INTRODUCTION. Journal of Pharmaceutical Sciences, Vol. 100, (2011) 2011 Wiley-Liss, Inc. and the American Pharmacists Association

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1 Differential Effect of Grapefruit Juice on Intestinal Absorption of Statins Due to Inhibition of Organic Anion Transporting Polypeptide and/or P-glycoprotein YOSHIYUKI SHIRASAKA, KENSUKE SUZUKI, TAKEO NAKANISHI, IKUMI TAMAI Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa , Japan Received 24 January 2011; revised 2 April 2011; accepted 6 April 2011 Published online 24 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI /jps ABSTRACT: The purpose of this study is to examine the contributions of organic anion transporting polypeptide (Oatp) and/or P-glycoprotein (P-gp) to grapefruit juice (GFJ) interaction with two statins, pravastatin and pitavastatin, which undergo negligible metabolism in rats. The two statins were found to be substrates of both Oatp1a5 and Oatp2b1, whereas pitavastatin, but not pravastatin, was a substrate of P-gp. The plasma concentration of pravastatin after oral administration was significantly decreased by GFJ and naringin, whereas that of pitavastatin was significantly increased. Naringin inhibited Oatp1a5- and Oatp2b1-mediated uptake of pravastatin and Oatp1a5-mediated, but not Oatp2b1-mediated, uptake of pitavastatin. Naringin also inhibited P-gp-mediated transport of pitavastatin. These results suggested that the decrease of pravastatin absorption in the presence of GFJ is due to the inhibitory effect of naringin on Oatp, whereas the increase of pitavastatin is due to the inhibition of P-gp. These observations are consistent with the results of in situ absorption studies. In conclusion, Oatp and/or P-gp contribute to the intestinal absorption of statins, and the differential effect of GFJ on pravastatin and pitavastatin absorption is at least partly accounted for by the different inhibitory effects of naringin on these transporters Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100: , 2011 Keywords: intestinal absorption; interaction; grapefruit juice; pitavastatin; pravastatin; naringin; OATP; P-glycoprotein INTRODUCTION Foods and/or beverages may affect drug absorption from the small intestine, potentially resulting in altered therapeutic efficacy and adverse effects. Grapefruit juice (GFJ) is a clinically important beverage because oral bioavailability of drugs that are metabolized by CYP3A4 was increased due to the mechanism-based inhibition of intestinal CYP3A4 Abbreviations used: AUC, area under the plasma concentration time curve; AP, apical; BL, basolateral; C max, maximum plasma concentration; crna, complementary RNA; GFJ, grapefruit juice; GI, gastrointestinal; HMG-CoA, 3-Hydroxy- 3-methylglutaryl coenzyme A; IC50, half maximal (50%) inhibitory concentration; MDR/Mdr, multidrug resistance; MES, 2-(N-morpholino)ethanesulfonic acid; OATP/Oatp, organic aniontransporting polypeptide; P app, apparent permeability; P-gp, P-glycoprotein; t max, time to reach maximum plasma concentration; t 1/2, apparent elimination half-life of the log-linear phase. Correspondence to: Ikumi Tamai (Telephone: ; Fax: ; tamai@p.kanazawa-u.ac.jp) Journal of Pharmaceutical Sciences, Vol. 100, (2011) 2011 Wiley-Liss, Inc. and the American Pharmacists Association by GFJ constituents. 1,2 Recent investigations indicated that GFJ drug interactions can occur in not only CYP3A4-mediated metabolism but also transporter-mediated absorption. 3 Indeed, ingestion of GFJ has been shown to affect drug absorption mediated by transporters such as organic anion transporting polypeptide (OATP) and P-glycoprotein (P-gp)/multidrug resistance 1 (MDR1). 3,4 GFJ increases the area under the plasma concentration time curve (AUC) of cyclosporin A after oral administration in healthy volunteers because of the inhibition of P-gp-mediated efflux. 5 Because P-gp normally functions to limit drug absorption, intestinal absorption of its substrate drugs may increase when P-gp is inhibited by GFJ. 5 7 GFJ may also inhibit OATP-mediated drug uptake, thereby leading to decreased absorption of its substrates in humans and rats At present, naringin, a major constituent flavonoid of GFJ, is thought to be an inhibitor of both OATP and P-gp expressed in intestine Interestingly, OATP shares some substrates with P-gp so that JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER

2 3844 SHIRASAKA ET AL. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011 GFJ may show a complex interaction with drugs at these transporters during intestinal absorption. 10,11 We have found that the intestinal absorption of the $ 1 -adrenoceptor antagonist talinolol was affected by naringin in a concentration-dependent manner because talinolol is a substrate of both Oatp1a5 and P-gp in rats. 10,11 At lower concentrations of naringin, Oatp1a5-mediated absorption of talinolol is inhibited, resulting in a decrease in the absorption. However, at higher concentrations of naringin, P-gp-mediated efflux is inhibited as well so that intestinal absorption of talinolol is eventually enhanced. Furthermore, an inconsistency in the effect of GFJ on the intestinal absorption of talinolol between humans and rats was reported. 8,16 We have postulated that this species difference in the effect of GFJ on talinolol absorption between humans and rats is caused by the differential effect (distinct IC 50, half maximal (50%) inhibitory concentration, values) of naringin on OATP/Oatp- and P-gp (MDR1/Mdr1)-mediated transport of talinolol in the two species. 8,11,16 Such differential effects of GFJ on transporters may make it difficult to predict the in vivo effect of GFJ on drug absorption processes, wherein both influx (OATP) and efflux (P-gp) transporters are involved, but the mechanisms have not been clarified in detail. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG- CoA) reductase inhibitors (statins) are widely used for the treatment of hypercholesterolemia. Among statins, pravastatin and pitavastatin undergo little metabolism and are mainly excreted in unchanged form to the bile. 17 Their absorptions are mediated by several transporters Indeed, there is considerable evidence, including our studies, that pravastatin and pitavastatin are transported by OATP/Oatp expressed in small intestines of human/rat, whereas pitavastatin is also transported by P-gp. 14,15,18 21 Therefore, GFJ may interfere with the pharmacokinetics of pravastatin and pitavastatin by modulating their intestinal absorption through the interaction of GFJ components with the statins at OATP and/or P- gp, resulting in a differential alteration of the absorption kinetics of these statins. In the present study, we aimed to clarify the mechanism of interaction of GFJ with pravastatin and pitavastatin in rats, using in vitro, in situ, andin vivo experimental techniques in order to test the hypothesis that OATP and P-gp are the major sites of GFJ statin interaction. Using rats as an experimental model, we focused on Oatp1a5 and Oatp2b1 to evaluate the contribution of Oatp family members to the GFJ interaction with these statins because Oatp1a5 and Oatp2b1 are expressed in rodent small intestine at relatively high levels Rat Oatp1a5 and Oatp2b1 share high sequence homology with human OATP1A2 and OATP2B1, respectively. Therefore, our results also provide a rationale for understanding the contributions of OATP and/or P-gp to the differential effect of GFJ on the intestinal absorption of pravastatin and pitavastatin in humans. MATERIALS AND METHODS Materials Pravastatin sodium was kindly provided by Kobayashi Kako Company, Ltd. (Fukui, Japan). Pitavastatin calcium and naringin were purchased from Toronto Research Chemicals (North York, Ontario, Canada) and Chromadex (Irvine, California), respectively. GFJ (Tropicana TM ; 100% pure at a normal strength) was purchased from a supermarket in Kanazawa city (Kanazawa, Japan). Rat Mdr1a complementary DNS (cdna) was a kind gift from Takeda Pharmaceutical Company, Ltd. (Osaka, Japan). LLC-PK1, porcine kideny proximal tubular epithelial, cells transfected with empty vector (Mock) were obtained from GenoMembrane, Inc. (Yokohama, Japan). Transwells (12 well/plate, 3.0 :m pores, 0.9 cm 2 membrane surface area) were purchased from Nippon Becton Dickinson Company, Ltd. (Tokyo, Japan). All other compounds and reagents were obtained from Sigma Aldrich Company (St. Louis, Missouri), Invitrogen (Carlsbad, California), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Nacalai Tesque, Inc. (Kyoto, Japan) or Applied Biosystems (Foster City, California). Pharmacokinetic Study in Rats Male Wistar rats (250 ± 20 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 a.m. 8:45 p.m. light) in an air-controlled room (temperature, 23.0 ± 2 C; humidity, 55 ± 5%). All animal experimentation was approved by the Committee of Kanazawa University for the Care and Use of Laboratory Animals, and was carried out in accordance with the Declaration of Helsinki and the