Advertisement
Research ArticleArticle

Transport of Rhodamine 123, a P-Glycoprotein Substrate, across Rat Intestine and Caco-2 Cell Monolayers in the Presence of Cytochrome P-450 3A-Related Compounds

Ryoko Yumoto, Teruo Murakami, Yuko Nakamoto, Risa Hasegawa, Junya Nagai and Mikihisa Takano
Journal of Pharmacology and Experimental Therapeutics April 1999, 289 (1) 149-155;

Abstract

Effects of cytochrome P-450 3A- and P-glycoprotein (P-gp)-related compounds, erythromycin, midazolam, ketoconazole, verapamil, and quinidine, on transport of rhodamine 123 (Rho-123), a P-gp substrate, were studied in rat intestine and in Caco-2 cells. Ileum was mainly used in rat studies because this segment showed greater P-gp-mediated Rho-123 transport. In an in vitro everted rat ileum, all the compounds examined significantly inhibited the transport of Rho-123 from serosal to mucosal surfaces across the intestine, with different inhibitory potencies among these compounds. In an in vivo rat study, the exsorption of Rho-123 from blood to the intestinal lumen, which was evaluated as exsorption clearance of Rho-123 under a steady-state plasma concentration of Rho-123, was also inhibited when these compounds were added to the intestinal lumen. Similarly, transepithelial transport of Rho-123 from the basolateral to apical side across Caco-2 cell monolayers was inhibited by these compounds. A linear relationship was observed in their inhibitory potencies on Rho-123 transport between in vitro and in vivo studies using rat ileum and between studies with rat ileum and Caco-2 cells. P-gp-mediated transport across the intestine was found to be inhibited not only by P-gp-related but also by all the cytochrome P-450 3A-related compounds examined. Within experimental error, the relative inhibitory potencies were the same between the studies with rat ileum (in vivo, in vitro) and those with Caco-2 cells. Thus, it is suggested that the function of P-gp and its sensitivity to these drugs may be similar in rat intestine and Caco-2 cells.

It has been demonstrated that the intestinal P-glycoprotein (P-gp), an ATP-dependent multidrug efflux pump, can be an active secretion system or an absorption barrier by transporting some drugs from intestinal cells into the lumen (Terao et al., 1996; Tsuji and Tamai, 1996). Substrates transported by P-gp include a variety of structurally and pharmacologically unrelated, hydrophobic compounds such as some anticancer agents, steroid hormones, calcium channel blockers, immunosuppressing agents, β-blockers, and so on (Hunter et al., 1993; Wils et al., 1994; Terao et al., 1996). P-gp is also expressed in other normal human and rodent tissues, including the adrenal gland, kidney, liver, colon, brain, testis, and eye (Thiebaut et al., 1987;Holash and Stewart, 1993). In these tissues, P-gp is reported to prevent the accumulation of xenobiotics by active efflux.

The P-gp-mediated transport of a substrate is modified not only by other P-gp substrates but also by cytochrome P-450 (CYP) 3A-related compounds because there are some overlapping substrate specificities between CYP3A and P-gp (Wacher et al., 1995; Schuetz et al., 1996a, b). CYP3A enzymes occur in hepatocytes and intestinal epithelial cells (Paine et al., 1996; Thummel et al., 1996) and metabolize many clinically important drugs, such as some immunosuppressing agents, antibiotics, calcium channel blockers, and anticancer agents (Porter and Coon, 1991; Guengerich, 1992; Lown et al., 1994). Some of these CYP3A substrates are recognized as substrates or inhibitors of P-gp. The CYP3A-mediated metabolism is also readily inhibited by the presence of CYP3A substrates and inhibitors, such as erythromycin, ketoconazole, itraconazole, cimetidine, and grapefruit juice, resulting in increased oral bioavailability and changes in the pharmacokinetics of the substrates (Olkkola et al., 1994; Wrighton and Ring, 1994;Kupferschmidt et al., 1995; Ameer and Weintraub, 1997). These complicated drug-drug interactions between P-gp substrates or between a P-gp substrate and a CYP3A substrate are receiving considerable attention in clinical pharmacotherapeutics.

Prediction of these drug-drug interactions preclinically would be valuable information for clinical pharmacotherapeutics. However, there is only a limited number of studies on the in vivo function of P-gp in normal tissues such as the intestine and on the relationship between in vivo and in vitro P-gp function under various experimental conditions. In the present study, we examined and compared the effects of CYP3A- and P-gp-related compounds, erythromycin, midazolam, ketoconazole, verapamil, and quinidine, on the transport of rhodamine 123 (Rho-123), a P-gp substrate, in rat intestine (in vitro and in vivo) and in Caco-2 cell monolayers. Presently, midazolam is categorized as a CYP3A substrate and others are inhibitors/substrates of both CYP3A and P-gp. Rho-123 has been used as a marker to study the function of P-gp in various multidrug-resistant cells and various normal tissues including the intestine (Hsing et al., 1992; Lee et al., 1994). Caco-2, a human colonic adenocarcinoma cell line expressing various functions of differentiated intestinal epithelial cells, is a useful in vitro system for studying the function of P-gp in the intestine (Hunter et al., 1993; Wils et al., 1994; Yee, 1997).

