Mrp2 inhibitors. The biliary excretion of PSP in Eisai hyperbilirubinemic rats (EHBR), hereditarily. Mrp2-defective rats, was signiËcantly lower than that in SD rats.
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Regular Article Mechanism of Active Secretion of Phenolsulfonphthalein in the Liver via Mrp2 (abcc2), an Organic Anion Transporter Shirou ITAGAKI1, Mitsuru SUGAWARA2, Michiya KOBAYASHI2, Katsumi MIYAZAKI2 and Ken ISEKI1,* 1Department
of Clinical Pharmaceutics & Therapeutics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 2Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Sapporo, Japan
Summary: Phenolsulfonphthalein (PSP) has been selected as a model drug that is eliminated from both the kidney and liver in rats. Although the renal PSP transport system has been studied, few details of the biliary excretion of PSP have been reported. We investigated the biliary excretion system for PSP in rats. It has been reported that the biliary excretion of many organic anions from hepatocytes into bile is mediated by a primary active transporter, referred to as multidrug resistance-associated protein 2 (Mrp2 W abcc2). The biliary excretion of PSP in SD rats was signiˆcantly decreased in the presence of Mrp2 inhibitors. The biliary excretion of PSP in Eisai hyperbilirubinemic rats (EHBR), hereditarily Mrp2-defective rats, was signiˆcantly lower than that in SD rats. Moreover, an eŒux experiment using Caco-2 cells was carried out to conˆrm Mrp2-mediated PSP transport. Mrp2 inhibitors signiˆcantly decreased PSP eŒux from Caco-2 cells. These results suggest that Mrp2 contributes to the biliary excretion of PSP in SD rats.
Key words: phenolsulfonphthalein; Mrp2; biliary excretion; organic anion
rats (EHBR)), the substrate speciˆcity of Mrp2 has been clariˆed.10) The human colon adenocarcinoma cell line Caco-2 has also been used as a model for MRP2 W Mrp2mediated eŒux, despite the fact that this cell line is derived from the intestine.11) Since the Caco-2 cells were grown in wells, basolateral transporters such as MRP1 (ABCC1) and MRP3 (ABCC3) make a minor contribution to the eŒux of drugs.12) The purpose of this study was to elucidate the transporter-mediated excretion system for PSP in the liver. The function and localization of Mrp2 suggest that this unidirectional transporter contributes to the secretion of PSP into the canaliculus lumen. We therefore studied the eŠect of co-administration of Mrp2 inhibitors on biliary excretion of PSP in SD rats. Moreover, to elucidate the contribution of Mrp2 to the transport of PSP, we compared the biliary excretion of PSP in SD rats and that in EHBR. We further examined the transport characteristics of PSP using Caco-2 cells, because these cells retain polarity of MRP2 expression.
Introduction More than 60 years ago, Smith et al.1) reported that phenolsulfonphthalein (PSP, phenol red) was eliminated from renal plasma in a single pass process through the kidney. PSP is known to be secreted into bile and urine in various species.2,3) In some studies, PSP has been selected as a model drug that is eliminated from both the kidney and liver in rats.4,5) The renal PSP transport system has been extensively studied.6,7) However, few details of the biliary excretion of PSP have been reported. Although, it has been reported that the biliary excretion of many xenobiotics is mediated by primary active transporters located on the bile canalicular membrane (such as multidrug resistance-associated protein 2 (Mrp2 W abcc2) and Mdr1 P-glycoprotein (P-gp W abcb1)8,9)), the mechanism of excretion of PSP across the canalicular membrane has not been elucidated. By comparing the transport properties in normal rats and those in mutants whose Mrp2 function is hereditarily defective (such as Eisai hyperbilirubinemic
Received; April 21, 2003, Accepted; August 22, 2003 *To whom correspondence should be addressed : Ken ISEKI, Ph. D., Department of Clinical Pharmaceutics & Therapeutics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan. Tel. & Fax. ＋81-11-706-3770, e-mail: ken-i＠pharm.hokudai.ac.jp
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Materials and Methods 1. Chemicals: Probenecid was purchased from Sigma Chemical Co. (St Louis, MO). Phenolsulfonphthalein, indomethacin and sulfobromophthalein (BSP) were purchased from Wako Pure Chemical (Osaka, Japan). Cyclosporin A (CYA) and pravastatin were kindly donated from Novartis Pharma (Tokyo, Japan) and Sankyo (Tokyo, Japan), respectively. All other reagents were of the highest grade available and used without further puriˆcation. 2. Animals: Male EHBR, aged 7 weeks (300–350 g in weight), were obtained from BMR Laboratories Inc. (Gifu, Japan). Male SD rats, aged 6 to 7 weeks (300– 350 g in weight), were obtained from NRC Haruna (Gunma, Japan). The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the ``Guide for the Care and Use of Laboratory Animals''. 