Species Difference in the Effect of Grapefruit Juice on Intestinal Absorption of Talinolol between Human and Rat

Transcription

1 /10/ $20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 1 Copyright 2010 by The American Society for Pharmacology and Experimental Therapeutics / JPET 332: , 2010 Printed in U.S.A. Species Difference in the Effect of Grapefruit Juice on Intestinal Absorption of Talinolol between Human and Rat Yoshiyuki Shirasaka, Erika Kuraoka, Hildegard Spahn-Langguth, Takeo Nakanishi, Peter Langguth, and Ikumi Tamai Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan (Y.S., E.K., T.N., I.T.); Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City, Egypt (H.L.); and School of Pharmacy, Johannes Gutenberg-University, Mainz, Germany (P.L.) Received August 4, 2009; accepted September 21, 2009 ABSTRACT Bioavailability of talinolol, a 1 -adrenergic receptor antagonist, was enhanced by coadministration with grapefruit juice (GFJ) in rats, whereas GFJ ingestion markedly reduced the absorption of talinolol in humans. Because our recent study indicated that the inhibitory effect of GFJ on organic anion-transporting polypeptide (Oatp)- and P-gp-mediated talinolol absorption depends on the concentration of naringin in ingested GFJ, the apparent inconsistent findings may be explained by the species difference in the affinity of naringin for OATP/Oatp and P-gp multidrug resistance 1 (MDR1/Mdr1) between humans and rats. Although human MDR1-mediated talinolol transport was not inhibited by 2000 M naringin, naringin inhibited human OATP1A2-, rat Oatp1a5-, and rat Mdr1a-mediated talinolol transport with IC 50 values of 343, 12.7, and 604 M, respectively, in LLC-PK1 cell and Xenopus laevis oocyte systems. P-Glycoprotein (P-gp), an ATP-binding cassette transporter, is an efflux transport protein encoded by the multidrug resistance (MDR) 1 gene (Mdr1 in rodents) and is well known to significantly affect the absorption kinetics of a number of orally administered drugs (Terao et al., 1996; Fromm, 2000; Naruhashi et al., 2001; Shirasaka et al., 2006, 2008a). In addition, organic anion-transporting polypeptide (OATP/Oatp), a family of membrane solute carrier (SLC) transporters, has recently been recognized to influence the intestinal absorption of several drugs in clinical use (Tamai et al., 2000; Dresser et al., 2002; Kobayashi et al., 2003; Nozawa et al., 2004; Maeda et al., 2007; Tani et al., 2008; Shirasaka et al., 2009). Due to the complicated absorption This work was supported in part by a Grant-in-aid for Scientific Research from Japan Society for the Promotion of Science [Research Project Number ]. Article, publication date, and citation information can be found at doi: /jpet Because the naringin concentration in commercially prepared GFJ was found to be approximately 1200 M, these results suggested that GFJ would reduce the intestinal absorption of talinolol through inhibition of OATP1A2-mediated talinolol uptake in humans, whereas an increase of talinolol absorption is mainly through inhibition of Mdr1a-mediated efflux in rats. The rat intestinal permeability of talinolol measured by the in situ closed loop method was indeed significantly increased in the presence of GFJ, whereas a significant decrease was observed with 6-fold diluted GFJ, in which the naringin concentration was approximately 200 M. The present study indicated that the species difference in the effect of GFJ on intestinal absorption of talinolol between humans and rats may be due to differences in the affinity of naringin for OATP/Oatp and MDR1/Mdr1 transporters between the two species. processes of drugs mediated by these influx and efflux transporters expressed in the human gastrointestinal (GI) tract, it may not be easy to predict intestinal absorption of orally administered drugs based upon previously established correlations between their physicochemical properties and membrane permeability (Tamai et al., 1997; Kikuchi et al., 2006; Ofer et al., 2006; Shirasaka et al., 2009). Talinolol (human bioavailability, 55 15%) is a long-acting and highly selective 1 -adrenergic receptor antagonist (Fig. 1A) (Trausch et al., 1995; Westphal et al., 2000; Weitschies et al., 2005). Our group previously reported an increase in the area under the plasma concentration-time curve (AUC) for talinolol when it was coadministered