Experimental Procedures

Materials.

Rho-123 was obtained from Kanto Chemical Co. (Tokyo, Japan). Verapamil hydrochloride and quinidine sulfate dihydrate were from Wako Pure Chemicals (Osaka, Japan). Erythromycin was from Merck (Darmstadt, Germany). Midazolam was from Nippon Roche (Tokyo, Japan). Ketoconazole was from Janssen Pharmaceutica N.V. (Beerse, Belgium). Cell culture medium and reagents were from Gibco Laboratories (Life Technologies Inc., Grand Island, NY). All other chemicals used were of the highest purity available.

In Vitro Transport Across Everted Rat Intestine.

Experiments with animals were performed in accordance with the “Guide for Animal Experimentation” from Hiroshima University and the Committee of Research Facilities for Laboratory Animal Sciences, Hiroshima University School of Medicine. Male Wistar rats weighing 220 to 300 g were fasted overnight with free access to water before the experiments. The whole small intestine was flushed with 50 ml of ice-cold saline with the animal under anesthesia with pentobarbital (30 mg/kg i.p. injection). The rat was exsanguinated, and the small intestine isolated was divided into five segments of an equal length. Each segment was everted, and a 10-cm-long everted sac was prepared. Rho-123 was dissolved at a concentration of 5 μM in pH 7.4 isotonic Dulbecco’s PBS (D-PBS) containing 25 mM glucose and different concentrations of DMSO. The Rho-123 solution (1 ml) was introduced into the everted sac (serosal side), and both ends of the sac were ligated tightly. The sac containing Rho-123 solution was immersed into 40 ml of D-PBS containing 25 mM glucose and the same concentration of DMSO as that in the serosal side. The medium was prewarmed at 37°C and preoxygenated with 5% CO2/95% O2 for 15 min. Under bubbling with a CO2/O2 mixture gas, the transport of Rho-123 from serosal to mucosal surfaces across the intestine was measured by sampling the mucosal medium periodically for 90 min. In an inhibition study, each inhibitor (P-gp- and/or CYP3A-related compounds) was added to the mucosal medium to give a designated final concentration (25–200 μM). The concentrations of DMSO inside and outside the sac were 0% for verapamil and quinidine, 0.2% for erythromycin, 0.5% for midazolam, and 4% for ketoconazole. Using these media, the transport of Rho-123 in the absence (control) or presence of an inhibitor was measured.

In Vivo Exsorption Across Rat Ileum.

Rats fasted overnight were anesthetized with pentobarbital (30 mg/kg i.p. injection) and affixed supine on a surface kept at 37°C to maintain their body temperature around 36°C. Cannulation (polyethylene tubing, PE-50) was made at a femoral vein for the administration of drug and a femoral artery for the sampling of blood, respectively. Physiological saline (1 ml) was injected i.v., followed by a constant-rate infusion at a rate of 2 ml/h/rat, via the cannula inserted at the femoral vein, as a stabilization period of about 30 min. During the stabilization period, the urinary bladder and bile duct were cannulated to collect the urine and bile samples, respectively. Also, the lumen of the ileum (20 cm long from the ileocecum) was flushed with 20 ml of saline prewarmed at 37°C, and the proximal end of the lumen was catheterized with an in-flow silicone cannula (outer diameter, 5 mm; inner diameter, 3 mm), which was connected to the perfusion system. The distal end of the ileum was also catheterized with an out-flow silicone cannula to collect intestinal effluents serially. Rho-123 was dissolved at a concentration of 100 μM in D-PBS containing 5% mannitol, in which mannitol was used to maintain sufficient and constant urinary flow rate. After the stabilization period, this Rho-123 solution was injected i.v. as a bolus with a volume of 4.36 ml/kg, followed by a constant infusion at a rate of 2 ml/h to attain a steady-state plasma concentration of Rho-123 at approximately 0.2 μM. At this time, the single perfusion of D-PBS containing 25 mM glucose into the ileal lumen was also started at a rate of 1 ml/min. After a steady-state plasma Rho-123 concentration was achieved (50 min after the initiation of Rho-123 infusion), the intestinal effluent, blood, urine, and bile samples were collected at designated time intervals for 40 min as a control phase. Then, the perfusate was changed to the buffer containing an inhibitor (25–100 μM). After a single perfusion for 20 min for stabilization, these biological fluids were further collected periodically for an additional 50 min as an inhibition phase. The perfusate used before and after the addition of an inhibitor contained 0% DMSO for verapamil and quinidine, 0.2% DMSO for erythromycin, 0.5% DMSO for midazolam, and 4% DMSO for ketoconazole. The exsorption from blood to ileal lumen and the biliary and urinary excretions of Rho-123 were expressed as an exsorption clearance (CLexp, ml/min/20 cm ileum) and excretion clearances (CLbile, CLurine, ml/min), respectively. For this calculation, the intestinal effluent, bile, and urine samples were collected every 10 min, and the sampling of blood was made at the intermediate time of each biological fluid collection. Total plasma clearance (CLtotal, ml/min) of Rho-123 was estimated by dividing the infusion rate with a steady-state plasma concentration.

