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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

In Vitro Substrate Identification Studies for P-glycoprotein-Mediated Transport: Species Difference and Predictability of in Vivo Results

Masayo Yamazaki, William E. Neway, Tomoyuki Ohe, I-Wu Chen, Janice F. Rowe, Jerome H. Hochman, Masato Chiba and Jiunn H. Lin
Journal of Pharmacology and Experimental Therapeutics March 2001, 296 (3) 723-735;

Abstract

Two different cellular assay models were assessed as in vitro systems for P-glycoprotein (P-gp) substrate identification: cellular accumulation studies with KB-V1, a human MDR1P-gp-overexpressing multidrug-resistant human epidermoid carcinoma cell line; and transcellular transport studies with L-MDR1 (or L-mdr1a), a human MDR1 (or mouse mdr1a)-transfected porcine renal epithelial cell line. The in vitro-in vivo correlation for P-gp-mediated transport activity was also examined by comparing in vitro data obtained from L-mdr1a cell studies and in vivo data frommdr1a (−/−)/(+/+) CF-1 mice studies for several compounds. The results are summarized as follows: 1) two in vitro assay systems routinely identified the substrate for humanMDR1 P-gp-mediated transport with similar quantitative results; 2) in vitro studies with L-MDR1 and L-mdr1a cells demonstrated that the P-gp substrate susceptibility is different between human and mouse for certain compounds (species difference); and 3) in vivo brain concentration ratios of mdr1a (−/−) to (+/+) CF-1 mice, either at a certain time point or up to 60 min, correlated well with the in vitro transcellular transport ratios from L-mdr1a cells (r2 = 0.968 and 0.926, respectively). This indicates that, at least in mice, the in vitro data are valid predictors of the in vivo contribution of P-gp: the contribution of P-gp to the distribution of the compound to the brain up to 60 min post i.v. administration. These results provide a rationale for predicting in vivo relevance of P-gp in human from in vitro data using human P-gp-expressing cells.

Drug-transporting P-glycoproteins (P-gps), which play a major role in multidrug resistance in cancer cells, are known as MDR1 P-gp in human and mdr1a (and mdr1b) P-gp in mice. These P-gps can exclude/extrude a wide range of structurally diverse anticancer drugs from the cell (Ambudkar et al., 1999). P-gp is also known to govern pharmacokinetics/tissue distribution of many drugs, e.g., cyclosporin A, digoxin, vinblastine, etc. (Schinkel et al., 1994, 1995,1997; Van Asperen et al., 1996).

Recently, it has been emphasized that drug interaction at the membrane transport level, in particular, interaction with P-gp, should be considered to avoid unexpected changes in pharmacokinetics of the drugs as well as undesirable clinical outcomes (Fromm et al., 1998; Greiner et al., 1999; Yu, 1999; Kim, 2000). To evaluate the contribution of P-gp to the tissue distribution of drugs, gene knockout mice {mdr1a (−/−), mdr1b (−/−), and double knockout mice [mdr1a/1b (−/−)]} have been used (Schinkel, 1999). Recently, a subpopulation of CF-1 mice [mdr1a (−/−), deficient in mdr1a-P-gp expression], was characterized at Merck Research Laboratories (Lankas et al., 1997; Umbenhauer et al., 1997). This mutant mouse subpopulation is an alternative tool for examining the role of mdr1a and likely more suitable than gene knockout mice; the expression levels of other gene products in the mdr family (mdr1b andmdr2) are not altered in mdr1a (−/−) CF-1 mice (Umbenhauer et al., 1997), whereas an increased expression ofmdr1b in kidney and liver has been reported inmdr1a gene knockout mice (Schinkel et al., 1994). Thus far, extensive studies with the gene knockout mice and mutant mice have revealed that mdr1a P-gp is a major contributor to the maintenance of the blood-brain barrier (Kwei et al., 1999; Schinkel, 1999).

However, in vivo studies with these mice might not be very practical for a rapid and large-scale screening of compounds. Moreover, in vivo studies with mice cannot provide any definitive information on human P-gp. Thus, in vitro assays with human P-gp-overexpressing cell lines and human P-gp-transfected cell lines are necessary to evaluate the potential of compounds to act as P-gp substrates/inhibitors.

The purposes of the present studies were to 1) determine whether two different in vitro assay systems for human MDR1 P-gp could consistently identify human P-gp substrates, 2) identify the possible species difference in MDR1 (mdr1a) P-gp substrate susceptibility between human and mouse, and 3) examine the in vitro-in vivo correlation in mdr1a P-gp-mediated transport. The two different in vitro assay systems used in this study were 1) KB-V1, a human MDR1 P-gp-overexpressing multidrug-resistant human epidermoid carcinoma cell line, and its parental cell line KB-3-1 (Akiyama et al., 1985; Shen et al., 1986); and 2) L-MDR1 (L-mdr1a), a human MDR1 (or mouse mdr1a)-transfected porcine renal epithelial cell line, and its parental cell line LLC-PK1 (Schinkel et al., 1995). By comparing the results obtained from cellular accumulation studies with KB cells and transcellular transport studies with P-gp-transfected cells, we tried to determine whether the two different in vitro assays could identify human MDR1 P-gp substrate with similar quantitative results. By comparing the results between L-MDR1 and L-mdr1a, we examined the possible species difference in P-gp substrate susceptibility in vitro between human and mouse. The test compounds used for in vitro studies were indinavir and ritonavir (human immunodeficiency virus protease inhibitors); the three benzodiazepine derivatives diazepam, 3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N′-(3-methylphenyl)-urea (cholecystokininB receptor antagonist, Chang et al., 1989), andN-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzeodiazepin-3-yl)-1H-indole-2-carboxamide (cholecystokininA receptor antagonist, Chang et al., 1986); MK-0826 (a carbapenem antibiotic, Kohler et al., 1999); MK-0991 (an antifungal agent, Bartizal et al., 1997); and four miscellaneous Merck compounds. In addition, digoxin and vinblastine, two prototypical P-gp substrates (Shen et al., 1986; Tanigawara et al., 1992; Schinkel et al., 1995; Van Asperen et al., 1996), and progesterone, a nonsubstrate but inhibitor (modulator) of P-gp (Yang et al., 1990; Ueda et al., 1992; Rao et al., 1994; Barnes et al., 1996), were examined. For in vivo experiments, seven compounds from those examined in vitro were administered i.v. to wild-type [mdr1a (+/+)] and mdr1a-deficient [mdr1a (−/−)] spontaneous mutant CF-1 mouse subpopulations. By comparing the plasma and brain concentrations in these two mouse subpopulations, we evaluated the in vivo contribution of mdr1a P-gp-mediated transport with regard to distribution to the brain. Finally, we compared the results from in vitro studies using L-mdr1a to those from in vivo studies to assess the validity of in vitro data in predicting the relative contribution of P-gp to the distribution of the compound to the brain.