Guideline of Kanazawa University for the Care and Use of Laboratory Animals. Prior to administration of statins, the right jugular veins of rats were cannulated and then groups of four rats were orally administered pravastatin (100 mg/ kg, 25 mg/ml) or pitavastatin (1 mg/kg, 0.25 mg/ml) solution [MES, 2-(N-morpholino)ethanesulfonic acid, -buffered solution, ph 6.5, in the absence or presence of 1000 :M naringin (2.32 mg/kg, 0.58 mg/ml) or MES-buffered 100% GFJ solution, ph 6.5] by gavage. Blood samples (250 :L) were collected from the cannula into heparinized tubes at designated times (up to 1440 min). Each blood sample was replaced with an equal volume of saline, and heparinized saline was used to maintain the patency of the catheter. Blood samples were centrifuged at at 1,800 g for 10 min. DOI /jps

3 DIFFERENTIAL EFFECT OF GRAPEFRUIT JUICE ON INTESTINAL ABSORPTION OF STATINS 3845 The resultant plasma was stored at 30 C until analysis. Plasma concentrations of pravastatin were analyzed using a noncompartmental method. The AUCs from 0 to 12h (AUC 0 12 ) for pravastatin and that from0to24h(auc 0 24 ) for pitavastatin were calculated using the linear trapezoidal rule. The maximum plasma concentration (C max ) and the time to reach maximum plasma concentration (t max ) were obtained directly from the experimental data. The apparent elimination half-life of the log-linear phase (t 1/2 )was calculated based on the terminal elimination rate constant determined by log-linear regression of the final data points (at least 3). Uptake Experiments in Xenopus Laevis Oocytes Xenopus oocytes were prepared and injected with 50 nl of Oatp1a5, Oatp2b1, OATP2B1, or OATP1A2 complementary RNA (crna) solution, or water, and then uptake experiments were performed as previously described. 10,19 In brief, the oocytes were incubated at room temperature for the designated time (180 min for pravastatin and 90 min for pitavastatin) in uptake buffer [88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO 3, 0.82mM MgSO 4, 0.33 mm Ca(NO 3 ) 2,0.41mMCaCl 2, and 10 mm 2-(Nmorpholino)ethanesulfonic acid; ph 6.5] containing pravastatin or pitavastatin. The uptake was terminated by washing the oocytes three times with ice-cold modified Barth s solution. Transporter-mediated uptake was obtained by subtracting the uptake rate by water-injected oocytes from that by oocytes expressing a transporter of interest. Uptake rate [:L/(min oocyte)] was calculated as the cell-to-medium ratio by dividing the uptake amount by the initial concentration of the substrate drug in the uptake medium. Transport Experiments in LLC-PK1 Cells LLC-PK1 cells stably expressing rat Mdr1a (LLC-PK1/Mdr1a) were established as described previously. 26,27 LLC-PK1/Mdr1a and LLC-PK1/mock cells were cultured on Transwells (Nippon Becton Dickinson Company, Ltd.) for transport study as described previously. 11,14 In brief, monolayers of LLC-PK1/Mdr1a and LLC-PK1/mock cells were preincubated in transport medium (Hanks balanced salt solution, ph 7.4) for 30 min at 37 C. To ensure cell monolayer integrity, only monolayers of LLC- PK1/Mdr1a and LLC-PK1/mock cells that exhibited transepithelial electrical resistance values higher than 200 cm 2 were used for transport experiments. Transcellular transport of pravastatin (10 : M) and pitavastatin (1 :M) was measured in the apical (AP) to basal (BL) and BL to AP directions, and the permeability ratios (BL to AP / AP to BL) were calculated. An aliquot of transport buffer was obtained from the donor side at 5 min for measurement of the initial concentration, and from the receiver side at 30, 60, 80, 100, and 120 min. Transport experiments were performed in the absence of a ph gradient (AP ph = BL ph = 7.4). All the experiments were performed at 37 C. The apparent permeability (P app, cm/s) of pitavastatin across cell monolayers was calculated using Eq. 1: P app = dq dt 1 AC D (1) where Q (:mol) is the amount of pitavastatin transported over a certain time period t (s), C D is the initial concentration of pitavastatin in the donor compartment (:M), and A is the membrane surface area (0.9 cm 2 ). in Situ Intestinal Closed-loop Experiment This animal experimentation was also approved by the Committee of Kanazawa University for the Care and Use of Laboratory Animals, and was carried out in accordance with the Declaration of Helsinki and the Guideline of Kanazawa University for the Care and Use of Laboratory Animals. Permeability of substrate drugs through rat intestinal membrane