In Vitro Transepithelial Transport Across Caco-2 Cell Monolayers.

The transepithelial transport of Rho-123 across cell monolayers, which were cultured in Transwell chamber (Costar, Cambridge, MA), in the absence or presence of an inhibitor was determined in the similar manner as reported previously (Nagai et al., 1995). Briefly, Caco-2 cells between passages 57 and 77 were cultured in a chamber for 16 to 21 days after seeding. Transepithelial electric resistance (TEER) of Caco-2 cell monolayers was monitored before transport studies using a Millicell ERS testing device (Millipore, Bedford, MA), and monolayers with TEER of more than 200 ohm·cm2 were used for the transport studies. Rho-123 was dissolved at a concentration of 5 μM in D-PBS containing 25 mM HEPES, 25 mM glucose, and various concentrations of DMSO (pH 7.4). The drug solution was placed either on the apical (1.5 ml) or basolateral (2.6 ml) side, and the other side was filled with buffer solution containing the same concentration of DMSO. For inhibition study, each inhibitor was added to both sides to give a designated final concentration. The concentrations of DMSO in transport media used were 0% for verapamil and quinidine, 0.05% for erythromycin, 0.1% for midazolam, and 1% for ketoconazole. The transepithelial transport of Rho-123 in the presence or absence of an inhibitor was measured at 37°C periodically for 90 min.

Analysis.

Concentrations of Rho-123 in various biological samples obtained from in vivo transport study were determined by HPLC with an instrument equipped with a fluorometric detector using a column of TSKgel ODS-80TM (Tosoh, Tokyo, Japan). Mobile phase used was a mixture of acetonitrile and 1% acetic acid (40:60, v/v) at a flow rate of 1 ml/min. Detection was made at wavelengths of 485 nm for excitation and 546 nm for emission. Concentrations of Rho-123 in the transport media obtained from in vitro transport studies using everted sac and Caco-2 cells were determined fluorometrically with Hitachi fluorescence spectrophotometer F-3000 (Tokyo, Japan) because no metabolism of Rho-123 was observed under these experimental conditions.

Differences among group mean values were assessed by one-way ANOVA and/or Student’s t test. A difference of P< .05 was considered statistically significant.

Results

Regional Differences in Rho-123 Transport Across Everted Rat Intestine In Vitro.

The transport of Rho-123 from serosal to mucosal surfaces across everted rat intestine in the absence or presence of verapamil (300 μM), a potent P-gp inhibitor, was determined at different anatomic regions of rat small intestine. As typical examples, the time courses of Rho-123 transport across the everted duodenum and lower ileum are shown in Fig.1. The transport of Rho-123 increased with time in a zero-order fashion after a lag time of approximately 10 min in both segments, and a greater transport was observed in the ileum. In both segments, the transport was significantly inhibited by verapamil. Thus, the expression of P-gp in rat intestine was functionally confirmed. The transport rate was calculated from the slope of cumulative Rho-123 amount with time (Fig.2). Verapamil decreased the transport rate of Rho-123 to nearly the same extent in all regions. The difference in the transport rate between the absence and presence of verapamil can be regarded as P-gp-mediated transport of Rho-123. Thus, a significant regional variation was observed in functional P-gp along the length of rat small intestine.

Figure 1
Figure 1

Transport of Rho-123 from serosal to mucosal surfaces across the everted duodenum (left) and lower ileum (right) in the absence (○) or presence (●) of verapamil at a concentration of 300 μM. The concentration of Rho-123 in the serosal side was 5 μM. Each value represents the mean ± S.E.M. Solid lines represent the regression line, respectively: ○, left, y = 0.00610x − 0.0964 (r = 0.992,n = 3); ●, left, y = 0.00297x − 0.0271 (r = 0.999,n = 3); ○, right, y = 0.0120x − 0.151 (r = 0.999,n = 5); and ●, right, y = 0.00452x − 0.0402 (r = 0.999,n = 3), where x and ydenote the time (min) and transported amount of Rho-123 (nmol/10 cm-sac), respectively. *Significantly different from that in the absence of verapamil at a level of P < .05.