with grapefruit juice (GFJ) in rats (Spahn-Langguth and Langguth, 2001). Although GFJ could affect both CYP3A4 and P-gp, P-gp has been suggested to be the primary site of interaction between talinolol and GFJ because talinolol is unlikely subject to metabolism based on the reports that less ABBREVIATIONS: P-gp, P-glycoprotein; AUC, area under the plasma concentration-time curve; AP, apical; BL, basolateral; GI, gastrointestinal; GFJ, grapefruit juice; MDR/Mdr, multidrug resistance; OATP/Oatp, organic anion-transporting polypeptide; P app, apparent permeability; SLC, solute carrier; TEER, transepithelial electrical resistance; LC/MS, liquid chromatography/mass spectrometry. 181

2 182 Shirasaka et al. Fig. 1. Chemical structure of talinolol (A) and naringin (B). than 1% of the administered dose is found as hydroxylated form in urine in humans, dogs, and rats (Klemm and Wenzel, 1975; Trausch et al., 1995; Schupke et al., 1996). On the other hand, in a human clinical study, it was reported that GFJ ingestion markedly reduced both AUC and oral bioavailability of talinolol (Schwarz et al., 2005). Because recent investigations interestingly indicated that naringin, the main constituent flavonoid of GFJ, has a significant inhibitory effect on not only P-gp-mediated efflux but also OATP-mediated uptake of drugs, the apparently contradictory findings concerning the effect of GFJ on intestinal absorption of talinolol might be explained by a complicated interaction of GFJ with both OATP and P-gp transporters in small intestine (Fig. 1B) (Dresser et al., 2002; Bailey et al., 2007; de Castro et al., 2007). Although naringin may affect the OATP and P-gp expressed in the liver, it is reasonable to consider that naringin is practically unable to interfere with OATP and P-gp in organs other than the GI tract because it is known that naringin is hardly detected in plasma samples after ingestion of GFJ. This fact is supported by previous report showing that neither naringin nor its aglycone naringenin, but a low concentration of naringenin glucuronides, was found in plasma from GFJ interaction studies in humans (Fuhr and Kummert, 1995). Therefore, it is considered that the alteration in talinolol AUC would be determined by interactions of naringin with OATP and P-gp in basically the GI tract. In our previous report, talinolol was shown to be a substrate for both rat Oatp1a5 and P-gp, which are localized at the apical (AP) membrane of enterocytes (Shirasaka et al., 2009). Then, it was described that a high concentration of naringin might inhibit Mdr1a-mediated talinolol efflux, resulting in an increase of the AUC of oral talinolol, whereas a lower concentration might inhibit only Oatp1a5 but not Mdr1a, resulting in a decrease of the AUC (Shirasaka et al., 2009). It seems likely that the interaction of GFJ with both OATP and P-gp is the cause of the complex nonlinear intestinal absorption of talinolol (Ofer et al., 2006; Shirasaka et al., 2009). Therefore, the apparently inconsistent effects of GFJ ingestion on talinolol absorption between humans and rats may be explained by species differences in the effects of naringin on the OATP/ Oatp and P-gp (MDR1/Mdr1) transporters. In the present study, we analyzed the inhibitory effects of naringin on human and rat OATP/Oatp- and MDR1/Mdr1- mediated talinolol transport by determining the kinetic parameters using LLC-PK1 cell and Xenopus laevis oocyte transporter expression systems and the in situ rat intestinal closed loop technique. Our results indicate that the inconsistency in GFJ effect on intestinal absorption of talinolol between human and rat can be ascribed to species differences in the affinity of naringin for OATP/Oatp and MDR1/Mdr1. Materials and Methods Materials. Talinolol racemate was kindly provided by Arzneimittelwerk Dresden (AWD, Radebeul, Germany). Naringin was purchased from Chromadex (Irvine, CA). Grapefruit juice (Tropicana; 100% pure at a normal strength) was purchased from a supermarket (Kanazawa, Japan). LLC-PK1/MDR1 and LLC-PK1/mock cells were kindly supplied by GenoMembrane, Inc. (Yokohama, Japan). Rat Mdr1a and Mdr1b complementary DNAs (cdnas) were kindly provided by Takeda Pharmaceutical Co., Ltd. (Osaka, Japan). Medium 199 was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Invitrogen (Carlsbad, CA). Transwells (12 well/plate, 