Drug Metabolism and Disposition Fast Forward
First published on February 14, 2008; DOI: 10.1124/dmd.107.020040
0090-9556/08/3605-916-922$20.00
DMD 36:916-922, 2008
Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo
Yoshiyuki Shirasaka,
Yoshie Masaoka,
Makoto Kataoka,
Shinji Sakuma, and
Shinji Yamashita
Faculty of Pharmaceutical Sciences, Setsunan University, Nagaotoge-cho, Hirakata, Osaka, Japan
(Received December 6, 2007;
accepted February 13, 2008)
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Abstract
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In a previous study, the concentration-dependent permeability of P-glycoprotein (P-gp) substrate drugs, quinidine, verapamil, and vinblastine, in several cell monolayers with different levels of P-gp expression was analyzed kinetically to obtain fundamental parameters for P-gp-mediated transport, Vmax and Km(app) values. Both Vmax and Km(app) values of each drug were found to show linear correlations with the expression level of P-gp. These findings imply the possibility of estimating the Vmax and Km(app) values of P-gp substrate drugs in the in vivo intestinal membrane on the basis of the P-gp expression level. In the present study, concentration-dependent drug permeability to the rat small intestines (upper jejunum and ileum) was simulated on the basis of Vmax and Km(app) values of each drug estimated from the P-gp expression level in the rat small intestines. To validate the predictability of these procedures, drug permeability in the rat small intestines was measured by the in situ single-pass perfusion method. It was confirmed that simulated permeability of each drug in the rat jejunum and ileum corresponded well with permeability measured by the in situ single-pass perfusion method. This study clearly demonstrated the potential to estimate the permeability of P-gp substrate drugs in the human intestine from its P-gp expression level and thus the possibility to predict the oral absorption of those drugs.
P-glycoprotein (P-gp), one of the most important efflux transporters in the field of biopharmacy, is an ATP-binding cassette (ABC) transporter encoded by the multidrug resistance 1 gene (MDR1/ABCB1). P-gp is expressed not only in cancer cells but also in many normal tissues (Loo and Clarke, 1999
). So far, many reports have described the effects of P-gp on the pharmacokinetic profiles of clinically important drugs, by denaturing absorption, distribution, and excretion. For example, P-gp located in the apical domain of the enterocytes of the gastrointestinal tract limits the uptake and absorption of its substrate drugs after oral administration (Hunter and Hirst, 1997; Meijer et al., 1997; Jonker et al., 1999). Because of the great impact of P-gp on the pharmacokinetic profiles of a variety of drugs, various in vitro experimental techniques are now used to identify the compounds subjected to P-gp-mediated efflux (Kim et al., 1998; Polli et al., 1999, 2001; Susanto and Benet, 2002; Shirasaka et al., 2006a,b). However, it is still difficult to quantitatively predict the effect of P-gp on in vivo intestinal absorption of such drugs.
In a previous study, we measured the apical (AP) to basal (BL) absorptive permeability of P-gp substrate drugs in several cultured cell monolayers with different expression levels of P-gp (Shirasaka et al., 2008). In all cell monolayers, AP to BL permeability of P-gp substrate drugs showed a sigmoid-type relation to their donor (AP) concentration and reached a maximal value at the higher concentration range because of the saturation of P-gp-mediated efflux. Concentration-dependent permeability of each drug was kinetically analyzed to obtain fundamental parameters for P-gp-mediated transport, Vmax and Km(app) values. Assuming that drug concentration in the vicinity of the drug binding site of P-gp is proportional to the apical concentration, the Km(app) value could be regarded as an apparent affinity of the drug to P-gp, which is defined on the basis of apical drug concentration. Interestingly, not only the Vmax value but also the Km(app) value was found to show linear correlation with the expression level of P-gp quantified by real-time quantitative PCR and Western blot analyses. From these results, we propose the following procedures to simulate the concentration-dependent permeability of P-gp substrate drugs in the human intestine:
- Measure the concentration-dependent permeability of the test compound in various cell monolayers with different expression levels of P-gp (at least three kinds of cell monolayers).
- Obtain Vmax and Km(app) values of the test compound in each monolayer by using kinetic models.
- Quantify the P-gp expression level in each monolayer (protein or mRNA level) and obtain the linear regression lines to Vmax and Km(app) values.
- Estimate Vmax and Km(app) values in the human intestinal membrane by incorporating its P-gp expression level into the linear regression line.
- Simulate the concentration-dependent permeability of the test compound in the human intestinal membrane by using the estimated parameters.
In the present study, concentration-dependent drug permeability to the rat small intestine (upper jejunum and ileum) was simulated on the basis of Vmax and Km(app) values of each drug obtained in a cell monolayer study and on P-gp expression levels in rat small intestine. To validate the predictability of the above procedures, drug transport in rat small intestine was measured by the in situ single-pass perfusion method.