Figure 2
Figure 2

Regional difference in Rho-123 transport from serosal to mucosal surfaces across the everted intestine in the absence (open column) or presence (hatched column) of verapamil at a concentration of 300 μM. The concentration of Rho-123 in the serosal side was 5 μM. Each value represents the mean ± S.E.M. of three to six trials. *Significantly different from that in the absence of verapamil in each segment at a level of P < .05. †Significantly different from that in the absence of verapamil across rat duodenum at a level of P < .05.

Effects of CYP3A-Related Compounds on Rho-123 Transport Across Everted Rat Ileum In Vitro.

As a typical example, the effect of midazolam, a CYP3A substrate, on Rho-123 transport across the everted rat ileum is shown in Fig. 3. The transport of Rho-123 from serosal to mucosal surfaces was inhibited by midazolam depending on its concentration in the mucosal medium. The inhibitory potencies of various inhibitors on Rho-123 transport were studied, and the results were expressed as the percentage of Rho-123 transport in the presence of an inhibitor relative to that in the absence of the inhibitor (Fig. 4). In these studies, various concentrations of DMSO (0–4%), which was used to increase the solubility of inhibitors in the mucosal and serosal media, were used among different inhibitors. However, no significant effect was found of DMSO on Rho-123 transport in the absence of inhibitors (data not shown). The transport of Rho-123 was significantly decreased in a dose-dependent fashion by all the CYP3A-related compounds examined.

Figure 3
Figure 3

Transport of Rho-123 from serosal to mucosal surfaces across the everted ileum in the absence (○) or presence (● and ▴) of midazolam at a concentration of 100 μM (●) or 200 μM (▴). The concentration of Rho-123 in the serosal side was 5 μM. Each value represents the mean ± S.E.M. The solid lines represent the regression line, respectively; ○, y = 0.0156x − 0.144 (r = 0.997,n = 5); ●, y = 0.00812x − 0.118 (r = 0.995,n = 4); and ▴, y = 0.00448x − 0.0642 (r = 0.991,n = 3), where x and ydenote the time (min) and transported amount of Rho-123 (nmol/10 cm-sac), respectively. *Significantly different from that in the absence of midazolam at a level of P < .05. †Significantly different from that in the presence of 100 μM midazolam at a level of P < .05.

Figure 4
Figure 4

Concentration-dependent inhibitory potencies of ketoconazole (○), midazolam (▵), erythromycin (■), quinidine (▴), and verapamil (●) on Rho-123 transport across the everted ileum. The concentration of Rho-123 in the serosal side was 5 μM. Each value represents the mean ± S.E.M. of three to six trials.

Effects of CYP3A-Related Compounds on Rho-123 Exsorption Across Rat Ileum In Vivo.

Under a steady-state plasma concentration of Rho-123, ileum was perfused with D-PBS alone, followed by D-PBS containing an inhibitor in a single perfusion, to evaluate the effect of an inhibitor on exsorption from blood to the lumen and on biliary and urinary excretions of Rho-123. As a typical example, the effect of midazolam is shown in Fig. 5, in which each parameter is expressed as a clearance value. In the absence of an inhibitor in the lumen, the CLtotal, CLexp, CLurine, and CLbile of Rho-123 remained constant during the experimental period. By perfusing the ileum with midazolam, the plasma level of Rho-123 increased a little, and the CLtotal of Rho-123 was decreased proportionally. The decreased CLtotal of Rho-123 would be due, at least in part, to the decreased CLexp, CLurine, and CLbile, as shown in Fig. 5. Similar inhibitory effects on Rho-123 clearances were observed in all compounds examined. The effects on CLexp of Rho-123 are summarized in Fig.6. As was the case for the in vitro everted sac study (Fig. 4), all compounds examined significantly reduced the in vivo CLexp value of Rho-123. The relationship was examined of their inhibitory potencies on Rho-123 transport between in vivo and in vitro studies. As shown in Fig.7, a linear relationship was observed between them (r = 0.947, P < .01).

Figure 5
Figure 5

Total plasma (CLtotal), exsorption from blood to lumen (CLexp), urinary excretion (CLurine), and biliary excretion (CLbile) clearances of Rho-123 in the absence (○) or presence (●) of luminal midazolam under a steady-state plasma concentration of Rho-123 in rats. Arrows denote the initiation of midazolam perfusion into the jejunal lumen at a concentration of 100 μM by a single perfusion at a rate of 1 ml/min. The plasma concentration of Rho-123 before perfusion of midazolam was approximately 0.2 μM. The solid line represents the mean of each clearance of three trials.