3.0- m pores, 0.9-cm 2 membrane surface area) were purchased from Nippon Becton Dickinson Co., Ltd. (Tokyo, Japan). Benzylpenicillin, streptomycin, G418, and gentamicin were purchased from Sigma-Aldrich (St. Louis, MO). All other compounds and reagents were obtained from Nacalai Tesque, Inc. (Kyoto, Japan), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Sigma-Aldrich, Bio-Rad Laboratories (Hercules, CA), or Applied Biosystems (Foster City, CA). Establishment of LLC-PK1 Transformants Stably Expressing Mdr1. LLC-PK1/Mdr1a and LLC-PK1/Mdr1b cells were prepared as described previously (Katoh et al., 2006; Takeuchi et al., 2006). In brief, LLC-PK1 cells ( cells) were transfected with 2 g of pcdna3.1 (Invitrogen) containing Mdr1a or Mdr1b using LipofectAMINE 2000 reagent. Two days later, selection with G418 (500 g/ml) was started, and then individual colonies were isolated with medium containing 150 ng/ml colchicine. Finally, transformants were screened for Mdr1a- and Mdr1b-mediated quinidine transport activities (data not shown). Established cell lines expressing Mdr1a and Mdr1b were designated LLC-PK1/Mdr1a and LLC- PK1/Mdr1b, respectively. LLC-PK1 Cell Culture. LLC-PK1/MDR1 and LLC-PK1/mock cells were cultured at 37 C in a humidified atmosphere of 5% CO 2 in air using Medium 199 supplemented with 14.3 mm NaHCO 3, 10% FBS, 100 units/ml benzylpenicillin, 100 g/ml streptomycin, and 500 g/ml G418. Cells were routinely subcultured at 90% confluence with trypsin (0.25%) EDTA (1 mm). For transport studies, LLC-PK1/ MDR1 and LLC-PK1/mock cells were plated onto Transwell filter membrane inserts at a density of cells/cm 2. Medium was replaced with fresh medium at 3 and 5 days after initiation of cell culture, and cell monolayers were used for transport studies at 6 days after seeding. Likewise, LLC-PK1/Mdr1a and LLC-PK1/ Mdr1b cells were cultured at 37 C in the same medium. For transport studies, LLC-PK1/Mdr1a and LLC-PK1/Mdr1b cells were plated onto Transwell filter membrane inserts at a density of cells/cm 2 and then cultured for 4 days until use for transport experiments. Transport Experiments. The cell monolayers were preincubated in transport medium (Hanks balanced salt solution; mm CaCl 2, 5.36 mm KCl, mm KH 2 PO 4, mm MgSO 4, mm NaCl, mm Na 2 HPO 4, 25 mm D-glucose, and 10 mm HEPES, ph 7.4) for 30 min at 37 C. After preincubation, the transepithelial electrical resistance (TEER) was measured routinely before and after each experiment with a Millicell-ERS system (Milli-

3 Species Difference in Talinolol-Grapefruit Juice Interaction 183 pore Corporation, Bedford, MA) to ensure cell monolayer integrity. LLC-PK1/MDR1, LLC-PK1/Mdr1a, LLC-PK1/Mdr1b, and LLC-PK1/ mock cells that exhibited TEER values higher than 200, 250, 250, and 200 cm 2, respectively, were used for transport experiments. Transport measurement was initiated by adding talinolol (10 M) to the donor side and transport medium to the receiver side. Transport of talinolol was observed in two directions, apical (AP) to basal (BL) and BL to AP. Samples were obtained from the donor side at 5 min for measurement of initial concentration and from the receiver side at 30, 60, 80, 100, and 120 min. Transport experiments were performed under ph gradient conditions (apical ph 6.5 and basal ph 7.4), except where otherwise indicated in Table 1 (apical ph basal ph 7.4). All experiments were performed at 37 C. After all of the experiments were completed, TEER was measured to ensure cell monolayer integrity, and data generated in cell monolayers in which viability had not been adversely affected by the experimental conditions were accepted. The apparent permeability (P app, cm/s) of talinolol across cell monolayers was calculated using the following equation: P app dq dt 1 A C D (1) where Q is the amount of talinolol transported over time t [therefore, dq/dt is the amount of talinolol transported within a given time period (micromolars per second)]. C D is the initial concentration of talinolol in the donor compartment (in micromolars), and A is the membrane surface area (0.9 cm 2 ). Uptake Experiments in X. laevis Oocytes. Preparation of oocytes, in vitro synthesis of OATP1A2 (SLCO1A2), OATP2B1 (SLCO2B1), and Oatp1a5 (Slco1a5) crnas, and uptake experiments were conducted as described previously (Nozawa et al., 2004; Shirasaka et al., 2009). In