was evaluated by the in situ intestinal closed-loop method. Rats (250 ± 20 g body weight) were fasted overnight and anesthetized with pentobarbital; then an intestinal loop (length 10 cm) was made at the lower ileum by cannulation with silicone tubing (internal diameter 3 mm, outside diameter 5 mm). The intestinal contents were removed by slow infusion of saline and air. Pravastatin (10 :M) or pitavastatin (1 :M) solution [phosphate-buffered solution, ph 6.5, in the absence or presence of naringin (1000 :M) or phosphatebuffered 100% GFJ solution, ph 6.5] was introduced into the intestinal loop and both ends of the loop were ligated. After a certain time period (60 min for pravastatin and 30 min for pitavastatin), the luminal solution in the loop was collected. Permeability values of pravastatin and pitavastatin were evaluated in terms of the percentage of dose absorbed, calculated by subtracting the remaining amount of statin from the administered amount. Equation 2 was used to calculate the permeability: Permeability = k av (2) 2Brl where k a is the first-order absorption rate constant of the substrate drug, estimated from the percentage of the dose absorbed during a defined period, V is the volume of pravastatin or pitavastatin solution applied to the loop, and r and l represent the radius and length of the used segment of intestine, respectively. The length was 10 cm and the radius was 0.18 cm (taken from the values of the radius of each intestinal segment reported by Fagerholm et al. 28 ). DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011

4 3846 SHIRASAKA ET AL. Liquid Chromatography Tandem Mass Spectrometry Analysis The concentrations of pravastatin and pitavastatin in all samples were quantified with a liquid chromatography tandem mass spectrometry (LC MS/MS) system consisting of a MDS-Sciex API 3200 TM triple quadrupole mass spectrometer (AB SCIEX, Foster City, CA) coupled with a LC-20AD ultra-fast LC system (Shimadzu Company, Kyoto, Japan). The LC MS/ MS analysis for pravastatin and pitavastatin was performed under the conditions described previously. 14,15 Mercury MS (C 18, mm, Luna 3 :m; Phenomenex, Torrance, California) and Mercury MS (C 18, mm, Luna 3 :m; Phenomenex) were used as the analytical column for pravastatin and pitavastatin, respectively. The gradient elution mobile phase consisted of ammonium acetate (12 mm; ph 4.5) and methanol for pravastatin, and 0.1% formic acid and acetonitrile for pitavastatin. The mass transitions (Q1/Q3) of m/z 442.2/209.0 and 422.1/290.1 were used for pravastatin and pitavastatin, respectively. Analyst software version 1.4 (AB SCIEX) was used for data manipulation. Statistical Analysis Data are given as the mean of values obtained in at least three experiments with standard error of the mean (SEM). 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 Effect of GFJ on Oral Absorption of Pravastatin and Pitavastatin in Rats When pravastatin was orally administered to rats at a dose of 100 mg/kg (25 mg/ml), AUC 0 12 and C max of pravastatin were 574 ± 97 (ng h)/ml and 112 ± 26 ng/ ml, respectively (Fig. 1a and Table 1). Simultaneous administration of GFJ with pravastatin significantly decreased its AUC 0 12 and C max to 12% and 16%, respectively (Fig. 1a and Table 1). Similarly, naringin (1000 :M) significantly decreased the AUC 0 12 and C max of pravastatin to 3% and 14%, respectively. GFJ and naringin did not significantly alter t 1/2 of pravastatin (Table 1). On the contrary, when pitavastatin was orally administered to rats at a dose of 1 mg/kg (0.25 mg/ml), AUC 0 24 and C max of pitavastatin were 179 ± 42 (ng h)/ml and 25.1 ± 6.0 ng/ml, respectively (Fig. 1b and Table 1). Upon simultaneous administration of GFJ with pitavastatin, the AUC 0 24 and C max significantly increased to 199% and 139%, respectively (Fig. 1b and Table 1). Similar results were obtained with naringin, which caused a significant increase in the AUC 0 24 and C max of pitavastatin to 383% and 192%, respectively. GFJ and naringin did not significantly alter t 1/2 of pitavastatin (Table 1). Uptake of Pravastatin and Pitavastatin by Xenopus Oocytes Expressing Oatp1a5, Oatp2b1, Oatp1a2, and Oatp2b1 Uptake of pravastatin and pitavastatin was measured using Xenopus oocytes expressing rat Oatp1a5 and Oatp2b1 (Figs. 2a and 2b). The uptakes of pravastatin (10 :M) and pitavastatin (1 :M) by Oatp1a5 and Oatp2b1 crna-injected oocytes were significantly higher than those by water-injected oocytes, suggesting that these statins are substrates of both Oatp1a5 and Oatp2b1. Naringin (1000 : M) significantly decreased the uptake of pravastatin by oocytes expressing Oatp1a5 and Oatp2b1, and it significantly decreased the uptake of pitavastatin by oocytes expressing Oatp1a5, but not Oatp2b1 (Figs. 3a and 3b). On the contrary, elacridar (1 :M) did not affect the uptake of both pravastatin and pitavastatin by oocytes expressing Oatp1a5 or Oatp2b1 (Figs. 3a and 3b). Therefore, elacridar at a concentration of 1 :M was used to specifically inhibit P-gp in the subsequent studies. Furthermore, the inhibitory effect of naringin on OATP1A2- and OATP2B1-mediated uptake of Figure 1. Mean plasma concentration time profiles of (a) pravastatin and (b) pitavastatin in rats after oral administration. Pravastatin (100 mg/kg, 25 mg/ml) or pitavastatin (1 mg/kg, 0.25 mg/ml) was orally administered as MES-buffered solution (ph 6.5) in the absence (open circles) or presence of 1000 : M naringin (closed squares) or GFJ (ph 6.5) (closed triangles). Data are shown as the mean ± SEM (n = 4). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011 DOI /jps

5 DIFFERENTIAL EFFECT OF GRAPEFRUIT JUICE ON INTESTINAL ABSORPTION OF STATINS 3847 Table 1. Pharmacokinetic Parameters of Pravastatin and Pitavastatin After Oral Administration in the Presence of GFJ or Naringin in Rats Pharmacokinetic Parameters Compound Condition AUC a (ng h/ml) C max (ng/ml) t max (h) t 1/2 (h) Pravastatin b Control 574 ± ± ± ± GFJ 67.4 ± ± ± ± Naringin c 17.4 ± ± ± ± 0.11 Pitavastatin d Control 179 ± ± ± ± GFJ 357 ± ± ± ± Naringin c 685 ± ± ± ± 0.48 a AUC from 0 to 12 h (AUC 0 12 ) and AUC from 0 to 24 h (AUC 0 24 ) were calculated for pravastatin and pitavastatin, respectively. b Pravastatin solution was orally administered at 100 mg/kg, 25 mg/ml, ph 6.5. c Applied concentration of naringin was 1000 :M (2.32 mg/kg, 0.58 mg/ml). d Pitavastatin solution was orally administered at 1 mg/kg, 0.25 mg/ml, ph 6.5. p < 0.05, significantly different from values without naringin. Data are presented as the mean ± SEM (n = 4). AUC, area under plasma concentration time curve; C max, peak plasma drug concentration; t max,timetoreachmaximum plasma concentration; t 1/2, elimination half-life. pravastatin and pitavastatin was studied (Figs. 4a and 4b). Naringin significantly reduced the OATP1A2-mediated pravastatin uptake, but did not affect the OATP2B1-mediated uptake. In case of pitavastatin, naringin significantly inhibited both OATP1A2- and OATP2B1-mediated uptake (Figs. 4a and 4b). Impact of P-gp on Transcellular Transport of Pravastatin and Pitavastatin in LLC-PK1 Cells To evaluate the effect of rat P-gp on transcellular transport of pravastatin and pitavastatin, absorptive (AP to BL) and secretory (BL to AP) permeability of pravastatin (10 :M) and pitavastatin (1 :M) were Figure 2. Uptake of (a) pravastatin and (b) pitavastatin by Xenopus oocytes expressing Oatp1a5 and Oatp2b1. Uptake of pravastatin (10 :M) or pravastatin (1 :M) by water-injected oocytes (open columns) or Oatp1a5 or Oatp2b1 crna-injected oocytes (closed columns) was measured at room temperature and ph 6.5. p < 0.05, significantly different from uptake by water-injected oocytes. Data are shown as the mean ± SEM (n = 8 10). DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011

6 3848 SHIRASAKA ET AL. Figure 3. Inhibitory effects of naringin and elacridar on uptake of (a) pravastatin and (b) pitavastatin by Xenopus oocytes expressing Oatp1a5 and Oatp2b1. Uptake of pravastatin (10 :M) or pitavastatin (1 :M) by oocytes expressing Oatp1a5 or Oatp2b1 was measured in the absence or presence of naringin (1000 :M) or elacridar (1 :M) at room temperature and ph 6.5. The inhibitory effects of naringin and elacridar on Oatp1a5- and Oatp2b1-mediated uptake of pravastatin and pitavastatin are presented as percent of control (closed columns). p < 0.05, significantly different from control (without naringin and elacridar). Data are shown as the mean ± SEM (n = 8 10). determined in LLC-PK1/Mdr1a and LLC-PK1/mock cells (Table 2). Permeability ratios (BL to AP/AP to BL) of pitavastatin were 3.35 and 1.55 in LLC-PK1/ Mdr1a and LLC-PK1/mock cells, respectively. In the presence of elacridar (1 :M) and naringin (1000 :M), the permeability ratios of pitavastatin decreased to 0.87 and 1.01 in LLC-PK1/Mdr1a cells, respectively. Accordingly, it was demonstrated that pitavastatin is a substrate of P-gp and its transport is completely inhibited by naringin at 1000 :M. On the contrary, the permeability of pravastatin in the AP to BL direction was relatively higher than that in the BL to AP direction in both LLC-PK1/ Mdr1a and LLC-PK1/mock cells, with permeability ratios of 0.41 and 0.59 in LLC-PK1/Mdr1a and LLC- PK1/mock cells, respectively, suggesting that pravastatin is not a substrate of rat P-gp. These findings may also imply that influx transporter is involved in transcellular transport of pravastatin in these cell lines, although it is not known whether OATP is Table 2. Transcellular Transport of Pravastatin and Pitavastatin in LLC-PK1/Mdr1a and LLC-PK1/Mock Cells P app ( 10 6 cm/s) Compound LLC-PK1 Cell Lines Condition AP to BL BL to AP Ratio a Pravastatin b Mdr1a Alone 1.58 ± ± Mock Alone 1.22 ± ± Pitavastatin c Mdr1a Alone 3.05 ± ± Naringin d 5.31 ± ± Mock + Elacridar e 5.53 ± ± Alone 4.51 ± ± a The permeability ratio (BL to AP/AP to BL). b Initial donor concentration of pitavastatin was 10 :M. c Initial donor concentration of pitavastatin was 1 :M. d Applied concentration of naringin was 1000 :M. e Applied concentration of elacridar was 1 :M. p < 0.05, significantly different from AP to BL permeability. Data are represented as means ± SEM (n = 3). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011 DOI /jps

7 DIFFERENTIAL EFFECT OF GRAPEFRUIT JUICE ON INTESTINAL ABSORPTION OF STATINS 3849 Figure 4. Inhibitory effects of naringin on uptake of (a) pravastatin and (b) pitavastatin by Xenopus oocytes expressing OATP1A2 and OATP2B1. Uptake of pravastatin (10 :M) or pitavastatin (1 : M) by oocytes expressing OATP1A2 and OATP2B1 was measured in the absence or presence of naringin (1000 :M) at room temperature and ph 6.5. The inhibitory effects of naringin on OATP1A2- and OATP2B1-mediated uptake of pravastatin and pitavastatin are presented as percent of control (closed columns). p < 0.05, significantly different from control (without naringin). Data are shown as the mean ± SEM (n = 8 10). endogenously expressed in LLC-PK1 cells, which originate from porcine kidney tubular cells. Effect of Gfj and Naringin on Intestinal Permeability of Pravastatin and Pitavastatin in Rats In order to clarify the effect of GFJ on Oatpand Mdr1-mediated intestinal absorption of pravastatin and pitavastatin, rat intestinal permeability of pravastatin and pitavastatin was measured by the in situ closed-loop method (Figs. 5a and 5b). Because Oatp1a5, Oatp2b1, and P-gp are highly expressed in the lower part of the small intestine in rats, a substantial contribution of both transporters to the intestinal absorption of pravastatin and pitavastatin was expected in ileum. 7,23,29 Accordingly, in the present study, the permeability of pravastatin and pitavastatin was measured in rat ileum. As shown in Figure 5a, the intestinal permeability of pravastatin was 4.20 ± cm/s in the absence of inhibitors. GFJ and naringin (1000 : M) significantly decreased the permeability to 1.68 ± and 1.91 ± cm/s, respectively. However, no significant change in permeability was observed in the presence of elacridar (1 : M), indicating that P-gp is not involved in the intestinal absorption of pravastatin. On the contrary, the permeability of pitavastatin (13.1 ± cm/s) in the absence of inhibitors was significantly increased by GFJ and naringin to 22.0 ± and 21.1 ± cm/s, respectively (Fig. 5b). Similarly, in the presence of elacridar, the permeability was significantly increased to 26.3 ± cm/s, suggesting that P-gp contributes to intestinal absorption of pitavastatin. DISCUSSION When pravastatin was orally administered to rats, pharmacokinetic parameters characterizing its intestinal absorption, AUC and C max, were significantly decreased by coadministration of GFJ and naringin, whereas those of pitavastatin were significantly increased (Fig. 1). Similarly, intestinal membrane permeability was affected by naringin in the in situ closed-loop method (Fig. 5). Because these statins are metabolically stable, the observed changes in intestinal absorption and permeability are considered to be due to the effect of GFJ and naringin on membrane transport processes. These statins are substrates of Oatp1a5 and Oatp2b1, and their transport DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011

8 3850 SHIRASAKA ET AL. Figure 5. Effects of GFJ, naringin, and elacridar on (a) pravastatin and (b) pitavastatin absorption in rat small intestine. Permeability of pravastatin (10 :M) or pitavastatin (1 :M) in rat small intestine (ileum) was determined by means of an in situ closed-loop method in the absence or presence of GFJ, naringin (1000 :M), or elacridar (1 :M) at 37 C and ph 6.5. p < 0.05, significantly different from permeability without naringin and elacridar. Data are shown as means ± SEM (n = 4). was inhibited by naringin except for the Oatp2b1- mediated transport of pitavastatin (Figs. 2 and 3). As shown in Table 2, pitavastatin, but not pravastatin, is transported by P-gp. Furthermore, the P-gp inhibitor elacridar increased intestinal permeability of pitavastatin, but not pravastatin (Fig. 5). Accordingly, a decrease in the intestinal absorption of pravastatin by GFJ and naringin may be explained by the inhibition of Oatp1a5 and/or Oatp2b1. An increase in the absorption of pitavastatin may be due to the inhibition of P-gp, although Oatp1a5-mediated transport is also affected by naringin. Several mechanisms can be considered to explain this apparently inconsistent observation for pitavastatin. First, P-gp may have a greater effect than Oatp1a5 on the intestinal absorption of pitavastatin so that the inhibition of P-gp would predominate over that of Oatp1a5. Second, Oatp2b1 may contribute more than Oatp1a5 to the intestinal absorption of pitavastatin. In this case, the inhibitory effect of naringin on Oatp1a5 would not greatly affect the influx transport of pitavastatin. Another possible explanation would be that pitavastatin absorption is mainly controlled by passive diffusion and P-gp-mediated efflux, with a low contribution of influx transporters. However, because nonlinear absorption has been observed following single oral administration of pitavastatin, it is likely that pitavastatin absorption is mediated by influx transporters including OATP/Oatp. 14,15,30 In Figures 1 and 5, the effect of naringin on the plasma concentration of pravastatin and pitavastatin after oral administration tends to be consistent, but not the same as the results obtained by the in situ closed-loop study. Several investigations have shown that the site-dependent permeability of drugs along the gastrointestinal (GI) tract may complicate the prediction of in vivo drug absorption Accordingly, our apparently inconsistent findings may be explained by the regional difference of Oatp- and/or P-gp-mediated absorption of pravastatin and pitavastatin along the GI tract. Meanwhile, the tested concentrations of pravastatin and pitavastatin in in vivo study are so high as compared with those in in situ and in vitro studies simply due to the low detection limit of those statins in plasma by means of even the highly sensitive LC MS/MS system (Fig. 1). We are certainly unable to rule out a possibility that the difference of dosage between the experiments affects each result. However, because orange juice was reported to increase the bioavailability of pravastatin (100 mg/kg, same as the dose in our study) due to induction of Oatp in rats, pravastatin is likely absorbed by OATP/Oatp even at a high dose. 35 Pitavastatin reportedly showed no inhibition of P-gp-mediated digoxin transport even at 100 :M. 36 Therefore, the P-gp-mediated transport of pitavastatin is expected not to be saturated at high doses. Taken together, our results are expected to be consistent even if the concentrations of statins are different in the experiments. On the contrary, pravastatin and pitavastatin were applied at a concentration of a 10-fold difference in our in in vitro and in situ studies. Because clinical dose of pravastatin is usually more than 10- foldas compared with that of pitavastatin (10 20 mg JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011 DOI /jps

9 DIFFERENTIAL EFFECT OF GRAPEFRUIT JUICE ON INTESTINAL ABSORPTION OF STATINS 3851 and 1 2 mg, respectively), we believe that the tested concentrations of both statins in the present study reflect their clinical states. The present findings are relevant to a clinical report describing an increase in the plasma pitavastatin concentration upon coadministration of pitavastatin with GFJ in humans (Fig. 1b). 37 However, in that clinical report, GFJ had shown only a small, although statistically significant, effect on the pharmacokinetics of pitavastatin. This can be explained by the attenuation of the inhibitory effect of GFJ on P-gp due to the inhibition of OATP because human OATP1A2- and OATP2B1-mediated transports of pitavastatin were inhibited by naringin (Fig. 4b). On the contrary, alteration of the pharmacokinetics of pravastatin in humans by GFJ has not yet been reported, although we observed GFJ interaction with pravastatin in rats in the present study (Fig. 1a). 38,39 As shown in Figure 4a, naringin inhibited OATP1A2- mediated, but not OATP2B1-mediated, transport of pravastatin. On the basis of the increasing evidence that intestinal absorption of various drugs is mediated by OATP2B1, OATP2B1 may be a major determinant of the intestinal absorption of pravastatin in humans, as compared with OATP1A2. 14,15,19 21,40 We also believe that species difference between humans and rats has contributed to the difficulty in understanding the intestinal drug absorption and interactions mediated by transporters (e.g., OATP and P-gp). We have shown that the species difference in the effect of GFJ on talinolol transport mediated by OATP/ Oatp and P-gp can explain the differential in vivo effect of GFJ on the oral absorption of talinolol between humans and rats. 12,16,21 Recent reports suggest that OATP1A2 exhibits a broad substrate specificity as compared with OATP2B This is consistent with our results showing that naringin inhibited OATP1A2-mediated, but not OATP2B1-mediated, transport of pravastatin (Fig. 4a). In contrast, naringin inhibited OATP2B1- as well as OATP1A2-mediated transport of pitavastatin (Fig. 4b). Figure 3 also shows that naringin inhibited Oatp2b1-mediated transport of pravastatin, but not pitavastatin, implying the substrate-dependent inhibitory effect of naringin on OATP2B1/Oatp2b1. We have previously shown that there are multiple binding sites with different affinity on OATP1B1. 44 Therefore, the substrate-dependent inhibitory effect of naringin on OATP2B1/Oatp2b1 might be explained by the presence of multiple binding sites that discriminate pravastatin and pitavastatin, although OATP2B1 reportedly exhibited a single functional Figure 6. Schematic diagram of differential effect of GFJ (naringin) on OATP/Oatp- and P-gp-mediated absorption between pravastatin and pitavastatin in rat and human. Pravastatin and pitavastatin are transported by Oatp1a5 and Oatp2b1 and by Oatp1a5, Oatp2b1 and P-gp, respectively, in rat (upside). Pravastatin and pitavastatin are transported by OATP1A2 and OATP2B1 and by OATP1A2, OATP2B1, and P-gp, respectively, in human (downside). Major (black arrows) and minor (gray arrows) pathways in intestinal absorption processes of pravastatin and pitavastatin is presumed by the present study. Naringin (GFJ) inhibits Oatp1a5- and Oatp2b1-mediated pravastatin transports and Oatp1a5- and P-gp-mediated pitavastatin transports in rat (upside). Naringin (GFJ) inhibits and OATP1A2-mediated pravastatin transport and OATP1A2-, OATP2B1-, and P-gp-mediated pitavastatin transports in human (downside). DOI /jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011

10 3852 SHIRASAKA ET AL. site for estrone-3-sulfate. 44 Taken together, substrate recognition by OATP2B1/Oatp2b1 is considered to be the key determinant for GFJ and naringin interactions with drugs. 14,15,19 21,40 CONCLUSION Our findings demonstrated that the differential involvement of Oatp and/or P-gp can explain the different effects of GFJ on the intestinal absorption of pravastatin and pitavastatin in rats (Fig. 6). Although contributions of other influx and/or efflux transporters cannot be ruled out, the present study strongly suggested that intestinal OATP/Oatp and P- gp contribute greatly to the intestinal absorption of drugs, and interactions at these transporters can at least partly explain the clinically observed drug beverage interactions (Fig. 6). ACKNOWLEDGMENTS This work was supported in part by a Grant-in- Aid for Scientific Research from the Japan Society for the Promotion of Science (research project number ). The authors would like to thank Kobayashi Kako Company, Ltd. (Fukui, Japan) for providing pravastatin sodium. The authors would also like to thank Dr. Satoru Asahi and Dr. Toshiyuki Takeuchi of Takeda Pharmaceutical Company, Ltd. (Osaka, Japan) for providing rat Mdr1a cdnas and for their technical advice. REFERENCES 1. Bailey DG, Malcolm J, Arnold O, Spence JD Grapefruit juice drug interactions. Br J Clin Pharmacol 46: Bailey DG, Spence JD, Munoz C, Arnold JM Interaction of citrus juices with felodipine and nifedipine. Lancet 337: Dresser GK, Bailey DG The effects of fruit juices on drug disposition: A new model for drug interactions. Eur J Clin Invest 33(Suppl 2): Greenblatt DJ Analysis of drug interactions involving fruit beverages and organic anion-transporting polypeptides. 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