Figure 6
Figure 6

Concentration-dependent inhibitory potencies of ketoconazole (○), midazolam (▵), erythromycin (■), quinidine (▴), and verapamil (●) on Rho-123 exsorption clearance across rat ileum in vivo under a steady-state plasma concentration of Rho-123. Each inhibitor was perfused into a 20-cm-long ileum at different concentrations by a single perfusion at a rate of 1 ml/min. The plasma concentration of Rho-123 before perfusion of an inhibitor was approximately 0.2 μM. Each value represents the mean ± S.E.M. of three or four trials.

Figure 7
Figure 7

In vitro versus in vivo inhibitory potencies of various compounds on Rho-123 transport across rat ileum. KTZ, ketoconazole; MDZ, midazolam; EM, erythromycin; QD, quinidine; VRP, verapamil. Each value represents the mean ± S.E.M. of three to five trials. The solid line represents the regression line:y = 0.816x + 19.3 (r = 0.947, P < .01,n = 7), where x and ydenote the in vivo exsorption clearance (percent of control) and the in vitro transport of Rho-123 (percent of control), respectively.

Effects of CYP3A-Related Compounds on Rho-123 Transepithelial Transport Across Caco-2 Cell Monolayers In Vitro.

Inhibitory potencies of CYP3A-related compounds on Rho-123 transepithelial transport were evaluated using Caco-2 cells. We first confirmed that the transport of Rho-123 from the basolateral to apical side was significantly higher than that from the apical to basolateral side. In addition, the basolateral-to-apical transport of Rho-123 was reduced and the apical-to-basolateral transport was increased by verapamil and quinidine (100 μM). The transport of Rho-123 in the presence of these inhibitors was almost the same in both directions (data not shown). These data indicate that P-gp is functionally expressed in these Caco-2 cells. We then studied the effect of midazolam on Rho-123 transport (Fig. 8). Midazolam significantly decreased the basolateral-to-apical transport of Rho-123, with an increase in the apical-to-basolateral transport. All other compounds also significantly reduced the basolateral-to-apical transport of Rho-123. Their inhibitory potencies on the basolateral-to-apical transport of Rho-123 were plotted against the concentrations of each inhibitor in the transport medium (Fig.9). The plotting of their inhibitory potencies observed in rat ileum (in vivo) against those in Caco-2 cells showed a linear relationship (Fig. 10) (r = 0.895, P < .01). Similarly, a linear correlation was also observed between the studies with in vitro rat ileum and those with Caco-2 cells (r = 0.757,P < .05). The rank orders of inhibitory potencies were ketoconazole < erythromycin, midazolam, quinidine < verapamil.

Figure 8
Figure 8

Transepithelial transport of Rho-123 in the basolateral (B)-to-apical (A) direction (left) and in the apical (A)-to-basolateral (B) direction (right) across Caco-2 cell monolayers in the absence (○) or presence (● and ▴) of midazolam at a concentration of 100 μM (●) or 200 μM (▴). The concentration of Rho-123 was 5 μM. Each value represents the mean ± S.E.M. of three trials. The solid lines represent the regression line, respectively: ○, left, y = 1.648x − 13.87 (r = 0.999); ●, left, y = 0.8289x + 0.5320 (r = 0.999); ▴, left, y = 0.6448x + 0.3993 (r = 0.999); ○, right, y = 0.3837x + 1.071 (r = 0.998); ●, right, y = 0.5269x + 2.024 (r = 0.999); and ▴, right, y = 0.5238x + 2.132 (r = 0.999), where x andy denote the time (min) and transported amount of Rho-123 (pmol/cm2), respectively.

Figure 9
Figure 9

Concentration-dependent inhibitory potencies of ketoconazole (○), midazolam (▵), erythromycin (■), quinidine (▴), and verapamil (●) on Rho-123 transport in the basolateral-to-apical direction across Caco-2 cell monolayers. The concentration of Rho-123 was 5 μM. Each value represents the mean ± S.E.M. of three to five trials.

Figure 10
Figure 10

Inhibitory potencies of various compounds on Rho-123 transport in the serosal (or basolateral)-to-mucosal (or apical) direction in rat ileum (in vivo) and in Caco-2 cells. KTZ, ketoconazole; MDZ, midazolam; EM, erythromycin; QD, quinidine; VRP, verapamil. Each value represents the mean ± S.E.M. of three to six trials. The solid line represents the regression line:y = 0.594x + 26.9 (r = 0.895, P < .01,n = 9), where x and ydenote the in vitro transport across Caco-2 cell monolayers (percent of control) and in vivo exsorption clearance of Rho-123 across rat ileum (percent of control), respectively.