brief, for standard experiments, defolliculated oocytes were injected with 50 nl of 50-ng crna solution or water as a control and then cultured for 3 days at 18 C in modified Barth s solution [88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO 3, 0.82 mm MgSO 4, 0.33 mm Ca(NO 3 ) 2, 0.41 mm CaCl 2, and 10 mm HEPES, ph 7.4] containing 50 g/ml gentamicin until use for experiments. To initiate uptake experiments, the oocytes were transferred to a 12-well culture plate and preincubated in uptake buffer (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO 3, 0.82 mm MgSO 4, 0.33 mm Ca(NO 3 ) 2, 0.41 mm CaCl 2, and 10 mm 2-(N-morpholino)ethanesulfonic acid, ph 6.5) containing talinolol at room temperature for the designated time. The uptake was terminated by washing three times with ice-cold modified Barth s solution. Kinetic parameters were estimated by means of nonlinear leastsquares analysis using KaleidaGraph (Synergy Software, Reading, PA). The affinity of talinolol for OATP1A2, OATP2B1, and Oatp1a5 (K m ) and the maximal velocity of OATP1A2-, OATP2B1-, and Oatp1a5-mediated talinolol uptake (V max ) were obtained by fitting to the following equation: V V max C K m C where V is the initial uptake rate of talinolol (picomole per minute per oocyte) and C is the concentration of talinolol in the medium (micromolar). Inhibition Kinetics. Kinetic parameters were estimated by means of nonlinear least-squares analysis using KaleidaGraph (Synergy Software). The inhibitory effect of naringin on talinolol transport was expressed as percentage of control, and the naringin concentration giving half-maximal inhibition (IC 50 ) was obtained by means of the following equation: % of Control 100 IC 50 IC 50 I where [I] is naringin concentration (micromolar). In Situ Intestinal Closed Loop Experiment. Male Wistar rats ( g body weight) were housed three per cage, with free access to commercial chow and tap water, and were maintained on a 12 h dark/light cycle (8:45 AM 8:45 PM light) in an air-controlled room (temperature, C; humidity, 55 5%). All animal experimentation was carried out in accordance with the Declaration of Helsinki and with the Guideline of Kanazawa University for the Care and Use of Laboratory Animals. The permeability of rat intestinal membrane was evaluated by the in situ intestinal closed loop method. Animals were fasted overnight and were anesthetized with pentobarbital. The abdominal cavity was opened, and an intestinal loop (length, 10 cm) was made at the lower ileum by cannulation with silicone tubing (inner diameter, 3 mm). The intestinal contents were removed by slow infusion of saline and air. Talinolol (10 M) solution [phosphate-buffered solution, ph 6.5 in the absence or presence of naringin (200 or 2000 M) or phosphate-buffered GFJ solution, ph 6.5] was introduced into the intestinal loop, and both ends of the loop were ligated. After a certain period of time (usually 5 min), the luminal solution in the loop was collected. The permeability of talinolol was evaluated in terms of the percentage of dose absorbed, calculated by subtracting the remaining amount of talinolol from the administered amount. The following equation was used to calculate the permeability: Permeability k a V d 2 rl where k a is the first-order absorption rate constant of talinolol estimated from the percentage of the dose absorbed during the defined period, V d is the volume of talinolol solution introduced into the loop, and r and l represent the radius and length of the used intestine, respectively; thus, 2 rl corresponds to the relevant intestinal luminal surface area. The length was 10 cm, and the value of the (2) (3) (4) TABLE 1 Impact of P-gp on transcellular transport of talinolol in LLC-PK1/MDR1, LLC-PK1/Mdr1a, LLC-PK1/Mdr1b, and LLC-PK1/mock cells P app Cell Lines Talinolol a Ratio c AP to BL BL to AP 10 6 cm/s LLC-PK1/MDR1 Alone * Cyclosporine A b LLC-PK1/Mdr1a Alone * Cyclosporine A b LLC-PK1/Mdr1b Alone * LLC-PK1/mock Alone * P 0.05, significantly different from permeability ratio in LLC-PK1/mock cells; P 0.05, significantly different from permeability ratio in the absence of cyclosporine A. a Initial donor concentration of talinolol was 10 M. b Applied concentration of cyclosporine A was 10 M. c The permeability ratio (BL to AP/AP to BL). Data are represented as the mean S.E.M. (n 3).