3. In vivo study: Three to six male SD rats and EHBR were used in all experiments. The rats were anesthetized with sodium pentobarbital (40 mg W kg weight, i.p.). The common bile duct of each rat was cannulated with a blunted 24-gauge hypodermic needle shaft to collect bile specimens. PSP (2.2 mmol W kg) and an inhibitory drug solution were injected through the femoral vein. Blood was collected at 1, 15, 30, 45, 60 min after injection. Plasma was prepared by centrifugation (850×g for 15 min) of blood samples. Methanol, corresponding to a double volume of plasma, was added to each plasma specimen. After centrifugation of the mixture (12,000×g for 15 min), the concentration of PSP in the supernatant was measured. Bile specimens were collected at 0–15, 15–30, 30–45, 45–60 min after injection. The whole contents of the bladder were withdrawn with a syringe at 60 min after injection. 4. Cell culture: Caco-2 cells were obtained form American Type Culture Collection (Rockville, MD). The cells were routinely maintained in plastic culture ‰asks (Falcon, Becton Dickinson and Co., Lincoln Park, NJ). These stock cells were subcultivated before reaching con‰uence. The medium used for growth of Caco-2 cells was Dulbecco's modiˆed Eagle's medium (Gibco, Life Technologies, Inc., Grand Island, NY) with 10z fetal bovine serum (ICN Biomedicals, Inc., Aurora, OH), 1z nonessential amino acids (Gibco) and 4 mM glutamine without antibiotics. The monolayer cultures were grown in an atmosphere of 5z CO2–95z O2 at 379 C. Cells reached con‰uency after 5–7 days in culture. The cells were harvested with 0.25 mM trypsin and 0.2z EDTA (0.5–1 min at 379 C), resuspended, and seeded into a new ‰ask. Cells between the 35th and 52nd passages were used in this study. For the eŒux study, Caco-2 cells were seeded at a cell
density of 4×105 cells W cm2 on 24-well plastic plates (Falcon, Becton Dickinson and Co., Lincoln Park, NJ). The cell monolayers were fed a fresh growth medium every 2 days and were then used at 10 to 15 days for the eŒux experiments. 5. EŒux studies: The incubation medium used for the eŒux study from the apical side was 10 mM HEPES buŠer (pH 7.4) containing 25 mM D-glucose, 137 mM NaCl, 5 mM KCl, 0.3 mM Na2 HPO4, 0.4 mM KH2 PO4, 1.0 mM CaCl2 and 0.4 mM MgSO4. The pH of the medium was adjusted with a solution of NaOH. After removal of the growth medium, cells were preincubated at 379C for 15 min with 0.5 mL of incubation medium. After removal of the medium, cells were incubated with 0.5 mL of incubation medium containing PSP (100 mM) for 3 h at 379 C. After incubation, cells were washed ˆve times with an ice-cold incubation medium and re-incubated in PSP-free medium for a certain time at 379 C. The amount of PSP eŒuxed in the medium was determined. To measure the amount of residual PSP in the cells, the medium was aspirated and the cells were washed rapidly ˆve times with an ice-cold incubation medium. The cells were suspended in 0.5 mL of an extraction solution (0.03 M phosphate buŠer (pH 7.0) W methanol＝50 W 50) for 1 h at room temperature. The extraction solution was used for the determination of PSP concentration after centrifugation at 6,100×g for 15 min. 6. Analytical procedures: Substrates were determined using an HPLC system equipped with a Hitachi L-6000 pump and L-4200H UV W VIS detector. The column was a Hitachi ODS Gel #3053 (4 mm i.d. ×250 mm). A mobile phase containing 20z acetonitrile and 50 mM H3 PO4 with pH adjusted to 3.0 by NaOH was used. Column temperature and ‰ow rate were 559C and 0.7 mL W min, respectively. Wavelength for detection of PSP was 432 nm. Protein concentration was measured by the method of Lowry et al.13) with bovine serum albumin as a standard. 7. Data Analysis: The area under the plasma concentration-time curve (AUC) was estimated by the trapezoidal rule using the plasma data from 0 to 60 min. The clearance values of bile and urine (CLbile and CLurine ) were determined by dividing the amounts of PSP excreted into urine and bile from 0 to 60 min by the AUC from 0 to 60 min. Analysis of variance (ANOVA) and unpaired Student's t-test were used for the statistical analysis, and a value of Pº0.05 was considered signiˆcant. Results Inhibitory eŠects of various drugs on the disposition of PSP in SD rats: The inhibitory eŠects of inhibitors of ATP-binding cassette (ABC) transporters on the excretion of PSP in SD rats were examined. It has been
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Fig. 1. Time proˆles for the plasma concentrations and the cumulative biliary excretions of PSP in SD rats. The time proˆles for plasma concenkg) in the presence or absence of CYA trations (A) and cumulative biliary excretions (B) were determined after i.v. injection of PSP (2.2 mmol W kg) and indomethacin (5 mg W kg). Keys: , control; ×, CYA; $, indomethacin. Each value represents the mean with S.D. of three to six (5 mg W determinations. ***Pº0.001, signiˆcantly diŠerent from that in the absence of inhibitors.