Discussion

Along the length of rat small intestine, the P-gp-mediated Rho-123 transport showed a regional variation, and the transport rate in the ileum was about 2.5- to 5.0-fold higher than other regions (Fig. 2). Our data are in good agreement with those of Trezise et al. (1992), in which they reported that the expression of multidrug resistance MDR1 mRNA varies in rat intestine, with moderate expression in the duodenum and the jejunum, maximal expression in the ileum, and then a decrease in expression through the proximal and distal colon. Similar results have also been reported in male CF I mice (Chianale et al., 1995). The transport rate of Rho-123 from serosal to mucosal surfaces in the ileum was double that of the transport rate measured in the jejunum. The level of mdr3 mRNA and P-gp found in the ileum was 6-fold higher than the level found in the duodenum.

The transport of Rho-123 in rat intestine was not completely inhibited even by the presence of verapamil at a concentration of 300 μM (Fig.2), although Chianale et al. (1995) reported that the transport in mice was almost completely inhibited by the presence of verapamil at a concentration of 100 μM. Data similar to ours have been reported byHsing et al. (1992) using male Sprague-Dawley rats. The mechanism for the transport of Rho-123 in the presence of such a high concentration of verapamil is not clear. However, in rat jejunum, the epithelial cell layers connected by tight junctions are relatively leaky with TEER of approximately 30 ohm·cm2 (Frömter and Diamond, 1972). Therefore, transport via the paracellular route may be involved in intestinal transport of Rho-123, which should be insensitive to verapamil.

All of the examined CYP3A-related compounds significantly reduced the exsorption of Rho-123 across the rat ileum. Similar inhibitory potencies were also observed in the jejunum in the present study (data not shown). Recently, we examined whether P-gp is involved in the transepithelial transport of midazolam, erythromycin, and ketoconazole themselves across Caco-2 cell monolayers. In that study, it was found that P-gp is involved in the transport of erythromycin, at least in part, but not of midazolam and ketoconazole (Takano et al., 1998). Thus, the interaction of such CYP3A-related compounds with P-gp can occur even if the compound is not a P-gp substrate.

In the present study, we measured the effect of various compounds on CLexp, CLurine, and CLbile of Rho-123 in vivo. However, to evaluate the inhibitory potencies of these compounds, including CYP3A substrates, on CLurine and CLbile values of Rho-123 in a quantitative manner, many other factors, such as the absorption rate and the loss of the compound itself in the intestine and/or liver by metabolism (first-pass metabolism), should also be taken into consideration. In addition, as reported by Sweatman et al. (1990), metabolites of Rho-123 such as Rho-110 (a deacylated metabolite of Rho-123) and/or its glucuronide conjugate were found in biological fluids except for luminal effluents, which makes the analysis more complex. Accordingly, in the present study, the effect of inhibitors on the CLexp value of Rho-123 alone was evaluated as a function of an inhibitor concentration. The inhibitory potencies of various compounds on Rho-123 exsorption in vivo (Fig. 6) were almost the same as those observed in an in vitro study (Fig. 3). A linear relationship between in vitro everted sac and the in vivo studies shown in Fig. 7 suggests that the P-gp-related drug-drug interactions in vivo can be predicted by in vitro everted sac studies.

Recently, we determined the inhibitory effects of erythromycin, midazolam, and ketoconazole on P-gp-mediated transepithelial transport of Rho-123 in Caco-2 cells (Takano et al., 1998). A linear relationship in the inhibitory potencies of CYP3A- and P-gp-related compounds between rat ileum (in vivo, in vitro) and Caco-2 cells (Fig. 10) suggests that the drug-drug interactions related to P-gp-mediated transport in human intestine could be predicted by in vivo or in vitro transport study using rat ileum, as well as by Caco-2 cells. Yee (1997)also suggested that Caco-2 cells can be used to predict in vivo intestinal absorption of various compounds, including P-gp substrates in humans. However, the regression line between rat ileum and Caco-2 cells was not directly proportional; it had an intercept on they-axis without passing through the origin (Fig. 10). The incomplete inhibition in rat ileum may be due, at least in part, to its leaky epithelia, in which paracellular transport of Rho-123 would occur as discussed previously. In contrast, the monolayers of Caco-2 cells used in the present study were less leaky (with TEER more than 200 ohm·cm2), and transport via a paracellular route should be almost negligible, as reported previously (Harada et al., 1997). Under such conditions, P-gp-mediated transepithelial transport of Rho-123 in Caco-2 cells can be almost completely inhibited by potent P-gp inhibitors like verapamil and quinidine.