4 184 Shirasaka et al. radius of small intestine reported by Fagerholm et al. (1997) was used (0.18 cm). LC/MS/MS Analysis. The concentration of talinolol in all samples was quantified with a liquid chromatography-tandem mass spectrometry (LC/MS/MS) system consisting of a MDS-Sciex API 3200 triple quadrupole mass spectrometer (Applied Biosystems) coupled with a LC-20AD high-performance liquid chromatography system (Shimadzu Co., Kyoto, Japan) (Shirasaka et al., 2009). Mercury MS (C 18, mm, 5 m; Phenomenex, Torrance, CA) was used as the analytical column, and the mobile phase was composed of 0.1% formic acid in water and acetonitrile. In the LC/MS/MS system, electrospray ionization in the positive ion mode was employed. The mass transition (Q1/Q3) of m/z 364.3/308.1 was used for talinolol. Analyst software version was used for data manipulation. Statistical Analysis. Data are given as the mean of values obtained in at least three individual experiments with S.E.M. Statistical analyses were performed with the unpaired Student s t test, and a probability of less than 0.05 (p 0.05) was considered to be statistically significant. Results Transcellular Transport of Talinolol in LLC-PK1/ MDR1, LLC-PK1/Mdr1a, and LLC-PK1/Mdr1b Cells. Absorptive (AP to BL) and secretory (BL to AP) permeability of talinolol (10 M) were measured in LLC-PK1/MDR1, LLC- PK1/Mdr1a, LLC-PK1/Mdr1b, and LLC-PK1/mock cells (Table 1). In LLC-PK1/MDR1, LLC-PK1/Mdr1a, and LLC-PK1/ Mdr1b cells, the permeability of talinolol in the BL to AP direction was higher than that in the AP to BL direction, whereas the permeability in LLC-PK1/mock cells was comparable in both directions. Permeability ratios (BL to AP/AP to BL) of talinolol were , , and in LLC-PK1/MDR1, LLC-PK1/Mdr1a, and LLC-PK1/ Mdr1b cells, respectively, all of which were significantly higher than that ( ) in LLC-PK1/mock cells. In the presence of cyclosporine A (10 M), a potent MDR1/Mdr1 inhibitor, the permeability ratio of talinolol was significantly decreased in LLC-PK1/MDR1 and LLC-PK1/Mdr1a cells, suggesting that talinolol is a substrate of both human and rat P-gp. Because the expression level of Mdr1b is low in the rat TABLE 2 Comparison of IC 50 values of naringin for OATP/Oatp and MDR1/ Mdr1-mediated talinolol transport between humans and rats Species Transporter Naringin IC 50 a M Human MDR (or not inhibited) OATP1A OATP2B (or not inhibited) Rat Mdr1a Oatp1a a IC 50 values were obtained from Figs. 2 and 6. Fig. 2. Inhibitory effects of naringin on MDR1- and Mdr1a-mediated transport of talinolol in LLC-PK1/MDR1 and LLC-PK1/Mdr1a cells. Transport experiments with talinolol (100 M) were performed in the absence or presence of various concentrations of naringin at 37 C and under apical ph 6.5 and basal ph 7.4. The inhibitory effect of naringin on MDR1- (A) and Mdr1a (B)-mediated transport of talinolol was represented as percentage of control (filled circles), assuming that cyclosporine A (10 M) completely inhibits P-gp-mediated efflux (corresponding to 0% control) (open circles). Data are shown as the mean S.E.M. (n 3). Fig. 3. Uptake of talinolol by X. laevis oocytes expressing OATP1A2. A, time course of talinolol (100 M) uptake by oocytes expressing OATP1A2. Uptake of talinolol by OATP1A2-cRNA-injected oocytes (filled circles) was compared with that by water-injected oocytes (open circles) at room temperature and ph 6.5. B, concentration dependence of talinolol uptake by oocytes expressing OATP1A2. Uptake of talinolol by waterinjected or OATP1A2-cRNA-injected oocytes was measured at various concentrations for 120 min at room temperature and ph 6.5. OATP1A2- mediated uptake was determined by subtracting the uptake by waterinjected oocytes from that by OATP1A2-cRNA-injected oocytes. Data are shown as the mean S.E.M. (n 8 10).