Materials and Methods

Chemicals

[G-3H]Digoxin (DG; 19.0 Ci/mmol), [1,2,6,7,16,17-N-3H]progesterone (138.0 Ci/mmol), (R)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N′-[3-methylphenyl-2,4,6-3H]urea (compound X, 75.6 Ci/mmol), (±)-(2,3-dihydro-[N-methyl-3H]-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-1H-indole-2-carboxamide (compound Y, 77.4 Ci/mmol), [methyl-3H]diazepam (83.0 Ci/mmol), and [carboxyl-14C]inulin (2.64 mCi/g) were purchased from NEN Life Science Products, Inc. (Boston, MA). [G-3H]Vinblastine sulfate (VBL; 15.5 Ci/mmol) was purchased from Amersham Life Sciences (Buckinghamshire, UK). [3H]Indinavir (1.0 Ci/mmol), [3H]ritonavir (1.1 Ci/mmol), and unlabeled ritonavir were from Moravek Biochemicals Inc. (Brea, CA). [3H]MK-0991 (3.44 μCi/μmol), [14C]MK-0826 (7.56 μCi/μmol), and all other radioactive Merck compounds {N-tertiary-butyl-4-(benzofuran-2-ylmethyl)-1-[2(S)-hydroxy-4(R)-[2(R)-hydroxy-indan-1(S)-yl[14C]carbamoyl]-5-phenyl-pentyl]-piperazine-2(S)-carboxamide (compound A, 29.97 μCi/μmol); 4-{5-[4-(3-chlorophenyl)-3-oxopiperazin-1-ylmethyl]-imidazol-1-ylmethyl}-[nitrile-14C]benzonitrile (compound B, 24.67 μCi/μmol);N-(6-amino-2-methylpyridin-3-ylmethyl)-2-(6-methyl-2-oxo-3-phenethylamino-2H-pyrazin-[5-14C]-1-yl)acetamide dihydrochloride monohydrate (compound C, 15.04 μCi/μmol); andN-(4-{1-methyl-2-(4-piperidinyl)-4-[3-(trifluoromethyl)phenyl]-1H-[2-14C]imidazol-5-yl}-2-pyridinyl)-N-[(1S)-1-phenylethyl]amine perchloric acid salt (compound D, 53.00 μCi/μmol)} were synthesized at Merck Research Laboratories. Radiochemical purity of the compounds used in the present study was greater than 94.2%. Unlabeled VBL, DG, progesterone, diazepam, cyclosporin A, and vincristine were purchased from Sigma Chemical Co. (St. Louis, MO). Unlabeled indinavir and compounds A–D were synthesized at Merck Research Laboratories (Rahway, NJ, and West Point, PA). Unlabeled compound X and compound Y were synthesized at the Development Laboratories (Hoddesdon, UK). All other reagents were analytical grade.

Cell Lines and Cell Cultures

KB-V1 and KB-3-1.

The drug-sensitive human epidermoid carcinoma cell line KB-3-1 and its VBL-selected MDR variant KB-V1 were kindly provided by Dr. Michael M. Gottesman (National Cancer Institute, Bethesda, MD) and used under license agreement. Both cell lines were maintained in Dulbecco's modified Eagle's medium (high glucose; Life Technologies, Gaithersburg, MD) containing 10% (v/v) fetal calf serum (FCS; BioWhittaker, Walkersville, MD), 50 units/ml penicillin (Life Technologies), 50 μg/ml streptomycin (Life Technologies), and 2 mM l-glutamine (Life Technologies) at 37°C in a humidified atmosphere of 5% CO2, 95% air. KB-V1 cells were grown in the continuous presence of VBL (1 μg/ml) (Shen et al., 1986). Confluent monolayers were subcultured every 7 days by treatment with trypsin-EDTA (0.25% trypsin, 1 mM EDTA·4Na; Life Technologies).

L-MDR1, L-mdr1a, and LLC-PK1.

Human MDR1transfectants L-MDR1, mouse mdr1a transfectants L-mdr1a, and their parental cell line LLC-PK1 pig kidney epithelial cells were kindly provided by Dr. Alfred H. Schinkel (The Netherlands Cancer Institute, Amsterdam) and used under license agreement. Cells were cultured in medium 199 (Life Technologies) supplemented with 2 mMl-glutamine, penicillin (50 units/ml), streptomycin (50 μg/ml), and 10% FCS (v/v) (Life Technologies) (Schinkel et al., 1995). For L-MDR1 and L-mdr1a, cells were maintained in the continuous presence of vincristine (640 nM) (Schinkel et al., 1995). Confluent monolayers were subcultured every 3 to 4 days by treatment with trypsin-EDTA. All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2, 95% air.

Drug Accumulation/Transport Studies

To test whether certain compounds are P-gp substrates, we compared the cellular accumulation in KB-3-1 and -V1, as well as the unidirectional transcellular transport across the monolayers of L-MDR1, L-mdr1a, and LLC-PK1. To quantitatively compare a number of compounds for their susceptibility as a transport substrate of P-gp, we fixed the concentrations of all the compounds at 1 μM with the exception of MK-0991 and MK-0826 (10 μM, because of their low permeability across membranes and low specific activities). A serum/protein free medium was used throughout the experiments to avoid any differences in protein binding among the compounds we tested. Experiments were conducted independently at least three times.