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Materials and Methods
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Chemicals. Verapamil hydrochloride and quinidine sulfate were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Vinblastine sulfate was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). For Western blot analysis, sample buffer (pH 6.8) consisting of 62.5 mM Tris-HCl, 25% glycerol, 2% SDS, and 0.01% bromphenol blue; Readygels J consisting of 7.5% resolving gels and Tris-HCl; and polyvinylidene difluoride membrane (0.2 µm pore size) were purchased from Bio-Rad (Hercules, CA). Skim milk was obtained from Becton Dickinson Bioscience (Bedford, MA). Anti-P-glycoprotein (C219) was purchased from Calbiochem (Darmstadt, Germany). Anti-GAPDH (6C5) was purchased from American Research Products, Inc. (Belmont, MA). Biotinylated protein ladder detection pack, anti-mouse IgG (H&L), HRP-linked, and LumiGLO chemiluminescent substrate kit were purchased from Cell Signaling Technology Inc. (Danvers, MA). All other compounds and reagents were obtained from Nacalai Tesque, Inc., Wako Pure Chemical Industries, Ltd., Sigma-Aldrich (St. Louis, MO), Bio-Rad, or Applied Biosystems (Foster City, CA).
In Situ Single-Pass Perfusion Experiment. Male Wistar rats were housed three per cage with free access to commercial chow and tap water and were maintained on a 12-h dark/light cycle (7:00 AM–7:00 PM light) in an aircontrolled room (temperature, 24.5 ± 1°C; humidity, 55 ± 5%). All animal experimentation reported in this manuscript was performed in accordance with the Declaration of Helsinki and with the Guide of Setsunan University for the Care and Use of Laboratory Animals. The permeability of rat intestinal membrane was evaluated by the in situ single-pass perfusion method. Rats (200–250 g b.wt.) fasted overnight were anesthetized with pentobarbital. The abdominal cavity was opened, an intestinal loop (10 cm in length) was made at two regions (upper jejunum and ileum) by cannulation with a silicone tube (3 mm i.d.), and then the intestinal contents were removed by a slow infusion of saline and air.
Following the above procedure, the test solution (phosphate-buffered solution, adjusted to pH 6.5) containing each compound and FD-4 (10 µM) was perfused with an infusion pump at a flow rate of 0.5 ml/min. The effluent was collected from 30 min after starting the perfusion to 90 min, at 10-min intervals, because steady-state absorption usually was achieved by 30 min under these conditions. Drug permeability was calculated according to the following equation:
 | (1) |
where Q is the flow rate and Cin and Cout are inlet and outlet drug concentrations, respectively. The effect of water transport during perfusion on Cout was corrected using the concentration ratio of a nonabsorbable marker (FD-4). r and l represent the radius and length of the used segment of intestine, respectively; thus, the value of 2
rl corresponds to its surface area. As a radius of each intestinal segment, the value reported by Fagerholm et al. (1997) was used (0.18 cm for jejunum and ileum).
Preparation of Intestinal Brush Border Membrane Vesicles. Brush border membrane vesicles were isolated from the upper jejunum and ileum of rats by the Ca2+ precipitation technique as described previously (Minami et al., 1993; Moore et al., 1996; Katai et al., 1999). Male Wistar rats (200–250 g b.wt.) fasted overnight were anesthetized with pentobarbital and euthanized by cervical dislocation and complete blood removal. The abdominal cavity was opened, and the entire small intestine was removed and thoroughly flushed with ice-cold saline. The two regions (upper jejunum and ileum, 10 cm in length) were placed on a glass plate maintained at 4°C and cut open; mucosa was harvested by scraping with a microscope glass slide. All subsequent procedures were performed at 4°C. The mucosal scrapings were added to 10 ml of buffer A (10 mM Tris-HCl, pH 7.4, 50 mM mannitol, and 5 mM EDTA · 2Na) and centrifuged at 500g for 10 min. The supernatant was collected and then adjusted to 10 mM CaCl2 by the addition of 100 µl of 1.0 M CaCl2 with constant stirring. After stirring for 15 min at 4°C, the solution was centrifuged at 3000g for 17 min to remove precipitated cellular material and organelles. The supernatant, which contained mainly brush border membranes, was then centrifuged at 35,000g for 30 min at 4°C. The pellet (BBM fractions) was then lysed with 100 µl of buffer L [50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1 mM dithiothreitol, and protease inhibitor cocktail (Complete Mini)] and 100 µl sample buffer (62.5 mM Tris-HCl, 25% glycerol, 2% SDS, and 0.01% bromphenol blue, pH 6.8) for Western blot analysis.
Western Blot Analysis. Briefly, BBM lysates were adjusted to a final protein concentration of 10 mg/ml in sample buffer with 5% 2-mercaptoethanol and were loaded at a volume of 10 µl (corresponding to 100 µg of protein) on Readygels J consisting of 7.5% resolving gels with Tris-HCl. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred electrophoretically onto a 0.2-µm pore polyvinylidene difluoride membrane. Blots were blocked overnight at 4°C with Tris-buffered saline/Tween 20 (0.15 M NaCl, 0.05% Tween 20, and 20 mM Tris-HCl, pH 7.5) containing 5 mM sodium azide and 5% (w/v) skim milk and then incubated with anti-P-glycoprotein (C219) and anti-GAPDH (6C5) for 2 h at 37 °C. Anti-mouse IgG (H&L), HRP-linked, was used as a secondary antibody. Detection was performed with a LumiGLO Chemiluminescent Substrate Kit to activate the horseradish peroxidase signal. Blots were then exposed to a cold camera imaging system (Lumino Imaging Analyzer, FAS-1000; TOYOBO Co., Ltd., Osaka, Japan). The protein level of P-gp was quantified by computer image analysis with image software (Gel-Pro Analyzer version 3.1, TOYOBO Co.) as described previously (Shirasaka et al., 2008). Then it was further normalized to the P-gp level per intestinal surface area (micrograms per square centimeter) by using total protein amounts in the BBM fraction on 10 cm for the small intestine in each region.
Kinetic Analysis. In a previous report, concentration-dependent permeability of P-gp substrate drugs to the cell monolayer was kinetically analyzed according to the following equation, assuming that AP to BL flux (VAB) can be expressed as the difference between passive (VPD) and P-gp-mediated flux (VP-gp) (Shirasaka et al., 2008):
 | (2) |
where CLPD, and Papp, PD are the permeation clearance and membrane permeability by passive diffusion, respectively, and S is the surface area of the membrane. Ca represents the drug concentration in the apical solution, and the Hill coefficient (r) was introduced to the equation to add flexibility to the fitted curve. P-gp-mediated efflux was defined by two parameters, maximal velocity of P-gp-mediated efflux (Vmax) and Km(app).
In the present study, on the basis of eq. 2, the concentration-dependent permeability of three drugs (quinidine, verapamil, and vinblastine) in rat small intestine was simulated from both in vitro and in situ data. Because the r values for all drugs were close to 1.0 in all cell monolayers in our previous study, the r value was fixed to 1.0 in all simulations. In addition, kinetic parameters for the concentration-dependent permeability of three drugs in rat intestinal membrane, Vmax and Km(app), were obtained by fitting the experimental data of in situ single-pass perfusion to eq. 2 (the r value was fixed to 1.0) by the nonlinear least-squares method using the MULTI program (Yamaoka et al., 1981).
Analytical Methods. The concentration of drugs in perfusate samples was analyzed with a reversed-phase high-performance liquid chromatography system (LC-20AD; Shimadzu Co., Kyoto, Japan) equipped with a liquid chromatography-mass spectrometry detector (LCMS-2010A; Shimadzu Co.). Mercury MS (Luna 5µ C18, 10 x 4.0 mm; Phenomenex, Torrance, CA) was used as an analytical column, and the mobile phase was composed of 0.1% formic acid in water and acetonitrile. Selected ion monitoring was used to detect protonated molecules of quinidine [m/z 325.10 (+)], verapamil [m/z 455.25 (+)], and vinblastine [m/z 811.60 (+)]. In some perfusate samples, the concentration of drugs was analyzed with the reversed-phase high-performance liquid chromatography system (LC-10AT; Shimadzu Co.) equipped with a variable wavelength ultraviolet detector (SPD-10AV; Shimadzu Co.). The column (J'sphere ODS-H80 75 mm x 4.6 mm; YMC, Kyoto, Japan) was used with a mobile phase consisting of 50 mM phosphate buffer (pH 2.5) and acetonitrile. Quinidine and verapamil were quantified at wavelengths of 230 and 235 nm, respectively. For the measurement of total protein, the absorbance of the cell suspension was photometrically measured at a wavelength of 562 nm by UV-visible spectrophotometer (UV-1650PC; Shimadzu Co.).
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FIG. 1. Kinetic model of AP to BL transport of P-gp substrate. A, AP to BL flux of drugs can be determined by subtraction of P-gp-mediated transport, VP-gp, from passive transport, VPD. B, the Km(app) value represents the donor concentration of the drug at which the decreased permeability by P-gp-mediated efflux [Papp(max) - Papp(min)] became half of its maximal value. Reproduced with permission from Shirasaka et al. (2008).
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Results
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Expression Level of P-gp in Rat Intestinal Membrane. In a previous report, concentration-dependent permeability of P-gp substrate drugs, quinidine, verapamil and vinblastine, in several cell monolayers with different levels of P-gp expression was kinetically analyzed according to eq. 2, assuming that AP to BL flux (VAB) can be expressed as the difference between passive (VPD) and P-gp-mediated flux (VP-gp) (Shirasaka et al., 2008). In eq. 2, the Km(app) value represents the donor concentration of the drug at which the decreased permeability by P-gp-mediated efflux [Papp(max) - Papp(min)] became half of its maximal value (Fig. 1, A and B); therefore, the Km(app) value could be regarded as the apparent affinity of drugs to P-gp. On the basis of eq. 2, kinetic parameters for the concentration-dependent permeability of all three drugs, Vmax and Km(app) values, were obtained (Shirasaka et al., 2008). It was demonstrated that both Vmax and Km(app) values of each drug are the proportional function of P-gp expression level (Fig. 2, A and B). Because the Km(app) value was defined on the basis of the apical concentration of drugs, this parameter can be regarded as a hybrid parameter of the real affinity of drugs to P-gp (Km) and the ratio of drug concentration in the apical solution (Ca) and in the vicinity of the drug binding site of P-gp (Cbind). If P-gp activity is high enough, the substrate drug that diffused to the vicinity of the drug binding site of P-gp from the apical solution would be pumped out efficiently, and thus Cbind is kept low even when the Ca becomes high. Therefore, in cells that highly express P-gp, higher Ca is required to saturate P-gp and then Km(app) values increase, depending on the P-gp expression level.
These findings imply the possibility of estimating the Vmax and Km(app) values of P-gp substrate drugs in the in vivo intestinal membrane from its P-gp expression level. To simulate the concentration-dependent permeability of P-gp substrate drugs in the rat small intestine, P-gp expression levels in the rat small intestine (upper jejunum and ileum) were first determined by Western blot analysis (Fig. 3A). P-gp expression levels in the jejunum and ileum quantified by computer image analysis were 77.7 ± 6.9 and 231.5 ± 36.1 µg/cm2 (mean ± S.E.), respectively, indicating that the P-gp expression level in the ileum was approximately 3-fold higher than that in the jejunum.
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FIG. 3. Western blot analysis of P-gp in rat small intestines (jejunum and ileum). Western blotting was performed in loading 100 µg of BBM lysates prepared from rat small intestines (lane 1, the molecular mass of standards; lane 2, blank; lanes 3, 5, and 7, jejunum; and lanes 4, 6, and 8, ileum). P-gp and GAPDH was detected using monoclonal anti-P-gp (C219) and monoclonal anti-GAPDH (6C5), respectively. Expression levels of P-gp were quantified using computer image analysis.
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Estimation of Vmax and Km(app) Values of P-gp Substrate Drugs in Rat Intestinal Membrane. Quinidine was used as an example to explain the process of estimating Vmax and Km(app) values in the rat intestinal membrane (Fig. 4, A and B; Table 1).
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FIG. 4. Estimation of Vmax and Km(app) values of quinidine in rat small intestines (jejunum and ileum). The expression level of P-gp in rat small intestines (77.7 µg/cm2 for jejunum and 231.5 µg/cm2 for ileum) was incorporated into the in vitro relationship between Vmax and P-gp expression levels (A) and the in vitro relationship between Km(app) and P-gp expression levels (B).
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TABLE 1 Passive permeability (Papp, PD) of quinidine, verapamil, and vinblastine in Caco-2 monolayers and rat small intestine (jejunum and ileum) Papp, PD to rat small intestines was measured by the in situ single-pass perfusion method with P-gp inhibitor (alprenolol) added to the perfusion medium. Data are presented as mean ± S.E. (n = 3). The ratio (rat/Caco-2) represents the ratio of passive permeability in Caco-2 monolayers [Papp, PD(Caco-2)] and in rat small intestines [Papp, PD(rat)] [i.e., Papp, PD(rat)/Papp, PD(Caco-2)].
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Expression levels of P-gp in the rat small intestine were incorporated in in vitro relations between the Vmax value and P-gp expression level (Y = 3.61 x 10-8 X) (Fig. 4A). Then, Vmax values of quinidine in rat small intestines were obtained as 0.280 x 10-5 and 0.835 x 10-5 µmol/min/cm2 in the jejunum and ileum, respectively, and the results are summarized in Table 2, where the Vmax value is expressed as a unit of micromoles per minute per 10 cm of gut.
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TABLE 2 Comparison between predicted and experimental kinetic parameters [Km(app)/Vmax] for P-gp-mediated transport of quinidine, verapamil, and vinblastine
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For the Km(app) value, passive diffusivity of drugs to the vicinity of the drug binding site of P-gp affects the relation between the Km(app) and P-gp expression levels because Km(app) is a hybrid parameter of real affinity (Km) and the diffusive process to the drug binding site of P-gp. To observe differences in diffusivity between cell monolayers and the rat small intestine, passive permeability of drugs to rat small intestines were measured by adding a P-gp inhibitor (alprenolol) to the perfusion medium. In Table 1, passive permeability (Papp, PD) of quinidine to rat jejunum and ileum was compared with that to Caco-2 monolayers. Quinidine showed 3.5 to 4.0 times higher Papp, PD to rat small intestines than to Caco-2 monolayers. In the process of estimating the Km(app) value in rat intestines from the relation between Km(app) in cell monolayers and P-gp expression level, the difference in passive permeability was used as the scaling factor by dividing the slope in Fig. 2B by the ratio of Papp, PD [Papp, PD(rat)/Papp, PD(Caco-2)] (Fig. 4B). This process of in vitro-in vivo scaling is reasonable because, for example, 4 times higher diffusivity has the same effect on Cbind with the condition that Ca increased 4 times (if diffusivity is the same). In Fig. 4B (Y = 1.21 x 10-2 X + 0.584 in jejunum and Y = 1.10 x 10-2 X + 0.584 in ileum), Km(app) values of quinidine in the rat jejunum and ileum were estimated to be 1.53 and 3.13 µM, respectively (Table 2).
For verapamil and vinblastine, the same procedures were used to estimate Vmax and Km(app) values in rat intestines. The results are summarized in Table 2.
Simulation of the Concentration-Dependent Permeability of P-gp Substrate Drugs in Rat Intestinal Membrane. By incorporating Vmax and Km(app) values of quinidine estimated in Fig. 4, A and B, and Papp, PD measured by the single-pass perfusion method in Table 1 to eq. 2, the concentration-dependent permeability of quinidine in the rat jejunum and ileum was simulated (Fig. 5, A and B, ——). To validate the accuracy of the simulation, the permeability of quinidine in rat small intestines was measured by in situ single-pass perfusion methods with different luminal concentrations (Fig. 5, A and B, 
). As shown in Fig. 5, A and B, simulated permeability in the rat jejunum and ileum coincided well with the permeability measured by the in situ single-pass perfusion method. In Table 2, predicted and experimentally obtained parameters, Vmax and Km(app) values, are summarized for P-gp-mediated transport of quinidine. These parameters were found to differ slightly between predicted and experimental values.
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FIG. 5. Simulation of concentration-dependent permeability of three P-gp substrates, quinidine, verapamil, and vinblastine, in rat small intestines (jejunum and ileum). Papp of quinidine (A and B), verapamil (C and D), and vinblastine (E and F) to rat small intestines in two regions (A, C, and E, jejunum; B, D, and F, ileum) was simulated by using estimated Km(app) and Vmax values and measured Papp, PD (——). Experimental Papp to rat small intestines was obtained by the in situ single-pass perfusion method (). Data are presented as means ± S.E. (n = 3).
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The same procedures were used to simulate the concentration-dependent permeability of verapamil and vinblastine in rat small intestines (Fig. 5, C–F; Table 2). It was confirmed that the simulated permeability of verapamil and vinblastine in the rat jejunum and ileum also coincided well with the permeability obtained by the in situ single-pass perfusion method. The permeability of vinblastine showed strong dependence on its luminal concentration, whereas that of verapamil was rather insensitive to the change in the concentration (Fig. 5, C–F).
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Discussion
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In a previous report, we proposed a new method to simulate the concentration-dependent permeability of P-gp substrate drugs in the human intestine from in vitro data of cultured cell monolayers such as Caco-2 cells (Shirasaka et al., 2008). In this study, the appropriateness of our method was assessed by simulating the permeability of three P-gp substrates to the rat small intestine. Although the fact that permeability of P-gp substrates to the rat small intestine was predicted from in vitro study using cell lines expressing human P-gp may come under intense scrutiny, several reports have shown the good correlation between the functional activity of human MDR1 and that of rat mdr1a/1b. Takeuchi et al. (2006) established the LLC-PK1 cell lines, which stably express various MDR1 (derived from human, monkey, canine, rat, and mouse) to investigate species differences in P-gp-mediated efflux activity. When the permeability ratios (BL to AP/AP to BL) of P-gp substrate drugs were compared among these cell lines, a good correlation was observed between human and rat MDR1 (both MDR1a and 1b). In particular, three P-gp substrates, quinidine, verapamil and vinblastine, which were used in this study, contributed to a good correlation. By using the series of same cell lines, Katoh et al. (2006) examined the species differences in maximal activity (Vmax/ Km) of P-gp to transport its substrate drugs and clarified the fact that maximal activity of rat MDR1b correlated well with that of human MDR1. In addition, the apparent Km value of quinidine to rat MDR1a was reported to be approximately 5 µM (Müller et al., 1994) and corresponded well with that of human MDR1 in other reports [5.42 µM by Adachi et al. (2001) and 7.88 µM by Shirasaka et al. (2006b)]. On the basis of these facts, it is possible to validate our method by simulating the concentration-dependent permeability of three P-gp substrate drugs to rat small intestine and then by comparing the results of simulation with the experimentally measured permeability of those drugs.
The expression level of P-gp in the rat ileum was revealed to be approximately 3-fold higher than that in the jejunum (Fig. 3, A and B), consistent with recent reports. Cao et al. (2005) reported that P-gp expression gradually increased along the rat GI tract from the duodenum to the colon and was 2.7-fold higher in the ileum than in the jejunum. Tian et al. (2002) also clarified that P-gp expression in the ileum was 2.31-fold higher than that in the jejunum.
By incorporating the expression level of P-gp in the rat jejunum and ileum into the in vitro correlation lines in Fig. 2, both Vmax and Km(app) values of three P-gp substrates in rat small intestines were estimated. It has already been demonstrated that the Km(app) value increases depending on the P-gp expression level because the degree of P-gp activity affects Cbind (Shirasaka et al., 2008). In addition, it is reasonable to consider that the Km(app) value decreases with increasing Papp, PD because the passive diffusivity of drugs to the vicinity of the drug-binding site of P-gp affects Cbind, thus affecting the relation between Km(app) and P-gp expression levels. In this study, to estimate the Km(app) value in rat small intestines, differences in the diffusivity between cell monolayers and rat small intestinal epithelium should be taken into account. To estimate the Km(app) value in rat small intestines, the relation to the P-gp expression level in Fig. 2B was corrected by dividing the slope by the ratio of passive permeability [Papp, PD(rat)/Papp, PD(Caco-2)] in Table 1 (Fig. 4B). Although the differences in the lipid composition of the cellular membrane between Caco-2 cells and rat enterocytes are not clear, it is reasonable to consider that these epithelial membranes showed almost the same passive diffusivity to small and lipophilic molecules such as the drugs used in this study; therefore, the differences in passive permeability between Caco-2 cell monolayers and rat small intestines shown in Table 1 might correspond to differences in the effective surface area of drug diffusion caused by villous structures of the intestinal membrane. Our method to correct of the relation between P-gp expression levels and Km(app) by the passive permeability of drugs enables the comparison of drug movement in the same area of the membrane (per cell).
By incorporating these parameters of rat intestinal P-gp flux and measured Papp, PD into eq. 2, the concentration-dependent permeability of three drugs in the rat jejunum and ileum was simulated (Fig. 5, A–F, ——). As a consequence, it was confirmed that the simulated permeability of all three drugs in the rat jejunum and ileum corresponded well with permeability measured by the in situ single-pass perfusion method, suggesting that our procedures to simulate the permeability of P-gp substrate drugs in vivo were successful (Fig. 5, A–F, ); however, in the ileum, simulated permeability of quinidine in the lower concentration range deviated slightly from the measured permeability. One of the plausible explanations for this inconsistency is that, because several cytochrome P-450 enzymes were reported to be expressed in the intestinal epithelial layer, cytochrome P-450-mediated metabolism of drugs during the intestinal absorption process might affect the permeability measured by the in situ single-pass perfusion method (Ching et al., 1995; Tracy et al., 1999; Galetin and Houston, 2006). In addition, the effects of other ABC transporters, such as breast cancer resistance protein and multidrug resistance-associated protein 2, are possible factors in the deviation (Englund et al., 2006; Han and Sugiyama, 2006). Han and Sugiyama (2006) revealed that the expression level of breast cancer resistance protein in the ileum was about twice as high as that in the duodenum and jejunum.
The present study indicated that the effect of P-gp on intestinal absorption of quinidine and vinblastine was significantly greater in the ileum than in the jejunum. This fact implies that regional differences in the expression level of P-gp affect the intestinal absorption of P-gp substrate drugs. However, in the case of verapamil, the impact of P-gp on its absorption in both jejunum and ileum was almost negligible. This finding might be explained by its high permeability (high Papp, PD), high affinity to P-gp (low Km), and/or low Vmax compared with other drugs, as shown in Fig. 2B (Borgnia et al., 1996; Adachi et al., 2001; Troutman and Thakker, 2003; Shirasaka et al., 2006b). Ogihara et al. (2006) investigated the influence of P-gp on the intestinal absorption of verapamil in each intestinal segment in wild-type and mdr1a/1b gene-deficient mice, whereas no significant effects of P-gp on the absorption of verapamil were observed in all intestinal segments. These results also suggested that the effect of P-gp on in vivo intestinal absorption of its substrate drugs varied widely among drugs.
In this study, rat intestinal permeability of P-gp substrate drugs were simulated from both in vitro and in situ data. Because the ratios of passive permeability [Papp, PD(rat)/Papp, PD(Caco-2)] in Table 1 were in a considerably tight range (3.59–14.3), it might be possible to evaluate Papp, PD(rat) from only the in vitro study, if the relation of passive permeability in rat intestine and in Caco-2 monolayers is analyzed precisely. This is the issue in a future project to develop a method for simulating the concentration-dependent permeability of P-gp substrate drugs to animal and also to human small intestine by knowing only the intestinal P-gp expression levels.
In conclusion, in this study, the concentration-dependent permeability of P-gp substrate drugs to the intestinal membrane was successfully simulated from the in vitro study with a cell culture system. This method enables quantitative evaluation of the effect of P-gp on the in vivo oral absorption of its substrate drugs. Regional differences in the intestinal absorption of P-gp substrate drugs can be simulated from the differences in P-gp expression levels. Because the expression level of P-gp in the human intestinal membrane can be quantified by using intestinal tissue samples obtained by biopsy, in the near future, a study of human intestine will be undertaken and presented in another report.
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Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.107.020040.
ABBREVIATIONS: P-gp, P-glycoprotein; ABC, ATP-binding cassette; MDR/mdr, multidrug resistance; AP, apical; BL, basal; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Address correspondence to: Dr. Shinji Yamashita, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan. E-mail: shinji{at}pharm.setsunan.ac.jp
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References
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Adachi Y, Suzuki H, and Sugiyama Y (2001) Comparative studies on in vitro methods for evaluating in vivo function of MDR1 P-glycoprotein. Pharm Res 18: 1660-1668.[CrossRef][Medline]
Borgnia MJ, Eytan GD, and Assaraf YG (1996) Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity. J Biol Chem 271: 3163-3171.[Abstract/Free Full Text]
Cao X, Yu LX, Barbaciru C, Landowski CP, Shin HC, Gibbs S, Miller HA, Amidon GL, and Sun D (2005) Permeability dominates in vivo intestinal absorption of P-gp substrate with high solubility and high permeability. Mol Pharm 2: 329-340.[CrossRef][Medline]
Ching MS, Blake CL, Ghabrial H, Ellis SW, Lennard MS, Tucker GT, and Smallwood RA (1995) Potent inhibition of yeast-expressed CYP2D6 by dihydroquinidine, quinidine, and its metabolites. Biochem Pharmacol 50: 833-837.[CrossRef][Medline]
Englund G, Rorsman F, Ronnblom A, Karlbom U, Lazorova L, Grasjo J, Kindmark A, and Artursson P (2006) Regional levels of drug transporters along the human intestinal tract: co-expression of ABC and SLC transporters and comparison with Caco-2 cells. Eur J Pharm Sci 29: 269-277.[CrossRef][Medline]
Fagerholm U, Lindahl A, and Lennernäs H (1997) Regional intestinal permeability in rats of compounds with different physicochemical properties and transport mechanisms. J Pharm Pharmacol 49: 687-690.[Medline]
Galetin A and Houston JB (2006) Intestinal and hepatic metabolic activity of five cytochrome P450 enzymes: impact on prediction of first-pass metabolism. J Pharmacol Exp Ther 318: 1220-1229.[Abstract/Free Full Text]
Han Y and Sugiyama Y (2006) Expression and regulation of breast cancer resistance protein and multidrug resistance associated protein 2 in BALB/c mice. Biol Pharm Bull 29: 1032-1035.[CrossRef][Medline]
Hunter J and Hirst BH (1997) Intestinal secretion of drugs: the role of P-glycoprotein and related drug efflux systems in limiting oral drug absorption. Adv Drug Deliv Rev 25: 129-157.[CrossRef]
Jonker JW, Wagenaar E, van Deemter L, Gottschlich R, Bender HM, Dasenbrock J, and Schinkel AH (1999) Role of blood-brain barrier P-glycoprotein in limiting brain accumulation and sedative side-effects of asimadoline, a peripherally acting analgesic drug. Br J Pharmacol 127: 43-50.[CrossRef][Medline]
Katai K, Miyamoto K, Kishida S, Segawa H, Nii T, Tanaka H, Tani Y, Arai H, Tatsumi S, Morita K, et al. (1999) Regulation of intestinal Na+-dependent phosphate co-transporters by a low-phosphate diet and 1,25-dihydroxyvitamin D3. Biochem J 343: 705-712.[CrossRef][Medline]
Katoh M, Suzuyama N, Takeuchi T, Yoshitomi S, Asahi S, and Yokoi T (2006) Kinetic analyses for species differences in P-glycoprotein-mediated drug transport. J Pharm Sci 95: 2673-2683.[CrossRef][Medline]
Kim RB, Fromm MF, Wandel C, Leake B, Wood AJ, Roden DM, and Wilkinson GR (1998) The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Invest 101: 289-294.[Medline]
Loo TW and Clarke DM (1999) Molecular dissection of the human multidrug resistance P-glycoprotein. Biochem Cell Biol 77: 11-23.[CrossRef][Medline]
Meijer DK, Smit JW, and Muller M (1997) Hepatobiliary elimination of cationic drugs: the role of P-glycoprotein and other ATP-dependent transporters. Adv Drug Deliv Rev 25: 159-200.[CrossRef]
Minami H, Kim JR, Tada K, Takahashi F, Miyamoto K, Nakabou Y, Sakai K, and Hagihira H (1993) Inhibition of glucose absorption by phlorizin affects intestinal functions in rats. Gastroenterology 105: 692-697.[Medline]
Moore AC, Gugger ET, and Erdman JW (1996) Brush border membrane vesicles from rats and gerbils can be utilized to evaluate the intestinal uptake of all-trans and 9-cis β-carotene. J Nutr 126: 2904-2912.[Abstract/Free Full Text]
Müller M, Mayer R, Hero U, and Keppler D (1994) ATP-dependent transport of amphiphilic cations across the hepatocyte canalicular membrane mediated by mdr1 P-glycoprotein. FEBS Lett 343: 168-172.[CrossRef][Medline]
Ogihara T, Kamiya M, Ozawa M, Fujita T, Yamamoto A, Yamashita S, Ohnishi S, and Isomura Y (2006) What kinds of substrates show P-glycoprotein-dependent intestinal absorption? Comparison of verapamil with vinblastine. Drug Metab Pharmacokinet 21: 238-244.[CrossRef][Medline]
Polli JW, Jarrett JL, Studenberg SD, Humphreys JE, Dennis SW, Brouwer KR, and Woolley JL (1999) Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor. Pharmacol Res 16: 1206-1212.[CrossRef]
Polli JW, Wring SA, Humphreys JE, Huang L, Morgan JB, Webster LO, and Serabjit-Singh CS (2001) Rational use of in vitro P-glycoprotein assays in drug discovery. J Pharmacol Exp Ther 299: 620-628.[Abstract/Free Full Text]
Shirasaka Y, Kawasaki M, Sakane T, Omatsu H, Moriya Y, Nakamura T, Sakaeda T, Okumura K, Langguth P, and Yamashita S (2006a) Induction of human P-glycoprotein in Caco-2 cells: development of a highly sensitive assay system for P-glycoprotein-mediated drug transport. Drug Metab Pharmacokinet 21: 414-423.[CrossRef][Medline]
Shirasaka Y, Onishi Y, Sakurai A, Nakagawa H, Ishikawa T, and Yamashita S (2006b) Evaluation of human P-glycoprotein (MDR1/ABCB1) ATPase activity assay method by comparing with in vitro transport measurements: Michaelis-Menten kinetic analysis to estimate the affinity of P-glycoprotein to drugs. Biol Pharm Bull 29: 2465-2471.[CrossRef][Medline]
Shirasaka Y, Sakane T, and Yamashita S (2008) Effect of P-glycoprotein expression levels on the concentration-dependent permeability of drugs to the cell membrane. J Pharm Sci 97: 553-565.[CrossRef][Medline]
Susanto M and Benet LZ (2002) Can the enhanced renal clearance of antibiotics in cystic fibrosis patients be explained by P-glycoprotein transport? Pharmacol Res 19: 457-462.[CrossRef]
Takeuchi T, Yoshitomi S, Higuchi T, Ikemoto K, Niwa S, Ebihara T, Katoh M, Yokoi T, and Asahi S (2006) Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1. Pharm Res 23: 1460-1472.[CrossRef][Medline]
Tian R, Koyabu N, Takanaga H, Matsuo H, Ohtani H, and Sawada Y (2002) Effects of grapefruit juice and orange juice on the intestinal efflux of P-glycoprotein substrates. Pharm Res 19: 802-809.[CrossRef][Medline]
Tracy TS, Korzekwa KR, Gonzalez FJ, and Wainer IW (1999) Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil. Br J Clin Pharmacol 47: 545-552.[CrossRef][Medline]
Troutman MD and Thakker DR (2003) Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium. Pharm Res 20: 1210-1224.[CrossRef][Medline]
Yamaoka K, Tanigawara Y, Nakagawa T, and Uno T (1981) A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn 4: 879-885.[Medline]
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Abstract
Materials and Methods
, quinidine; 
, verapamil; 
, vinblastine. Data were derived from Shirasaka et al. (2008