5 Species Difference in Talinolol-Grapefruit Juice Interaction 185 intestine, the Mdr1a-mediated talinolol transport was further studied (Li et al., 1999; Takara et al., 2003). Inhibitory Effect of Naringin on MDR1- and Mdr1a- Mediated Transport of Talinolol. The concentration dependence of the inhibitory effect of naringin on MDR1/ Mdr1a-mediated AP to BL transport of talinolol (10 M) is shown in Fig. 2. The inhibitory effect of naringin was represented as percentage of control, and the IC 50 value was calculated assuming that cyclosporine A (10 M) completely inhibits P-gp-mediated efflux (corresponding to 0% control). MDR1-mediated talinolol transport was not affected by naringin over the whole concentration range from 10 to 2000 M (Fig. 2A; Table 2). In contrast, naringin inhibited Mdr1amediated transport of talinolol with an IC 50 value of M (Fig. 2B; Table 2). Uptake of Talinolol by X. laevis Oocytes Expressing OATP1A2, OATP2B1, and Oatp1a5. To investigate whether talinolol is transported by OATP1A2, OATP2B1, and Oatp1a5, we first examined the time course of talinolol uptake by X. laevis oocytes expressing OATP1A2, OATP2B1, and Oatp1a5. The uptake of talinolol (100 M) was significantly increased compared with that by water-injected oocytes in each case, as shown in Figs. 3A, 4A, and 5A, respectively. Further analysis of the concentration dependence Fig. 4. Uptake of talinolol by X. laevis oocytes expressing OATP2B1. A, time course of talinolol (100 M) uptake by oocytes expressing OATP2B1. Uptake of talinolol by OATP2B1-cRNA-injected oocytes (filled circles) was compared with that by water-injected oocytes (open circles) at room temperature and ph 6.5. B, concentration dependence of talinolol uptake by oocytes expressing OATP2B1. Uptake of talinolol by waterinjected or OATP2B1-cRNA-injected oocytes was measured at various concentrations for 120 min at room temperature and ph 6.5. OATP2B1- mediated uptake was determined by subtracting the uptake by waterinjected oocytes from that by OATP2B1-cRNA-injected oocytes. Data are shown as the mean S.E.M. (n 8 10). Fig. 5. Uptake of talinolol by X. laevis oocytes expressing Oatp1a5. A, time course of talinolol (100 M) uptake by oocytes expressing Oatp1a5. Uptake of talinolol by Oatp1a5-cRNA-injected oocytes (filled circles) was compared with that by water-injected oocytes (open circles) at room temperature and ph 6.5. B, concentration dependence of talinolol uptake by oocytes expressing Oatp1a5. Uptake of talinolol by waterinjected or Oatp1a5-cRNA-injected oocytes was measured at various concentrations for 60 min at room temperature and ph 6.5. Oatp1a5-mediated uptake was determined by subtracting the uptake by water-injected oocytes from that by Oatp1a5-cRNA-injected oocytes. Data were reproduced from Shirasaka et al. (2009) with permission from Springer Science and Business Media.Data are shown as the mean S.E.M. (n 8 10). showed that the OATP1A2-, OATP2B1-, and Oatp1a5- mediated talinolol uptakes were saturable, with K m and V max values of M and pmol/oocyte/120 min, M and pmol/oocyte/120 min, and M and pmol/oocyte/60 min, respectively (Figs. 3B, 4B, and 5B). These results suggested that talinolol is a substrate of OATP1A2, OATP2B1, and Oatp1a5. Inhibitory Effect of Naringin on Talinolol Uptake by X. laevis Oocytes Expressing OATP1A2, OATP2B1, and Oatp1a5. The inhibitory effect of naringin on OATP1A2-, OATP2B1-, and Oatp1a5-mediated uptake of talinolol (100 M) was examined using X. laevis oocytes expressing OATP1A2, OATP2B1, and Oatp1a5, respectively. As shown in Fig. 6 and Table 2, naringin inhibited OATP1A2- and Oatp1a5-mediated uptake of talinolol (100 M) with IC 50 values of and M, respectively. However, OATP2B1-mediated talinolol uptake was not affected by naringin throughout the concentration range from 10 to 2000 M. Concentration-Dependent Effects of Naringin and GFJ on Intestinal Permeability of Talinolol in Rats. To determine whether naringin and GFJ inhibit intestinal absorption of talinolol in a concentration-dependent manner, the rat intestinal permeability of talinolol was measured by