Cellular Accumulation in KB Cells

Measurement of the accumulation of test compounds in KB-3-1 and -V1 cell monolayers was carried out as described (Fojo et al., 1985) with minor modifications. In preparing the cell monolayers, 5 ml of a cell suspension containing 1 × 106 cells in Dulbecco's modified Eagle's medium (low glucose without phenol red; Life Technologies) with 10% FCS was plated in four wells of six-well tissue culture dishes (Nunc A/S, Roskilde, Denmark) the day before use. Blank wells were prepared simultaneously using the same media alone to assess background. After an overnight incubation at 37°C, cell monolayers were washed once with serum-free Hanks' balanced salt solution (Life Technologies) with 10 mM Hepes (pH 7.4). This medium is called “transport medium”. Three milliliters of transport medium containing test compound was then added to each well. Incubation in the drug-containing medium was carried out for 2 h, after which the reaction was terminated by removing the medium and rinsing twice with 5 ml of cold phosphate-buffered saline. Cells were lysed in 1 ml of 1 N NaOH at room temperature and transferred to scintillation vials containing 0.5 ml of 2 N HCl and 15 ml of scintillation cocktail (Ready Safe, Beckman, CA). The total radioactivity was measured by liquid scintillation counter (LS6500; Beckman, Fullerton, CA). Cellular accumulation was presented as mean ± S.D. after subtracting background.

Transepithelial Transport across L-MDR1, L-mdr1a, and LLC-PK1 Cells

Transepithelial transport study was carried out as described (Kim et al., 1998) with minor modifications. L-MDR1, L-mdr1a, and LLC-PK1 cells were plated at a density of 4 × 105 cells/12-mm well on porous (3.0-μm) polycarbonate membrane filters (Transwell; Costar Corp., Cambridge, MA). Cells were supplemented with fresh media on the second day and used for the transport studies on the fourth day after plating. Transepithelial resistance was measured in each well using a Millicell ohmmeter (model ERS; Millipore Corp., Bedford, MA); wells registering a resistance of 300 Ω or greater, after correcting for the resistance obtained in control blank wells, were used in the transport experiments. About 1 to 2 h before the start of the transport experiments, the medium in each compartment was replaced with the transport medium. The transport experiment was then initiated (t = 0) by replacing the medium in each compartment with 700 μl of transport medium with and without test compounds. After 1, 2, 3, and 4 h, 200-μl aliquots were taken from the opposite compartment and replaced with fresh transport medium. Samples were placed in scintillation vials containing 5 ml of scintillation cocktail, and total radioactivity was measured by a liquid scintillation counter. The appearance of radioactivity in the opposite compartment was presented as a fraction of the total radioactivity added at the beginning of the experiment. Directional transport was measured in triplicate/quadruplicate and presented as mean ± S.D. The paracellular flux was monitored by the appearance of inulin [14C]carboxylic acid (0.2 mCi/ml) in the opposite compartment and was <0.5% of total radioactivity per hour.

Animals

Two subpopulations of male CF-1 mice (body weights of approximately 25–35 g), of which genotype for mdr1a was (+/+) and (−/−), respectively, were provided by Safety Assessment, Merck Research Laboratories (West Point, PA), and Charles River Laboratories (Hollister, CA). All animal care and treatment procedures were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee before initiating these studies.

In Vivo Studies

All the dosing solutions were prepared in either saline or 25 to 50% dimethyl sulfoxide (except for indinavir, 0.05 M citric acid) to provide 2 to 20 mg/kg as a total dose. The volume of dosing solution was 4 μl/g of body weight. Three mice at each time point for both groups, mdr1a (+/+) and mdr1a (−/−) mice, were used in this study. After an i.v. injection into the tail vein, the mice were sacrificed at 15, 30, and 60 min, and blood and brain were collected for analyses. Plasma samples were separated immediately by centrifugation. Brain samples were homogenized with 4 volumes of water. Samples were kept frozen at −20°C until analyzed.

High Performance Liquid Chromatography Analysis of Indinavir, Ritonavir, Vinblastine, and Compounds A and D

Plasma samples were added to 10 volumes of acetonitrile, and brain homogenates were added to 2 volumes of acetonitrile containing respective internal standards. After being vortex mixed and centrifuged, the supernatant was evaporated to dryness under nitrogen. The residue was reconstituted in high performance liquid chromatography mobile phase (75:25 acetonitrile and H2O mixture containing 0.01% trifluoroacetic acid), and an aliquot was injected onto Vydac protein peptide C18 column (4.6 × 250 mm, 5 μm), with the mobile phase delivered isocratically at a flow rate of 1.0 ml/min. The eluate was monitored at 220 nm.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of Compound X and Diazepam

Plasma samples were extracted with 10 volumes of acetonitrile, and brain samples were extracted twice with 8 volumes of acetonitrile. All samples were dried under nitrogen, and the residue was reconstituted in 30% acetonitrile. Quantification was achieved by LC-MS, a PerkinElmer Sciex API 150EX mass spectrometer (Foster City, Canada). Chromatography was conducted using Betasil C18 column (3.0 × 50 mm, 5 μm; Keystone Scientific, Inc., Bellefonte, PA), with a mobile phase of 45:55 mixture of 5 mM ammonium acetate (pH 4.2) and acetonitrile (flow rate 0.6 ml/min). The nebulizer probe was set at 425°C. Detection of compound X and diazepam was carried out by selected ion monitoring, whereby the quadrupole transmitted the [M + H]+ ion of compound X atm/z 399.2 and diazepam atm/z 285.2, respectively.

Results

Cellular Accumulation in KB Cells.

The P-gp marker substrates showed greater cellular accumulation in KB-3-1 than in KB-V1 by factors of 4 for DG and 40 for VBL, whereas progesterone showed no difference (Fig. 1). Among the compounds tested, compounds A–D, indinavir, and ritonavir exhibited greater accumulation in KB-3-1 than in KB-V1 by more than a factor of 4 (Fig.2). Cellular accumulations in KB-V1 cells were restored greatly in the presence of cyclosporin A (10 μM) for these six compounds (data not shown). In contrast, compound X demonstrated a relatively small increase (1.6–2-fold) in its accumulation in KB-3-1 in comparison with that in KB-V1 (Fig.3). For compound Y, diazepam, MK-0826, and MK-0991, no significant difference was observed in their cellular accumulation in KB-3-1 cells in comparison with that in KB-V1 cells (Fig. 3). These results suggest that compounds A–D, indinavir, and ritonavir are substrates for human P-gp. Compound X appears to be a poor substrate, whereas compound Y, diazepam, MK-0826, and MK-0991 are not likely to be substrates of human P-gp.

