n = 9). The absolute values of mean mitoxantrone fluorescence intensity in the vehicle (0.1% DMSO)-treated H460/MX20 cells and MCF-7/MX100 cells (controls) were 5.9 ± 0.6 (n = 9) and 4.3 ± 0.5 (n = 9), respectively. *, p < 0.05; ***, p < 0.001 compared with the vehicle control.
), or in combination with 10 mg/kg DHP-014 (
) or 20 mg/kg DHP-014 (