6 186 Shirasaka et al. Fig. 6. Inhibitory effects of naringin on talinolol uptake by X. laevis oocytes expressing OATP1A2, OATP2B1 and Oatp1a5. OATP1A2- (A), OATP2B1- (B), and Oatp1a5-mediated (C) uptake of talinolol (100 M) was determined by subtracting the uptake by water-injected oocytes from that by OATP1A2, OATP2B1, and Oatp1a5 crna-injected oocytes, respectively. Uptake of talinolol by water-injected or OATP1A2, OATP2B1, and Oatp1a5-cRNA-injected oocytes was measured in the absence or presence of various concentrations of naringin for 60 min at room temperature and ph 6.5. Data are shown as the mean S.E.M. (n 8 10). the rat in situ closed loop method (Figs. 7 and 8). It was reported that the expression level of P-gp in ileum is higher than that in jejunum of rats, and the expression level of Oatp1a5 is also higher in ileum (Walters et al., 2000; Tian et al., 2002; Cao et al., 2005; Shirasaka et al., 2008b). Therefore, because it was expected that the impact of Oatp1a5 and P-gp on the intestinal absorption of talinolol would be more clearly observable in ileum, in the present study we measured the rat intestinal permeability of talinolol in the ileum. As shown in Fig. 7, the permeability of talinolol in rat small intestine was cm/s in the absence of naringin. In Fig. 7. Concentration-dependent effects of naringin on talinolol absorption in rat small intestine. Permeability of talinolol (10 M, ph 6.5) in rat small intestine was determined by means of an in situ closed loop method in the absence or presence of naringin (200 and 2000 M) for 5 min at 37 C., P 0.05,, P 0.01, significantly different from permeability without naringin., P 0.01, significantly different from permeability with 200 M naringin. Data are shown as the mean S.E.M. (n 3). Fig. 8. Concentration-dependent effects of GFJ on talinolol absorption in rat small intestine. Permeability of talinolol (10 M, ph 6.5) in rat small intestine was determined by means of an in situ closed loop method in the presence of GFJ or 6-fold-diluted GFJ for 5 min at 37 C., P 0.05,, P 0.01, significantly different from permeability without GFJ., P 0.01, significantly different from permeability with 6-fold-diluted GFJ. Data are shown as the mean S.E.M. (n 3). the presence of 200 M naringin, the permeability was significantly decreased to cm/s. In the presence of 2000 M naringin, the permeability was significantly increased to cm/s. On the other hand, concomitant administration of 100% pure GFJ significantly increased the permeability of talinolol in rat small intestine to cm/s (Fig. 8). Because the concentration of naringin in the GFJ was found to be 1198 M, the permeability of talinolol was further measured in the presence of 6-fold-diluted GFJ, in which naringin concentration was expected to be approximately 200 M. Coadministration of 6-fold-diluted GFJ significantly decreased the permeability of talinolol to a level similar to that observed in the presence of 200 M naringin (Fig. 7), i.e., cm/s (Fig. 8).

7 Species Difference in Talinolol-Grapefruit Juice Interaction 187 Discussion Our recent study indicated that the inhibitory effect of GFJ on Oatp1a5- and P-gp-mediated talinolol absorption in rats depends on the naringin concentration in ingested GFJ (Shirasaka et al., 2009). Therefore, to understand the apparently contradictory effects of GFJ on intestinal absorption of talinolol between human and rat, we further explored the differential effects of GFJ on human and rat OATP/Oatp and MDR1/Mdr1 (Spahn-Langguth and Langguth, 2001; Schwarz et al., 2005). Talinolol was found to be a substrate for MDR1, OATP1A2, and OATP2B1 in humans and Mdr1a, Mdr1b, and Oatp1a5 in rats (Table 1; Figs. 3, 4, and 5). As shown in Fig. 6, A and B, naringin reduced OATP1A2- but not OATP2B1-mediated talinolol uptake in humans. The IC 50 value of naringin for OATP1A2-mediated talinolol uptake was 343 M (Fig. 6A; Table 2). The MDR1-mediated transport of talinolol was not affected by naringin throughout the concentration range from 10 to 2000 M (Fig. 2A; Table 2). Since de Castro et al. (2006) reported that naringin concentration ranges from 174 to 1492 M among 14 different brands of GFJ, our findings suggested that the naringin concentration in ingested GFJ may not be high enough to cause significant inhibition of MDR1-mediated talinolol efflux in human intestine. Therefore, in humans, GFJ would predominantly inhibit OATP1A2, but not MDR1, and thus talinolol absorption would be decreased in humans by GFJ. On the other hand, naringin inhibited both Oatp1a5- and Mdr1a-mediated transports of talinolol with IC 50 values of and M, respectively, in rats (Figs. 2B and 6C; Table 2). Therefore, ingested GFJ would inhibit both Mdr1a- and Oatp1a5- mediated transport of talinolol, thereby increasing talinolol absorption in rats, mainly due to the inhibition of the Mdr1a-mediated efflux. The difference in the IC 50 values of naringin for Oatp1a5- and Mdr1a-mediated talinolol transports may result in a complicated concentration-dependent effect of naringin on the intestinal absorption of talinolol in rats. As shown in Fig. 7, rat intestinal permeability of talinolol measured by the in situ closed loop method was significantly decreased in the presence of 200 M naringin, presumably because inhibition of Oatp1a5 would predominate over inhibition of P-gp at that concentration. In contrast, a significant increase was observed in the presence of 2000 M naringin, presumably due to inhibition of both Oatp1a5- and Mdr1a-mediated talinolol transport. Although we did not obtain direct evidence that both Oatp1a5 and Mdr1a are major transporter molecules involved in intestinal absorption of talinolol, the results of the present in situ study are consistent with a functional role of both Oatp1a5 and Mdr1a in intestinal absorption of talinolol. However, we cannot rule out the possibility that other influx transporters susceptible to naringin are also involved in the absorption of talinolol. Assuming that talinolol absorption is regulated by Oatp1a5 and Mdr1a, based on the results obtained in Fig. 7, it appears that P-gp activity plays a more important role than Oatp1a5 activity in the intestinal absorption of talinolol in rats, although Oatp1a5-mediated transport must also contribute substantially to the high intestinal permeability of talinolol. These considerations are consistent with increased absorption of talinolol upon coadministration of GFJ in rats, as described in a previous report (Spahn-Langguth and Langguth, 2001). It is noteworthy that concomitant administration of 100% pure GFJ significantly increased the permeability of talinolol, whereas the permeability was significantly decreased when talinolol was coadministered with 6-folddiluted GFJ in rats (Fig. 8). The naringin concentration in Fig. 9. Schematic diagram of species differences in the effect of GFJ (naringin) on OATP/Oatp- and MDR1/ Mdr1-mediated talinolol absorption between human and rat. Inhibitory effects of GFJ ( 600 M naringin) on Oatp1a5- and Mdr1a-mediated talinolol transport and OATP1A2-, OATP2B1-, and MDR1-mediated talinolol transport in rat and human, respectively (% control) (left). GFJ inhibits both Oatp1a5- and Mdr1a-mediated talinolol transport and OATP1A2-mediated talinolol transport in rat and human, respectively (right).

8 188 Shirasaka et al. the GFJ used was 1198 M (so that 6-fold-diluted GFJ contains approximately 200 M naringin); therefore, it is reasonable that these findings correspond well with the results obtained by coadministration of naringin (200 or 2000 M), as shown in Fig. 7. It was reported that GFJ did not have an influence on the transport of mannitol (a marker for paracellular transport) across intestinal epithelial Caco-2 cells and had little effect on the TEER (Lim and Lim, 2006). The present study also indicates that naringin have no effect on the TEER in LLC-PK1 cells. From these observations, it was considered that an increased intestinal permeability for talinolol by coadministration with naringin (2000 M) or GFJ in rats was not caused by the disruption of tight junction barrier and paracellular permeability. Meanwhile, although various components of GFJ (e.g., the flavonoids naringin and naringenin and the furanocoumarins bergamottin, 6,7 dihydroxybergamottin, and 6,7 -epoxybergamottin) have been suggested to contribute to GFJ-drug interaction, the results obtained from Figs. 7 and 8 support the view that naringin is the major inhibitor of OATP/Oatp and P-gp transporters in GFJ and is primarily responsible for altering the absorption kinetics of orally administered drugs (Schmiedlin-Ren et al., 1997; Bailey et al., 1998, 2007; de Castro et al., 2006, 2007, 2008). In conclusion, our findings indicate that the species difference in the effect of GFJ on intestinal absorption of talinolol between humans and rats may be due to the differential effect (differences in the IC 50 values) of naringin on OATP/Oatp- and MDR1/Mdr1-mediated transport of talinolol in the two species. 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9 Species Difference in Talinolol-Grapefruit Juice Interaction 189 ability of the beta 1-adrenoceptor antagonist talinolol in man. Biopharm Drug Dispos 16: Walters HC, Craddock AL, Fusegawa H, Willingham MC, and Dawson PA (2000) Expression, transport properties, and chromosomal location of organic anion transporter subtype 3. Am J Physiol Gastrointest Liver Physiol 279:G1188 G1200. Weitschies W, Bernsdorf A, Giessmann T, Zschiesche M, Modess C, Hartmann V, Mrazek C, Wegner D, Nagel S, and Siegmund W (2005) The talinolol double-peak phenomenon is likely caused by presystemic processing after uptake from gut lumen. Pharm Res 22: Westphal K, Weinbrenner A, Giessmann T, Stuhr M, Franke G, Zschiesche M, Oertel R, Terhaag B, Kroemer HK, and Siegmund W (2000) Oral bioavailability of digoxin is enhanced by talinolol: evidence for involvement of intestinal P-glycoprotein. Clin Pharmacol Ther 68:6 12. Address correspondence to: Ikumi Tamai, Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa , Japan. tamai@p.kanazawau.ac.jp