The present study analyzed primarily the exsorption or basolateral-to-apical transport of Rho-123 in the presence of inhibitors. However, as shown in Fig. 8, these inhibitors simultaneously increased the transport in the apical-to-basolateral direction of Rho-123 by inhibiting the P-gp function. The increase in the absorption of Rho-123 in the presence of inhibitors was also observed in vivo. For example, in the presence of midazolam at a concentration of 100 μM in the perfusate, the absorption clearance of Rho-123, which was estimated by the difference in Rho-123 concentrations between perfusate and intestinal effluent in a single perfusion, increased to 0.13 ± 0.03 ml/min/20 cm, whereas essentially no absorption of Rho-123 in the absence of an inhibitor was detected. Similarly, other inhibitors such as verapamil also increased the absorption of Rho-123 (data not shown), suggesting the possible enhancement of intestinal absorption of P-gp substrates by the coadministration of CYP3A-related compounds.

In conclusion, P-gp-mediated transport across the intestine was found to be inhibited not only by P-gp-related compounds but also by the CYP3A-related compounds examined. Within experimental error, the relative inhibitory potencies of these compounds were the same between the studies with rat ileum (in vivo, in vitro) and those with Caco-2 cells. Thus, it is suggested that the function of P-gp and its sensitivity to inhibitors may be similar in rat intestine and Caco-2 cells and that these experimental models (rat intestine, Caco-2 cells) may be useful to further study the function of P-gp in human intestine.

Footnotes

  • Send reprint requests to: Mikihisa Takano, Ph.D., Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail:takanom{at}pharm.hiroshima-u.ac.jp

  • ↵1 This work was supported in part by a grant-in-aid from the Tokyo Biochemical Research Foundation.

  • Abbreviations:
    CYP
    cytochrome P-450
    P-gp
    P-glycoprotein
    Rho-123
    rhodamine 123
    TEER
    transepithelial electric resistance
    D-PBS
    Dulbecco’s PBS
    CLtotal
    total plasma clearance
    CLexp
    exsorption clearance
    CLurine
    urinary excretion clearance
    CLbile
    biliary excretion clearance
    • Received July 22, 1998.
    • Accepted November 2, 1998.