Figure 1
Figure 1

Cellular accumulation of marker compounds of P-gp-mediated transport in KB-3-1 and KB-V1 cells (2-h incubation). Left, digoxin (1 μM); middle, vinblastine (1 μM); right, progesterone (1 μM). Open and closed columns represent the cellular accumulation in KB-3-1 and KB-V1 cells, respectively. Results shown are mean ± S.D.

Figure 2
Figure 2

Cellular accumulation of compounds A–D, indinavir, and ritonavir at 2 h in KB-3-1 and KB-V1 cells. Concentration of each compound is 1 μM. Results shown are mean ± S.D.

Figure 3
Figure 3

Cellular accumulation of compound X, compound Y, diazepam, MK-0826, and MK-0991 in KB-3-1 and V1 cells. Concentration of compound X, compound Y, and diazepam is 1 μM and for MK-0826 and MK-0991 is 10 μM. Results shown are mean ± S.D.

Transcellular Transport across L-MDR1, L-mdr1a, and LLC-PK1.

The two marker substrates of P-gp, DG and VBL, exhibited basal-to-apical (B-to-A) directed vectorial transport in L-MDR1 and L-mdr1a cells (Fig. 4). In the parental LLC-PK1 cells, smaller but significant polarized transport was also observed. This is most likely due to the directional transport by the endogenous porcine P-gp in LLC-PK1 cells (Schinkel et al., 1995; Kim et al., 1998). Similar marked enhancement in B-to-A directed transport in LLC-PK1 has been reported for VBL by Smit et al. (1998). For the negative marker progesterone, no polarized transport was observed in these three cell lines (Fig. 4). These findings confirm that transcellular transport experiments using these three cell lines can be used as a powerful tool for P-gp substrate identification studies.

Figure 4
Figure 4

Transcellular transport of marker compounds of P-gp-mediated transport across L-MDR1, L-mdr1a, and LLC-PK1 cell monolayers. Top, digoxin (1 μM); middle, vinblastine (1 μM); bottom, progesterone (1 μM). Open circles and closed triangles represent translocation from B-to-A compartment and A-to-B compartment of each compound, respectively. Data shown are mean ± S.D.

Merck compounds A–D showed typical transport characteristics of both human MDR1 and mouse mdr1a P-gp substrates (Fig.5); that is, B-to-A directed vectorial transport was observed in both L-MDR1 and L-mdr1a, but no significant net flux was observed in LLC-PK1. There was no significant difference in the percentage of net transport amount between L-MDR1 and L-mdr1a for these four compounds. In contrast, no significant net flux was observed in the three cell lines for diazepam, MK-0826, and MK-0991 (Fig. 6).

Figure 5
Figure 5

Transcellular transport of compounds A–D across L-MDR1, L-mdr1a, and LLC-PK1 cell monolayers. Concentration of each compound is 1 μM. Data shown are mean ± S.D.

Figure 6
Figure 6

Transcellular transport of diazepam, MK-0826, and MK-0991 across L-MDR1, L-mdr1a, and LLC-PK1 cell monolayers. Concentration of diazepam is 1 μM and of MK-0826 and MK-0991 is 10 μM. Data shown are mean ± S.D.

Compound X was noticeably transported by the L-MDR1 cell line, but more substantially by the L-mdr1a cells (Fig.7). Regarding the highly concentrated B-to-A directed transport observed for compound X in L-mdr1a, similar findings have also been reported by Schinkel et al. (1996). They also found that P-gp can accumulate substrates against a macroscopic concentration gradient, since at the end of the experiment (4 h) 4-fold more (80%) of the compounds was present on the apical side of the cell monolayer than on the basal side (20%), where the drugs were originally added.

Figure 7
Figure 7

Transcellular transport of compound X and compound Y across L-MDR1, L-mdr1a, and LLC-PK1 cell monolayers. Concentration of each compound is 1 μM. Data shown are mean ± S.D.

Compound Y was not detectably transported by either L-MDR1 or LLC-PK1, whereas there was only a low but significant level of transport across L-mdr1a in a B-to-A direction (Fig. 7). For compound X and compound Y, vectorial transport in P-gp-transfected cells was completely inhibited in the presence of 100 μM verapamil (data not shown).

Human immunodeficiency virus protease inhibitors indinavir and ritonavir demonstrated B-to-A directed vectorial transport in both L-MDR1 and L-mdr1a cell lines (Fig. 8). In addition, the B-to-A directed vectorial transport was observed also in the parental cells for these two compounds. The present results for indinavir from L-MDR1 and LLC-PK1 were consistent with the previous report by Kim et al. (1998). Cyclosporin A (10 μM) inhibited vectorial transport of indinavir and ritonavir in L-MDR1, L-mdr1a, and LLC-PK1 (data not shown).

Figure 8
Figure 8

Transcellular transport of indinavir and ritonavir across L-MDR1, L-mdr1a, and LLC-PK1 cell monolayers. Concentration of each compound is 1 μM. Data shown are mean ± S.D.

Species Difference in the Susceptibility of P-gp-Mediated Transport between Human MDR1 and Mouse mdr1a.

Figure9 illustrates a summary of the transcellular transport data obtained with the two P-gp transfectants L-MDR1 and L-mdr1a. There was a fairly good 1:1 correlation in the transcellular transport ratio [B-to-A versus apical-to-basal (A-to-B) at 3 h] between L-MDR1 and L-mdr1a, indicating that the susceptibility of P-gp between human MDR1 and mouse mdr1a was very consistent for most of the compounds. However, compound X exhibited a substantial (∼3-fold) difference in its transported amount ratios between human and mouse (the values were higher formdr1a than for L-MDR1). Furthermore, indinavir, DG, and compound C showed a higher (2–3-fold) transport ratio in L-MDR1 than in L-mdr1a. Indeed, in any of the experiments (n = 5–8), not only B-to-A/A-to-B ratios but also the permeability coefficients for L-MDR1 were always higher than those for L-mdr1a for indinavir, and vice versa for compound X. These results clearly demonstrate the existence of species difference in the susceptibility of P-gp-mediated transport for certain compounds between human and mouse.

Figure 9
Figure 9

Comparison of the basal-to-apical versus apical-to-basal transcellular transport ratios obtained from L-mdr1a and L-MDR1 cell monolayers. Data are shown in mean ± S.E (n = 3–8). Inset, ○, progesterone; ▪, diazepam; ▾, compound Y; ▴, MK-0826; ♦, MK-0991.

Validation of in Vitro Assay Models for MDR1 P-gp Substrate Identification.

Table 1 summarizes the in vitro results of MDR1 P-gp substrate identification obtained with two different assay models (cellular accumulation study with KB cells and transcellular transport study with P-gp-transfected LLC-PK1 cells). Both assay models routinely identified P-gp substrate with similar quantitative results, with a few exceptions. VBL exhibited an extremely high ratio in its cellular accumulation between KB-3-1 and KB-V1, which can likely be explained by the fact that KB-V1 was derived from the KB-3-1 through selection in increasing concentrations of VBL (Shen et al., 1986). Also, it would be conceivable that KB-V1 has another genetic alteration during selection, which causes such an exceedingly low accumulation of VBL.

View this table:
Table 1

Comparison of cellular accumulation ratio of KB-3-1/KB-V1 and transcellular transport ratio of L-MDR1 and LLC-PK1

In Vivo Study with mdr1a (+/+) and (−/−) CF-1 Mouse Subpopulations.

Seven compounds from those examined in vitro were administered i.v. to mdr1a (+/+) and (−/−) CF-1 mice subpopulations, and plasma and brain concentrations of the unchanged (parent) compounds were measured at 15, 30, and 60 min time points. For all the compounds tested, there were no significant differences in the plasma concentration between mdr1a (+/+) and (−/−) subpopulations, irrespective of the time point (Table2). However, brain concentrations (or AUCbrain values up to 60 min) showed a variety of differences between wild-type and mutant mice, depending on the compound. For example, compounds A and D displayed a 13- and 10-fold difference, respectively, whereas diazepam displayed no significant difference in its AUCbrain (0–60 min) values of mdr1a (+/+) and (−/−) CF-1 mice.

View this table:
Table 2

Plasma and brain concentrations of unchanged drug in mdr1a(−/−) and (+/+) CF-1 mice subpopulations after intravenous injection of indinavir, ritonavir, diazepam, compound X, vinblastine, and compounds A and D

Comparison of in Vitro and in Vivo Results of Mousemdr1a P-gp Substrate Identification: In Vitro-in Vivo Correlation.

A plot of the in vivo Kp,brain ratio formdr1a (−/−) and (+/+) CF-1 mice after i.v. administration versus in vitro B-to-A/A-to-B transport ratio in L-mdr1a cells showed a strong correlation (r2 = 0.968,p < 0.001) for all the seven compounds (Fig.10a). A positive correlation (r2 = 0.926, p < 0.01) is also observed if AUCbrain (0–60 min) is plotted as an ordinate for mouse in vitro-in vivo comparison (Fig. 10b). However, results from L-MDR1 and mouse in vivo did not correlate as strong (Fig.11).

Figure 10
Figure 10

Comparison of the basal-to-apical versus apical-to-basal transcellular transport ratio obtained from L-mdr1a in vitro and brain-to-plasma concentration ratio (Kp,brain) (a) or AUCbrain (0–60 min) ratio (b) of mdr1a (−/−)/(+/+) CF-1 mice in vivo. The x-axes represent B-to-A versus A-to-B transcellular transport ratio obtained from L-mdr1a cells at 3-h incubation. The y-axis shows brain-to-plasma concentration ratio (Kp,brain) at 30 min (a) or AUCbrain (0–60 min) ratio (b) of mdr1a (−/−)/(+/+) CF-1 mice after i.v. injection, respectively. AUC brain (0–60 min) was calculated using a trapezoidal rule using the data of brain concentration at 15, 30, and 60 min. 1, diazepam; 2, indinavir; 3, compound X; 4, vinblastine; 5, ritonavir; 6, compound D; and 7, compound A.

Figure 11
Figure 11

Comparison of the basal-to-apical versus apical-to-basal transcellular transport ratio obtained from L-MDR1 in vitro and brain to plasma concentration ratio (Kp,brain) (a) or AUCbrain (0–60 min) ratio (b) of mdr1a (−/−)/(+/+) CF-1 mice in vivo. The x-axes represent B-to-A versus A-to-B transcellular transport ratio obtained from L-MDR1 cells at 3-h incubation. The y-axis shows brain-to-plasma concentration ratio (Kp,brain) at 30 min (a) or AUCbrain (0–60 min) ratio (b) of mdr1a (−/−)/(+/+) CF-1 mice after i.v. injection, respectively. No significant correlation (atp < 0.1) were observed between L-MDR1 ratio in vitro and either Kp,brain ratio (a) or AUCbrain ratio (b) of mdr1a (−/−)/(+/+) CF-1 mice in vivo, respectively. 1, diazepam; 2, indinavir; 3, compound X; 4, vinblastine; 5, ritonavir; 6, compound D; and 7, compound A.

Discussion

The identification of compounds as substrates of P-gp-mediated transport is currently one of the key issues in the drug discovery and development process, in particular, for central nervous system-acting compounds. A quantitative characterization of drug candidates with regard to P-gp-mediated transport is crucial in predicting drug disposition and drug interactions. Presently, in vitro assay models with either P-gp-overexpressing cells or P-gp-transfected cells are most commonly used to assess the potential for a compound to act as a P-gp substrate (Kim, 2000). There are some limitations, however, in these two assays to obtain a definitive answer. For instance, most of the P-gp substrates are lipophilic; thus, cellular accumulation results are not always reliable due to extensive nonspecific binding to surface membranes and intracellular components. Regarding transcellular transport studies, it is difficult to determine whether a compound is a P-gp substrate when it exhibits non-negligible B-to-A directed transport in LLC-PK1 cells. Furthermore, it has never been examined systematically whether these two in vitro assay models for P-gp-mediated transport can routinely identify P-gp substrates with similar quantitative results. We thus first examined this issue using human MDR1-overexpressing multidrug-resistant cell lines (KB-V1 and its parental cell line KB-3-1) and humanMDR1-transfected LLC cell line (L-MDR1). In an attempt to compare the results of the two assay models simply, we chose the two following ratios as indices of magnitude (susceptibility): cellular accumulation ratio of KB-3-1 and KB-V1, and B-to-A versus A-to-B transcellular transport amount ratio.

In transcellular transport studies, there are several indices that may be capable of representing the functional activity of P-gp. We selected transport amount ratios in P-gp transfectants as a measure of apparent P-gp pump potency because it was most likely that they would reflect the in vivo brain concentration ratios of mdr1a(−/−)/(+/+) CF-1 mice. The ratios balance passive diffusion and the effect of P-gp and they may better reflect the contribution of P-gp to restricting flux across the blood-brain barrier.

As far as human MDR1 P-gp is concerned, both assay systems consistently identified P-gp substrates with similar quantitative results, with a few exceptions (Table 1). Our results have confirmed that both assays are useful in P-gp substrate identification. When we find non-negligible B-to-A directed transport in LLC-PK1, as we observed for indinavir and ritonavir in this study (Fig. 8), cellular accumulation data are especially valuable since they can reinforce the results derived from transcellular transport studies alone. For in vitro P-gp substrate identification studies, it would be prudent to consider the results from at least two different assay models for any compounds of interest if we find the vectorial transport in the parental cells.

Our second objective was to examine species difference in the P-gp susceptibility of compounds between MDR1- andmdr1a-encoded P-gps. Currently, gene knockout mice andmdr1a-deficient CF-1 mice are the only practical models available for investigating the in vivo contribution of P-gp to the tissue distribution of compounds. If species difference does exist regarding P-gp susceptibility, it is inappropriate to predict in vivo relevance of P-gp in humans simply from that obtained from mice studies. Indeed, we have observed the difference in the susceptibility of P-gp for several compounds between human and mouse based on the in vitro results from P-gp transfectants, L-MDR1 and L-mdr1a (Fig. 9). The species difference in P-gp susceptibility we found was not due to the difference in the expression levels of respective P-gps in each cell line because Western blotting using C219 monoclonal antibody showed similar amounts of expressed P-gp in these two cell lines (data not shown). Furthermore, we conducted the transcellular transport study with several compounds at a time, some of which demonstrated a higher B-to-A/A-to-B ratio in L-MDR1 than in L-mdr1a (e.g., indinavir) and vice versa (e.g., compound X).2 Regarding a simple comparison of B-to-A/A-to-B transport amount ratios obtained from two different cell lines (L-MDR1 and L-mdr1a) to discuss species difference in the susceptibility of P-gp, we must be aware of the caveat: these data could be affected by at least two other unknowns. One is a difference in total protein expression, which may affect the passive permeability properties of the membrane, and the other is a difference in the density of functional transporter in both cell lines. The former is likely not the case since 1) total protein concentration in the well was the same between L-MDR1 and L-mdr1a; and 2) both paracellular flux (assessed using inulin) and transcellular transport of diazepam, which were examined simultaneously with indinavir, compounds A and X, showed no significant difference between the two cell lines. The latter does not seem to be the case inasmuch as the species dependence is compound-specific. Although difference in the density of functional P-gp in the two cell lines would affect the relative transport ratios, this should be consistent for all of the P-gp substrates. Therefore, the disparity between indinavir and compound X indicates a species-dependent difference in substrate specificity.

In an attempt to find what causes the difference in the degree of the susceptibility between human and mouse P-gps, we conducted kinetic studies with compound A (which showed no significant difference in the percentage of net transport amount between L-MDR1 and L-mdr1a; Figs. 5and 9) and compound X (which exhibited 3-fold greater net transport in L-mdr1a than in L-MDR1; Figs. 7 and 9) simultaneously. As a result, we obtained somewhat similar Km andVmax values for compound A in the two cell lines, whereas we found a 3-fold greaterVmax value for L-mdr1a than for L-MDR1 with no difference in Km for compound X (M. Yamazaki, T. Ohe, W. E. Neway, J. H. Hochman, M. Chiba, and J. H. Lin, unpublished observations). This indicates that the difference in Vmax might cause the species difference in the susceptibility of P-gp for compound X. However, this is only one piece of information; it is unclear that this is the case for all other compounds that exhibit species difference in P-gp-mediated transport. It should also be noted that the resultantKm andVmax are apparent parameters, not the intrinsic parameters for P-gp itself. Further investigation with more compounds will be required to determine conclusively what causes species difference in P-gp susceptibility.

Investigations on the differences among P-gps encoded byMDR1, mouse mdr1a, and mdr1b genes regarding drug resistance profiles and sensitivity to known modulators have revealed that the three P-gp isoforms possess specific and distinguishable functional characteristics (Tang-Wai et al., 1995). With respect to transport, a higher susceptibility in mdr1athan in MDR1 has been reported for several compounds from in vitro experiments with L-MDR1 and L-mdr1a cells (Schinkel et al., 1996). This is the case for compound X in the present study. Compound X was shown to be a good substrate for mouse mdr1a P-gp, whereas data from both L-MDR1 and KB cells consistently indicated that it is a poor substrate of human MDR1 P-gp (Figs. 3 and 7). The in vivo CF-1 mice study demonstrated approximately a 6-fold difference in brain distribution ratio between mdr1a (−/−) and (+/+) CF-1 mice for compound X, as judged by the AUCbrain (0–60 min) ratio (Table 2). These results suggest that if the conclusion is based solely on the in vitro and/or in vivo mouse P-gp assays for compound X, there might be 1) an overestimation in the contribution of P-gp-mediated transport into the brain in humans, and/or 2) a “false-positive” prediction in drug-drug interactions in terms of P-gp-mediated transport in human. The opposite might be true for indinavir. Thus, when predicting the role of P-gp for a compound in human from that in mice, we should consider the possibility of the species difference in P-gp susceptibility, which we have demonstrated in the present study.

To what extent we can predict in vivo results from in vitro findings is a crucial question that continues to be a challenge. As shown in Fig.10, in vitro transcellular transport ratio with L-mdr1a exhibits a fairly good correlation with in vivo brain concentration ratio betweenmdr1a (−/−) and (+/+) CF-1 mouse subpopulations. Based on the present results, it is likely that we can predict the in vivo contribution of P-gp to the brain distribution of the novel compound of our interest by using in vitro transport data relative to the P-gp contribution for known compounds having both in vitro and in vivo results.

In the present study, we concentrated on evaluating plasma and brain concentrations of the compounds up to 60 min to measure unchanged (parent) concentrations of each compound. Generally, most of the in vivo data obtained so far using knockout mice and mutant CF-1 mice are assessed at longer time points than those of the present studies. It is unclear for the moment whether the good correlation between in vitro and in vivo results will be evident after longer periods of administration and studies investigating this are in progress.

In conclusion, the present study has revealed the following: 1) two different in vitro experimental systems routinely identified the substrate for human MDR1 P-gp-mediated transport with similar quantitative results; 2) in vitro studies in L-MDR1 and L-mdr1a demonstrated that P-gp substrate susceptibility is different between human and mouse for several compounds; and 3) in vivo brain concentration ratio for mdr1a (−/−) and (+/+) CF-1 mice correlated well with in vitro B-to-A/A-to-B transport ratio in L-mdr1a cells. Thus, in vitro L-mdr1a transcellular transport data are valid predictors of the in vivo contribution of P-gp, the contribution of P-gp to the distribution of the compound to the brain in mice. Consequently, these results provide a rationale for predicting in vivo relevance of P-gp with respect to the distribution of compounds to the brain in human from in vitro data using human P-gp-expressing cells. Since we observed the species difference in the susceptibility in P-gp-mediated transport between human and mouse for certain compounds, it would be prudent to interpret any in vivo data obtained from mice along with in vitro results from both mouse and human P-gp transfectants.

Acknowledgments

We acknowledge Dr. Alfred H. Schinkel in The Netherlands Cancer Institute for permission to useMDR1-/mdr1a-transfected LLC-PK1 cell lines. We also thank Dr. Michael M. Gottesman in National Cancer Institute for permission to use KB-V1 and 3-1 cells. We express our gratitude to Janice E. Brunner for expert technical assistance with in vivo experiments. We thank Dr. Yasuyuki Ishi'i for fruitful discussion. We also thank Brendan C. Bender for helpful discussions and critical reading of the manuscript. We are grateful to Joy A. Nishime and Ruth Munilla for help in the preparation of the manuscript.

Footnotes

  • Send reprint requests to: Masayo Yamazaki, Ph.D., WP75-200, Merck Research Laboratories, West Point, PA 19486. E-mail:Masayo_Yamazaki{at}Merck.com

  • ↵1 Current address: Department of Drug Metabolism, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, 300-2611, Japan.

  • ↵2 We have confirmed that several compounds show mouse ≫ human (one compound exhibited ∼10-fold difference in the ratio between L-mdr1a versus L-MDR1) and/or human ≫ mouse, and we have found that the results were consistent irrespective of the B-to-A/A-to-B ratio or the absolute difference in directional transport. Due to the restriction of the disclosure of their chemical structures, we cannot include the data for these compounds in this manuscript.

  • We have chosen transport amount ratios at 3 h as a “practical” measure for P-gp activity for the following reason. We would like to compare the in vitro transport results in terms of P-gp for a series of compounds with diverse physicochemical properties. Some of the test compounds such as MK-0826 and MK-0991 showed extremely low membrane permeability. Furthermore, we observed a very small difference in dpm values between A-to-B samples of compound A and D and background (approximately <100 dpm for samples versus ∼25 dpm for background) at the first time point (1 h). This is presumably due to relatively low specific activity of 3H/14C labels we used and extensive efflux by P-gp. At a longer time period (at 3 h), dpm counts of the samples for the A-to-B direction were more consistent for both compounds. To increase the reliability of the data in A-to-B direction, however, we may have underestimated the intrinsic permeability for B-to-A direction significantly. However, it was likely not the case for the present study: we calculated the permeability coefficient, percentage of transport, and the ratio between B-to-A and A-to B at 1 and 3 h for several compounds that were most likely to be affected by nonlinearity and by “not adhering to sink condition” among the compounds tested. As a result, differences in any of the parameters calculated at 1 h and at 3 h were at most <25%, demonstrating that there were no significant differences in using data evaluated at 1 or 3 h for the present study.

  • Abbreviations:
    P-gp
    P-glycoprotein
    MDR
    multidrug resistance
    DG
    digoxin
    VBL
    vinblastine
    FCS
    fetal calf serum
    B-to-A
    basal-to-apical
    A-to-B
    apical-to-basal
    AUC
    area under the curve
    Kp,brain
    brain-to-plasma concentration ratio
    LC-MS
    liquid chromatography-mass spectrometry
    • Received August 3, 2000.
    • Accepted November 24, 2000.

References

  1. ↵
    1. Akiyama S,
    2. Fojo A,
    3. Hanover JA,
    4. Pastan I,
    5. Gottesman MM
    (1985) Isolation and genetic characterization of human KB cell lines resistant to multiple drugs. Somat Cell Genet 4:193–200.
  2. ↵
    1. Ambudkar SV,
    2. Dey S,
    3. Hrycyna CA,
    4. Ramachandra M,
    5. Pastan I,
    6. Gottesman MM
    (1999) Biochemical, cellular and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 39:361–398.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Barnes KM,
    2. Dickstein B,
    3. Cutler GB,
    4. Fojo T,
    5. Bates SE
    (1996) Steroid transport, accumulation, and antagonism of P-glycoprotein in multidrug-resistant cells. Biochemistry 35:4820–4827.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Bartizal K,
    2. Gill CH,
    3. Abruzzo GK,
    4. Flattery AM,
    5. Kong L,
    6. Scott PM,
    7. Smith JG,
    8. Leighton CE,
    9. Bouffard A,
    10. Dropinski JF,
    11. et al.
    (1997) In vitro preclinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872). Antimicrob Agents Chemother 41:2326–2332.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Chang RSL,
    2. Chen TB,
    3. Bock MG,
    4. Freidinger RM,
    5. Chen R,
    6. Rosegay A,
    7. Lotti VJ
    (1989) Characterization of the binding of [3H]L-365,260: A new potent and selective brain cholecystokinin (CCK-B) and gastrin receptor antagonist radioligand. Mol Pharmacol 35:803–808.
    OpenUrlAbstract
  6. ↵
    1. Chang RSL,
    2. Lotti VJ,
    3. Chen TB,
    4. Kunnel KA
    (1986) Characterization of the binding of [3H]-(±)-L-364,718: A new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors. Mol Pharmacol 30:212–217.
    OpenUrlAbstract
  7. ↵
    1. Fojo A,
    2. Akiyama S,
    3. Gottesman MM,
    4. Pastan I
    (1985) Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines. Cancer Res 45:3002–3007.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Fromm MF,
    2. Kim RB,
    3. Stein CM,
    4. Wilkinson GR,
    5. Roden DM
    (1998) Inhibition of P-glycoprotein-mediated drug transport: A unifying mechanism to explain the interaction between digoxin and quinidine. Circulation 99:552–557.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Greiner B,
    2. Eichelbaum M,
    3. Fritz P,
    4. Kreichgauer HP,
    5. Von Richter O,
    6. Zundler J,
    7. Kroemer HK
    (1999) The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest 104:147–153.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Kim RB
    (2000) Transporters and drug disposition. Curr Opin Drug Discov Dev 3:94–101.
    OpenUrlPubMed
  11. ↵
    1. Kim RB,
    2. Fromm MF,
    3. Wandel C,
    4. Leake B,
    5. Wood AJJ,
    6. Roden DM,
    7. Wilkinson GR
    (1998) The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Invest 96:1698–1705.
    OpenUrl
  12. ↵
    1. Kohler J,
    2. Dorso KL,
    3. Young K,
    4. Hammond GG,
    5. Rosen H,
    6. Kropp H,
    7. Silver LL
    (1999) In vitro activities of the potent, broad-spectrum carbapenem MK-0826 (L-749,345) again broad-spectrum β-lactamase-and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli clinical isolates. Antimicrob Agents Chemother 43:1170–1176.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Kwei GY,
    2. Alvaro RF,
    3. Chen Q,
    4. Jenkins HJ,
    5. Hop CEAC,
    6. Keohane CA,
    7. Ly VT,
    8. Strauss JR,
    9. Wang RQ,
    10. Wang Z,
    11. Pippert TR,
    12. Umbenhauer DR
    (1999) Disposition of ivermectin and cyclosporin A in CF-1 mice deficient in mdr1a P-glycoprotein. Drug Metab Dispos 27:581–587.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Lankas GR,
    2. Cartwright ME,
    3. Umbenhauer DR
    (1997) P-glycoprotein deficiency in a subpopulation of CF-1 mice enhances avermectin-induced neurotoxicity. Toxicol Appl Pharmacol 143:357–365.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Rao US,
    2. Fine RL,
    3. Scarborohugh GA
    (1994) Antiestrogens and steroid hormones: Substrates of the human P-glycoprotein. Biochem Pharmacol 48:287–292.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Shen D,
    2. Cardarelli C,
    3. Hwang J,
    4. Cornwell M,
    5. Richert N,
    6. Ishii S,
    7. Pastan I,
    8. Gottesman MM
    (1986) Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins. J Biol Chem 261:7762–7770.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Schinkel AH
    (1999) P-glycoprotein, a gatekeeper in the blood-brain barrier. Adv Drug Deliv Rev 36:179–194.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Schinkel AH,
    2. Mayer U,
    3. Wagenaar E,
    4. Mol CAAM,
    5. Van Deemter L,
    6. Smit JJM,
    7. Van der Valk MA,
    8. Voordouw AC,
    9. Spits H,
    10. Van Tellingen O,
    11. et al.
    (1997) Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci USA 94:4028–4033.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Schinkel AH,
    2. Smit JJM,
    3. Van Tellingen O,
    4. Beijnen JH,
    5. Wagenaar E,
    6. Van Deemter L,
    7. Mol CAAM,
    8. Van der Valk MA,
    9. Robanus-Maandag EC,
    10. Te Riele HPJ,
    11. et al.
    (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77:491–502.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Schinkel AH,
    2. Wagenaar E,
    3. Mol CAAM,
    4. Van Deemter L
    (1996) P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest 97:2517–2524.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Schinkel AH,
    2. Wagenaar E,
    3. Van Deemter L,
    4. Mol CAAM,
    5. Borst P
    (1995) Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J Clin Invest 96:1698–1705.
  22. ↵
    1. Smit JW,
    2. Weert B,
    3. Schinkel AH,
    4. Meijer DKF
    (1998) Heterologous expression of various P-glycoproteins in polarized epithelial cells induces directional transport of small (type 1) and bulky (type 2) cationic drugs. J Pharmacol Exp Ther 286:321–327.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Tang-Wai DF,
    2. Kajiji S,
    3. DiCapua F,
    4. de Graaf D,
    5. Roninson IB,
    6. Gros P
    (1995) Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators. Biochemistry 34:32–39.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Tanigawara Y,
    2. Okamura N,
    3. Hirai M,
    4. Yasuhara M,
    5. Ueda K,
    6. Kioka N,
    7. Komano T,
    8. Hori R
    (1992) Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). J Pharmacol Exp Ther 263:840–845.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Ueda K,
    2. Okamura N,
    3. Hirai M,
    4. Tanigawara Y,
    5. Saeki T,
    6. Kioka N,
    7. Komano T,
    8. Hori R
    (1992) Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem 267:24248–24252.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Umbenhauer DR,
    2. Lankas GR,
    3. Pippert TR,
    4. Wisei LD,
    5. Cartwright ME,
    6. Hall SJ,
    7. Beare CM
    (1997) Identification of a P-glycoprotein-deficient subpopulation in the CF-1 mouse strain using a restriction fragment length polymorphism. Toxicol Appl Pharmacol 146:88–94.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Van Asperen J,
    2. Schinkel AH,
    3. Beijnen JH,
    4. Nooijen WJ,
    5. Borst P,
    6. Van Tellingen O
    (1996) Altered pharmacokinetics of vinblastine in mdr1a P-glycoprotein-deficient mice. J Natl Cancer Inst 88:994–999.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Yang CPH,
    2. Cohen D,
    3. Greenberger LM,
    4. Hsu SIH,
    5. Horwitz SB
    (1990) Differential transport properties of two mdr gene products are distinguished by progesterone. J Biol Chem 265:10282–10288.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Yu DK
    (1999) The contribution of P-glycoprotein to pharmacokinetic drug-drug interactions. J Clin Pharmacol 39:1203–1211.
    OpenUrlCrossRefPubMed
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

In Vitro Substrate Identification Studies for P-glycoprotein-Mediated Transport: Species Difference and Predictability of in Vivo Results

Masayo Yamazaki, William E. Neway, Tomoyuki Ohe, I-Wu Chen, Janice F. Rowe, Jerome H. Hochman, Masato Chiba and Jiunn H. Lin
Journal of Pharmacology and Experimental Therapeutics March 1, 2001, 296 (3) 723-735;
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