Kinetic parameters of PSP after i.v. administration to rats CYA (5 mg W kg)
Indomethacin (5 mg W kg)
624±29.8*** 88.3±7.53*** 0.14±0.01*** 187±85.2*** 0.30±0.15***
522±15.5*** 163±17.4*** 0.31±0.03*** 142±29.7*** 0.27±0.06***
385±47.0 118±16.9*** 0.31±0.04*** 805±42.3 2.10±0.14
Control AUC (nmol min W mL) kg) Xbile 0–1h (nmol W min W kg) CLbile (mL W Xurine 0–1h (nmol W kg) min W kg) CLurine (mL W
390±26.7 311±23.4 0.80±0.08 929±146 2.38±0.35
kg) was injected through the femoral vein with or without inhibitors. Blood and bile specimens were collected at the speciˆed times. PSP (2.2 mmol W Xbile0–1h and Xurine0–1h represent the cumulative amounts excreted into bile and urine over a period of 60 min, respectively. The kinetic parameters were calculated from the values in Figs. 1, 2 and 3. Each value represents the mean with S.D. of three to six determinations. ***Pº0.001, signiˆcantly diŠerent from control.
reported that CYA is a mixed inhibitor of P-gp and MRPs14,15) and that indomethacin is an inhibitor of MRPs but not of P-gp.16) The plasma concentrations of PSP in the presence of these inhibitors were slightly higher than that in the absence of these inhibitors (Fig. 1A). The calculated AUC value was signiˆcantly higher in the presence of these drugs (Table 1). Figure 1B shows the time proˆles of the biliary excretion of PSP. In the presence of CYA and indomethacin, the cumulative amounts of PSP excreted into the bile were signiˆcantly decreased. These drugs reduced the CLbile value of PSP (Table 1). Biliary excretion of PSP in EHBR: Figure 2 shows the time proˆles of the plasma concentration and the cumulative amount of PSP excreted into the bile in EHBR. Plasma concentrations of PSP in SD rats and in EHBR were similar (Fig. 2A). In contrast, the amount of biliary excretion of PSP in EHBR was markedly lower than that in SD rats (Fig. 2B). CYA and indomethacin reduced the cumulative biliary excretion of PSP in SD rats to the level of that in EHBR (Table 1).
The calculated CLbile value in EHBR was signiˆcantly lower than that in SD rats (Table 1). Urinary excretion study: The amount of urinary excretion of PSP over a period of 1 hr after intravenous injection was determined (Fig. 3). The urinary excretion of PSP was signiˆcantly decreased by CYA and indomethacin in SD rats. These inhibitory eŠects were dose-dependent. The values of CLurine for PSP in the presence of these drugs were signiˆcantly lower than the control values (Table 1). The CLurine value in EHBR was similar to that in SD rats (Table 1). EŠect of CYA on the eŒux of PSP in Caco-2 cells: EŒux experiments were performed to determine whether CYA aŠects Mrp2-mediated PSP excretion. As indicated in Fig. 4A, CYA signiˆcantly limited the loss of cellular PSP in PSP-preloaded Caco-2 cells. After a 60-min eŒux period, Caco-2 cells retained about 20z and 40z of the initial accumulation in the absence and presence of CYA, respectively. The amount of PSP eŒux measured in the extracellular medium was signiˆcantly decreased in the presence of CYA, suggesting
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Fig. 2. Time proˆles of the plasma concentration and the cumulative biliary excretion of PSP in SD rats and EHBR. The time proˆles of plasma concentrations (A) and cumulative biliary excretions (B) were determined after i.v. injection of PSP in SD rats (open symbols) and EHBR (closed symbols). Each value represents the mean with S.D. of three to six determinations. ***Pº0.001, signiˆcantly diŠerent from SD rats.
Fig. 3. Urinary excretion of PSP in SD rats and EHBR. PSP was injected through the femoral vein with (closed bars) or without (open bars) inhibitors. The ratio of the excreted amount of PSP over a period of 60 min in a urine sample to the percent of the injected amount was determined. Each value represents the mean with S.D. of three to six determinations. *Pº0.05, **Pº0.01, ***Pº0.001, signiˆcantly diŠerent from that in the absence of inhibitors.
that CYA increased PSP retention in Caco-2 cells by inhibiting PSP excretion (Fig. 4B). Since the eŒux of PSP was linear for the initial 15 min, eŒux over a period of 15 min was subsequently used for inhibition study. EŠects of Mrp2 inhibitors on the initial eŒux of PSP in Caco-2 cells: The eŠects of various compounds that are known to be inhibitors of Mrp2 on the initial eŒux
of PSP in Caco-2 cells were determined (Table 2). As shown in Table 2, four known Mrp2 inhibitors, probenecid, methotrexate, pravastatin and BSP,17–20) signiˆcantly decreased the initial eŒux of PSP. However, verapamil, a substrate of P-gp,21) and 4,4?diisothiocyanostilbene-2,2?-disulfonic acid (DIDS), an inhibitor of organic anion transporters,22) did not aŠect the initial eŒux of PSP.
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Fig. 4. EŠects of CYA on cellular retention (A) and eŒux (B) of PSP in Caco-2 cells. Cells were incubated for 3 hours with 100 mM PSP at 379C. After incubation, the cells were washed, and cellular retention and eŒux of PSP were measured at the speciˆed times in the presence (closed circles) or absence (open circles) of 10 mM CYA at pH 7.4. Each point represents the mean with S.D. of four determinations. **Pº0.01, ***Pº0.001, signiˆcantly diŠerent from that in the absence of CYA.
Table 2. EŠects of various compounds on the initial eŒux of PSP in Caco-2 cells Relative secretion (z of control)
Compound Control Probenecid (1 mM) CYA (10 mM) MTX (50 mM) pravastatin (1 mM) BSP (1 mM) verapamil (1 mM) DIDS (1 mM)
100 54.4±3.56*** 81.8±3.54** 65.5±5.24*** 72.6±5.47*** 75.6±5.67*** 102±5.43 103±7.37
PSP-preloaded Caco-2 cells were incubated for 15 min in PSP-free buŠer in the presence or absence (control) of various compounds at pH 7.4. The control value for initial eŒux of PSP was 12.7± 0.83 pmol W min W mg protein. Each value represents the mean with S.D. of four determinations. **Pº.01, ***Pº.001, signiˆcantly diŠerent from the control.
Discussion PSP is excreted into bile and urine in a variety of species.2,3) Thus, PSP has been selected as a model drug that is eliminated from both the kidney and liver in rats.4,5) The mechanism by which PSP is excreted from the kidney has been studied.6,7) However, there appears to have been few studies on biliary excretion of PSP. Normally, transport across the canalicular membrane is the rate-limiting step for the biliary excretion of most bile constituents and for the formation of hepatocellular bile. It is driven primarily by ATP-dependent export pumps, which function unidirectionally and belong to the superfamily of ABC transporters. ABC transporters for amphipaths, Mdr1 P-glycoprotein and Mrp2, have
been shown to be localized to the canalicular membrane.8,9) These transporters confer multidrug resistance against a wide spectrum of cytotoxic agents. In the present study, we systematically examined the mechanisms of biliary excretion of PSP by performing an inhibition experiment. The plasma concentration and AUC value of PSP in the presence of CYA, which is an inhibitor of ABC transporters,14,15) were higher than those in the absence of CYA (Fig. 1A). Generally, a substrate of P-gp is thought to be a lipophilic and neutral or cationic drug.23) On the other hand, Mrp2 recognizes glucuronide conjugates and non-conjugated organic anions.24) Therefore, it is likely that Mrp2 plays a role in the biliary excretion of PSP, due to the fact that PSP has the structure of an amphipathic anion. The plasma concentration of PSP was increased by indomethacin, a well-known inhibitor of Mrps but not of P-gp (Fig. 1A).16) The results of the inhibitory experiment showed that the AUC value of PSP was signiˆcantly increased (Table 1). Additionally, CYA and indomethacin signiˆcantly reduced the biliary excretion of PSP (Fig. 1B). As a consequence, CYA and indomethacin reduced the CLbile value of PSP in SD rats (Table 1). These results suggest that Mrp2 mediates the biliary excretion of PSP. The clearance process involves uptake across the basolateral membrane and excretion across the bile canalicular membrane. The sinusoidal uptake also plays an important role in the clearance process. It is possible that CYA and indomethacin may inhibit the uptake process. In the second part of this study, we examined the contribution of Mrp2 to PSP transport. EHBR are
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genetically deˆcient in Mrp2.10) For this reason, the biliary excretion of Mrp2 substrate is much lower in EHBR. By using EHBR, the substrate speciˆcity of Mrp2 has been characterized. The cumulative biliary excretion of PSP was markedly reduced in EHBR and the CLbile was also reduced in EHBR compared with those in SD rats (Table 1), suggesting that Mrp2 mediates the biliary excretion of PSP. These results provide evidence that the canalicular transport of PSP is mediated by Mrp2. As shown in Fig. 2B, the biliary excretion of PSP was signiˆcantly decreased, but not stopped in EHBR. A primary active transporter other than Mrp2, which is still maintained in EHBR, may be responsible for the residual biliary excretion of PSP.25,26) It has been reported that the transport function for organic anions on the kidney was maintained in Mrp2 mutant rats and that the contribution of Mrp2 to urinary excretion was minor.12,27–29) Actually, the AUC values of PSP in SD rats and EHBR in the present study were almost the same. This could be the result of maintained urinary excretion mechanisms in EHBR. On the other hand, the AUC values of PSP in SD rats were signiˆcantly increased in the presence of CYA and indomethacin. We focused on the eŠects of CYA and indomethacin on the urinary excretion of PSP in SD rats. CYA and indomethacin reduced the CLurine value of PSP in SD rats (Table 1). These results suggest that the increased AUC values in the presence of CYA and indomethacin were due to the inhibition of biliary and urinary excretion of PSP. The CLurine values were not signiˆcantly diŠerent between SD rats and EHBR (Table 1). Finally, we carried out in vitro cell study. It has been reported that Caco-2 cells are useful for studying the function of Mrp2 in a cultured cell line and for examining the actions of inhibitors of MRP2 W Mrp2-mediated transport.11) It has been reported that MRP2 was expressed at a higher level than another MRPs in Caco-2 cells.11,30) Since the Caco-2 cells were grown in wells, basolateral transporters such as MRP1 and MRP3 make a minor contribution to the eŒux of drugs.12) We therefore chose this cell line to conˆrm the occurrence of Mrp2-mediated PSP transport. Mrp2 is also expressed in the apical membrane of rat hepatoma-derived cells, WIF-B.31) However, in the present study, we measured eŒux from the whole cells into the outer medium. In this case, the possibility of contribution of basolateral transporters to the eŒux of PSP cannot be excluded. We determined both the cellular retention and the amount of eŒux to the outer medium of PSP in the presence or absence of CYA using Caco-2 cells (Fig. 4). CYA reduced the rate of decrease in intracellular PSP corresponding to the reduction in PSP eŒux rate. The initial eŒux of PSP in Caco-2 cells was decreased in the presence of Mrp2 inhibitors (Table 2).17–20) The concen-
tration of CYA, pravastatin and BSP was at least 2.5fold higher than Ki or Km values of MRP2 for these compounds. It has been reported that Km value of MRP2 for methotrexate was 1 mM. Thus, it is possible that another transport system may be responsible for the PSP excretion in Caco-2 cells. Verapamil and DIDS, which are typical inhibitors of P-gp and organic anion transporters, respectively,21,22) had no eŠect on the eŒux of PSP from Caco-2 cells. The hypothesis that PSP is a substrate for Mrp2 was further supported by these results. In conclusion, the results of this study suggest that PSP is a substrate for Mrp2 and that Mrp2 contributes to the biliary excretion of PSP in rats. References 1)
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