References

  1. ↵
    1. Ameer B,
    2. Weintraub RA
    (1997) Drug interactions with grapefruit juice. Clin Pharmacokinet 33:103–121.
    OpenUrlPubMed
  2. ↵
    1. Chianale J,
    2. Vollrath V,
    3. Wielandt AM,
    4. Miranda S,
    5. Gonzalez R.,
    6. Fresno AM,
    7. Quintana C,
    8. Gonzalez S,
    9. Andrade L,
    10. Guzman S
    (1995) Differences between nuclear run-off and mRNA levels for multidrug resistance gene expression in the cephalocaudal axis of the mouse intestine. Biochim Biophys Acta 1264:369–376.
    OpenUrlPubMed
  3. ↵
    1. Frömter E,
    2. Diamond J
    (1972) Route of passive ion permeation in epithelia. Nature [New Biol] 235:9–13.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Guengerich FP
    (1992) Characterization of human cytochrome P450 enzymes. FASEB J 6:745–748.
    OpenUrlAbstract
  5. ↵
    1. Harada S,
    2. Murakami T,
    3. Higashi Y,
    4. Takano M,
    5. Yata N
    (1997) Enhancing mechanism of decanoic acid for the permeation of water-soluble compounds through Caco-2 cell monolayers. Pharm Sci 3:175–178.
  6. ↵
    1. Holash JA,
    2. Stewart PA
    (1993) The relationship of astrocyte-like cells to the vessels that contribute to the blood-ocular barriers. Brain Res 629:218–224.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Hsing S,
    2. Gatmaitan Z,
    3. Arias IM
    (1992) The function of Gp170, the multidrug-resistance gene product, in the brush border of rat intestinal mucosa. Gastroenterology 102:879–885.
    OpenUrlPubMed
  8. ↵
    1. Hunter J,
    2. Hirst BH,
    3. Simmons NL
    (1993) Drug absorption limited by P-glycoprotein-mediated secretory drug transport in human intestinal epithelial Caco-2 cell layers. Pharm Res 10:743–749.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Kupferschmidt HHT,
    2. Ha HR,
    3. Ziegler WH,
    4. Meier PJ,
    5. Krähenbühl S
    (1995) Interaction between grapefruit juice and midazolam in humans. Clin Pharmacol Ther 58:20–28.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Lee JS,
    2. Paull K,
    3. Alvarez M,
    4. Hose C,
    5. Monks A,
    6. Grever M,
    7. Fojo AT,
    8. Bates SE
    (1994) Rhodamine efflux patterns predict p-glycoprotein substrates in the National Cancer Institute drug screen. Mol Pharmacol 46:627–638.
    OpenUrlAbstract
  11. ↵
    1. Lown KS,
    2. Kolars JC,
    3. Thummel KE,
    4. Barnett JL,
    5. Kunze KL,
    6. Wrighton SA,
    7. Watkins PB
    (1994) Interpatient heterogeneity in expression of CYP3A4 and CYP3A5 in small bowel: Lack of prediction by the erythromycin breath test. Drug Metab Dispos 22:947–955.
    OpenUrlAbstract
  12. ↵
    1. Nagai J,
    2. Takano M,
    3. Hirozane K,
    4. Yasuhara M,
    5. Inui K
    (1995) Specificity of p-aminohippurate transport system in the OK kidney epithelial cell line. J Pharmacol Exp Ther 274:1161–1166.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Olkkola KT,
    2. Backman JT,
    3. Neuvonen PJ
    (1994) Midazolam should be avoided in patients receiving the systemic antimycotics ketoconazole or itraconazole. Clin Pharmacol Ther 55:481–485.
    OpenUrlPubMed
  14. ↵
    1. Paine MF,
    2. Shen DD,
    3. Kunze KL,
    4. Perkins JD,
    5. Marsh CL,
    6. McVicar JP,
    7. Barr DM,
    8. Gillies BS,
    9. Thummel KE
    (1996) First-pass metabolism of midazolam by the human intestine. Clin Pharmacol Ther 60:14–24.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Porter TD,
    2. Coon MJ
    (1991) Cytochrome P-450: Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem 266:13469–13472.
    OpenUrlFREE Full Text
  16. ↵
    1. Schuetz EG,
    2. Beck WT,
    3. Schuetz JD
    (1996a) Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. Mol Pharmacol 49:311–318.
    OpenUrlAbstract
  17. ↵
    1. Schuetz EG,
    2. Schinkel AH,
    3. Relling MV,
    4. Schuetz JD
    (1996b) P-glycoprotein: A major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and humans. Proc Natl Acad Sci USA 93:4001–4005.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Sweatman TW,
    2. Seshadri R,
    3. Israel M
    (1990) Metabolism and elimination of rhodamine 123 in the rat. Cancer Chemother Pharmacol 27:205–210.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Takano M,
    2. Hasegawa R,
    3. Fukuda T,
    4. Yumoto R,
    5. Nagai J,
    6. Murakami T
    (1998) Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells. Eur J Pharmacol 358:289–294.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Terao T,
    2. Hisanaga E,
    3. Sai Y,
    4. Tamai I,
    5. Tsuji A
    (1996) Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier. J Pharm Pharmacol 48:1083–1089.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Thiebaut F,
    2. Tsuruo T,
    3. Hamada H,
    4. Gottesman MM,
    5. Pastan I,
    6. Willingham MC
    (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 84:7735–7738.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Thummel KE,
    2. O’Shea D,
    3. Paine M F,
    4. Shen DD,
    5. Kunze KL,
    6. Perkins JD,
    7. Wilkinson GR
    (1996) Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clin Pharmacol Ther 59:491–502.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Trezise AE,
    2. Romano PR,
    3. Gill DR,
    4. Hyde SC,
    5. Sepulveda FV,
    6. Buchwald M,
    7. Higgins CF
    (1992) The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression. EMBO J 11:4291–4303.
    OpenUrlPubMed
  24. ↵
    1. Tsuji A,
    2. Tamai I
    (1996) Carrier-mediated intestinal transport of drugs. Pharm Res 13:963–977.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Wacher VJ,
    2. Wu CY,
    3. Benet LZ
    (1995) Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: Implications for drug delivery and activity in cancer chemotherapy. Mol Carcinog 13:129–134.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Wils P,
    2. Phung-Ba V,
    3. Warnery A,
    4. Lechadeur D,
    5. Raeissi S,
    6. Hidalgo IJ,
    7. Scherman D
    (1994) Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers. Biochem Pharmacol 48:1528–1530.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Wrighton SA,
    2. Ring BJ
    (1994) Inhibition of human CYP3A catalyzed 1′-hydroxy midazolam formation by ketoconazole, nifedipine, erythromycin, cimetidine, and nizatidine. Pharm Res 11:921–924.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Yee S
    (1997) In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in human: Fact or myth? Pharm Res 14:763–766.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Transport of Rhodamine 123, a P-Glycoprotein Substrate, across Rat Intestine and Caco-2 Cell Monolayers in the Presence of Cytochrome P-450 3A-Related Compounds

Ryoko Yumoto, Teruo Murakami, Yuko Nakamoto, Risa Hasegawa, Junya Nagai and Mikihisa Takano
Journal of Pharmacology and Experimental Therapeutics April 1, 1999, 289 (1) 149-155;
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement