Introduction

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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 and mdr2) are not altered in mdr1a (/) CF-1 mice (Umbenhauer et al., 1997), whereas an increased expression of mdr1b in kidney and liver has been reported in mdr1a 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 (cholecystokinin_B receptor antagonist, Chang et al., 1989), and N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzeodiazepin-3-yl)-1H-indole-2-carboxamide (cholecystokinin_A 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

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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); and N-(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 MDR1 transfectants 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 mM L-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  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 at m/z 399.2 and diazepam at m/z 285.2, respectively.

	Results

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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.

View larger version (14K): [in this window] [in a new window]   Fig. 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.

View larger version (24K): [in this window] [in a new window]   Fig. 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.

View larger version (22K): [in this window] [in a new window]   Fig. 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.

View larger version (39K): [in this window] [in a new window]   Fig. 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.

View larger version (38K): [in this window] [in a new window]   Fig. 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.

View larger version (36K): [in this window] [in a new window]   Fig. 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.

View larger version (31K): [in this window] [in a new window]   Fig. 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.

View larger version (29K): [in this window] [in a new window]   Fig. 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. Figure 9 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 for mdr1a 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.

View larger version (24K): [in this window] [in a new window]   Fig. 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.

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 (Table 2). 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: [in this window] [in a new window]   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 After intravenous injection, mice were killed at 15, 30, and 60 min and plasma and brain concentrations of unchanged drug were measured by either high performance liquid chromatography or LC-MS. Three mice were analyzed in each group. Plasma concentrations (in µg/ml) are shown as mean ± S.D. Brain concentrations of each compound in the pooled samples of three mice were measured and expressed in µg/g of brain. Kp,brain represents the brain-to-plasma concentration ratio. AUCbrain (0-60 min) was calculated using trapezoidal rule and is shown in µg/g · min.

Comparison of in Vitro and in Vivo Results of Mouse mdr1a P-gp Substrate Identification: In Vitro-in Vivo Correlation. A plot of the in vivo Kp,brain ratio for mdr1a (/) 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).

View larger version (21K): [in this window] [in a new window]   Fig. 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.

View larger version (21K): [in this window] [in a new window]   Fig. 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 (at p < 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

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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 human MDR1-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- and mdr1a-encoded P-gps. Currently, gene knockout mice and mdr1a-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. 5 and 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 K_m and V_max values for compound A in the two cell lines, whereas we found a 3-fold greater V_max value for L-mdr1a than for L-MDR1 with no difference in K_m 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 V_max 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 resultant K_m and V_max 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 by MDR1, 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 mdr1a than 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 between mdr1a (/) 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.