Long-Lasting Inhibition of the Intestinal Absorption of Fexofenadine by Cyclosporin a in Rats

RESEARCH ARTICLE Long-Lasting Inhibition of the Intestinal Absorption of Fexofenadine by Cyclosporin A in Rats KEI SUZUKI, YOSHIHISA SHITARA, KOUSUKE FUKUDA, TOSHIHARU HORIE Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Received 31 December 2011; revised 21 March 2012; accepted 10 April 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23174 ABSTRACT: The purpose of the present study is to examine the long-lasting inhibition of intestinal organic anion transporting polypeptides (Oatps) by cyclosporin A (CsA) in rats using fexofenadine (FEX) as a probe drug. We examined the pharmacokinetics of FEX after its intravenous or oral administration to rats at 3 or 24 h after the oral administration of CsA. When FEX was administered at 3 h after the administration of CsA, its plasma concentration increased regardless of whether it was administered intravenously or orally. When FEX was intravenously administered at 24 h after the oral administration of CsA, its plasma concentration was increased; however, that observed after its oral administration was not significantly different from the vehicle-treated control. When FEX was administered at 3 h after the administration of CsA, the hepatic availability (Fh ) and the fraction absorbed in the intestine as an unchanged form (Fa ·Fg ) of FEX were increased, resulting in increased bioavailability (=Fa ·Fg ·Fh ). At 24 h after the administration of CsA, the Fh of FEX was increased, whereas its bioavailability was decreased, suggesting that its Fa ·Fg was decreased because of the long-lasting inhibition. In conclusion, CsA has long-lasting inhibitory effects on Oatps in the rat intestine as well as in the liver. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: intestinal absorption; drug interactions; OATP; transporters; membrane transport; bioavailability; cyclosporin A; fexofenadine

INTRODUCTION Organic anion transporting polypeptides (OATPs in humans/Oatps in rodents) are responsible for the membrane transport of a wide variety of endogenous and exogenous compounds including therapeutic reagents.1–5 In humans, OATP1A2, OATP1B1, OATP1B3, and OATP2B1 are involved in transport of a large number of drugs, that is, they play important roles in their intestinal absorption, hepatic uptake, and tissue distribution.6–7 OATP1A2 and OATP2B1 are reportedly expressed in the human intestine, whereas Oatp1a1, Oatp1a4, Oatp1a5, and Oatp2b1 mRNAhave been detected in the rat intestine.4,8–11 In addition, OATP1A2 and OATP2B1 have been demonstrated to be localized on the apical membrane of the human intestine and are suggested to be involved in the intestinal uptake of fexofenadine (FEX).4,8 In rats, FEX is a substrate of Oatp1a5, although this transCorrespondence to: Toshiharu Horie (Telephone: + 81-43-2262886; Fax: + 81-43-226-2886; E-mail: [email protected]) Journal of Pharmaceutical Sciences © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association

porter has not been confirmed to play a role in FEX uptake in the intestine in vivo.11–12 On the contrary, P-glycoprotein (P-gp) is involved in the intestinal efflux of FEX in rats.11 Recently, a number of drug–drug interactions (DDIs) caused by transporter inhibition have been reported in clinical situations.13–16 Many such interactions have been reported to be associated with the inhibition of OATP1B1 in the liver, which results in reduced hepatic clearance of drugs and increase in their systemic exposure.3,15 In the intestine, there have also been many examples of DDIs involving the inhibition of P-gp-mediated drug efflux.17–18 More recently, Dresser et al.12 reported that the concomitant intake of fruit juice inhibits the intestinal absorption of FEX by inhibiting OATP-mediated intestinal uptake. After this report, the plasma concentrations of talinolol, aliskiren, and montelukast were reported to be reduced by the concomitant intake of grapefruit juice (GFJ).19–21 Azithromycin and clarithromycin are also substrates of human and rat OATP/Oatp.22–23 Garver et al.22 reported that intestinal Oatps might be involved in the absorption of azithromycin and JOURNAL OF PHARMACEUTICAL SCIENCES

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clarithromycin and the coadministration of rifamycin SV reduced their intestinal absorption in rats, resulting in reduced plasma concentrations of these drugs. In the case of talinolol, species differences in the effects of GFJ on its intestinal absorption were observed between rats and humans, that is, its plasma concentration was increased in rats when it was coadministered with GFJ.19,24–25 This can be explained by the different affinities of naringin, an ingredient of GFJ, for OATP/Oatp and multidrug resistance 1 (MDR1 for humans/Mdr1 for rodents: P-gp) between the two species. Previously, we reported that cyclosporin A (CsA) causes long-lasting inhibition of the hepatic uptake of sulfobromophthalein (BSP) in rats, resulting in a DDI between these drugs.26 This can be explained by the inhibition of Oatps because BSP is a substrate of these transporters. In addition, we reported that CsA is a long-lasting inhibitor of human OATP1B1 and OATP1B3.27 In the present study, we attempted to examine the effect of CsA on Oatp-mediated uptake in the rat intestine, focusing on long-lasting inhibition. We used FEX as a probe drug to examine Oatpmediated intestinal uptake, although P-gp is also involved in its intestinal efflux, and examined the effect of concomitant administration of CsA on the pharmacokinetics of FEX in rats.

MATERIALS AND METHODS Reagents Fexofenadine hydrochloride was purchased from Tokyo Chemical Industry Company, Ltd. (Tokyo, Japan). CsA was purchased from Wako Pure Chemicals (Osaka, Japan). Glipizide was purchased from Enzo Life Sciences, Inc. (Farmingdale, New York). All other reagents were of analytical grade. Animals All studies were conducted in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the National Institutes of Health (Bethesda, Maryland) and the Guidelines for Animal Studies of Chiba University. All protocols were approved by the Chiba University Institutional Animal Care and Use Committee. Seven-to-eight-weekold Sprague–Dawley rats were purchased from Nihon SLC (Shizuoka, Japan). The animals were housed in an air-conditioned room (25◦ C) under a 12-h light— dark cycle for at least 1 week before use. Food (the MF diet; Oriental Yeast, Tokyo, Japan) and water were provided ad libitum. Determination of Plasma FEX Concentration in Rats Prior to the study, CsA (10 mg/kg dissolved in olive oil) or vehicle was orally administered to the rats. AfJOURNAL OF PHARMACEUTICAL SCIENCES

ter overnight fasting, femoral artery of the rat was cannulated with polyethylene tube (PE-45; Natsume Seisakusho, Tokyo, Japan) under light ether anesthesia. In the rats in which FEX was administered intravenously, the femoral vein was also cannulated with PE-31 tubing (Natsume Seisakusho). After the operation, the rats were kept in Ballman cages with free access to water. FEX was administered intravenously as a bolus (1 mg/kg body weight) or orally (10 mg/kg body weight) at 3 or 24 h after the administration of CsA or vehicle. Blood samples were collected before the administration of FEX and 5, 10, 20, 30, 45, 60, 90, and 120 min or 5, 10, 20, 30, 45, 60, 90, 120, and 240 min after its intravenous or oral administration, respectively. Then, plasma samples were prepared by centrifugation of the blood samples (15,000 g × 5 min) using a bench-top centrifugator (Sigma 1-13; Sigma Laborzentrifugen, Osterode am Harz, Germany). The samples were stored at −20◦ C until the analysis. The concentration of FEX was determined using liquid chromatography–tandem mass spectrometry (LC– MS–MS). Determination of the Blood Concentration of CsA in Rats CsA (10 mg/kg) was orally administered to rats. At 3, 12, and 24 h after the administration of CsA, blood samples were collected from jugular vein of the rats and treated with ethylenediamine-N,N,N ,N tetraacetic acid, disodium salt, dihydrate (1 mg/mL). The samples were stored at −20◦ C until the analysis. The blood concentrations of CsA were determined by radioimmunoassay using the CYCLO-Trac radioimmunoassay kit (DiaSorin, Stillwater, Minnesota) according to the manufacturer’s instructions. LC–MS–MS Analysis of FEX The Agilent 1100 Series high-performance liquid chromatography system (Agilent Technologies, Santa Clara, California) was used in combination with QTRAP (AB Sciex, Foster City, California) for the LC–MS–MS analysis of FEX. Five hundred microliters of acetonitrile and 50 ng glipizide as an internal standard were added to 0.1 mL of the plasma sample and mixed well, before being centrifuged at 12,000 gat 4◦ C for 5 min (Model 3740; Kubota, Tokyo, Japan). R The supernatant was filtrated through a Millex -LH filter unit (pore size: 0.45 :m; Millipore Corporation, Bedford, Massachusetts) and evaporated to dryness. Then, the sample was dissolved in the mobile phase (acetonitrile–0.2% formic acid in water = 1:1), inR  jected into a Shodex ODP2 HP-2D column (Showa Denko, Tokyo, Japan, 5 :m, 2.0 × 150 mm2 ), and separated at 40◦ C at the flow rate of 0.2 mL/min. Selected reaction monitoring was used to detect FEX (positive ion mode, m/z 502.1 → 466.2) and glipizide (internal DOI 10.1002/jps

LONG-LASTING INHIBITION OF OATP IN RAT INSTESTINE BY CSA

standard: m/z 446.0 → 321.1). Using this method, linearity was observed for 0.1–10 ng of FEX.

Table 1. Primers Used for the Semiquantitative Real-Time PCR Analysis of Transporter Expression in Rat Ileal Mucosa Samples Oatp2b1

Intestinal RNA Extraction and Real-Time Reverse-Transcription Polymerase Chain Reaction Total RNA was extracted from ileal mucosa samples obtained from the rats at 24 h after the oral administration of CsA (10 mg/kg) or vehicle using Trizol (Life Technologies, Carlsbad, California) according to the protocol supplied by the manufacturer with some modification. The ileum mucosa (100 mg) was lysed with 1 mL of the reagent and incubated at room temperature for 5 min. Then, 0.2 mL chloroform was added and shaken vigorously using a vortex mixer, before being centrifuged at 12,000 g for 15 min at 4◦ C using the type 3740 centrifugator (Kubota). The upper aqueous phase was transferred into a fresh tube and mixed with 0.4 mL isopropyl alcohol. After incubation at room temperature for 10 min, the mixture was centrifuged at 12,000 g for 15 min at 4◦ C. Then, the precipitated RNA was washed with 70% ethanol and dissolved in diethylpyrocarbonate-treated water. The concentration and purity of RNA were determined spectrometrically. Then, complementary DNA was synthesized using the TaKaRa RNA PCR kit (AMV) ver. 3.0 (Takara Bio, Otsu, Japan), according to the manufacturer’s instruction. Total RNA (400 ng) was mixed with reaction buffer, 5 mM MgCl2 , deoxyribonucleotide triphosphate (dNTP) mixture (1 mM each), ribonuclease (RNase) inhibitor (1 U/:L), avian myeloblastosis virus (AMV) reverse-transcriptase XL (0.25 U/:L), and random 9-mers (2.5 :M) in a final volume of 10 :L. The reaction mixture was incubated at 30◦ C for 10 min followed by 50◦ C for 30 min and then heated at 99◦ C to inactivate the enzyme. Then, the sample was mixed with SYBR Green Real Time PCR Master Mix Plus (Toyobo, Osaka, Japan) and 30 :M of the primers described in Table 1, before being subjected to real-time semiquantitative polymerase chain reaction (PCR)using the ECO RealTime PCR system (Illumina, San Diego, California). All primers were commercially synthesized by Nihon Gene Research Laboratories (Sendai, Japan). The reaction mixture was incubated at 50◦ C for 2 min and heated at 95◦ C for 10 min. Then, real-time PCR was carried out by 40 cycles of denaturation at 95◦ C for 10 s and annealing/extension at 63◦ C for 1 min. Data Analysis From the plasma concentration–time profile of FEX, its area under the plasma concentration–time curve (AUCp ) to the last sampling point or infinity was calculated using a computer program, Napp (The University of Tokyo Hospital, Tokyo, Japan) by logtrapezoidal method. The total body clearance of FEX based on its plasma concentration (CLtot,p ) was calcuDOI 10.1002/jps

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Mdr1a Mdr1b Villin

5 5 5 5 5 5 5 5

(Forward) (Reverse) (Forward) (Reverse) (Forward) (Reverse) (Forward) (Reverse)

acg act ttg ccc acc ata gc 3 cca cgt aaa ggc gta gca tga 3 tct ggc ggc cat tat cca t 3 tca gag tac ggt tgt ttc cta cat t 3 tga atc cca aag tga cac tgg t 3 ata ctt ctg cga att gat ctc ctt a 3 aga gat ccg aga cca gca 3 tcg gag tca gac aca tgg 3

lated by the following equation: CLtot,p =

Dosei.v. AUCp,0−inf , i.v.

(1)

where Dosei.v. and AUCp,0–inf,i.v. represent the amount of FEX intravenously administered to rats and plasma AUCp of FEX from zero to infinity after its intravenous administration, respectively. Bioavailability (F) of FEX was calculated by the following equation: F=

(AUCp,0−inf , p.o. /Dosep.o. ) (AUCp,0−inf ,i.v. /Dosei.v. )

(2)

where Dosep.o. and AUCp,0–inf,p.o. represent the amount of FEX orally administered to rats and plasma AUC of FEX from zero to infinity after its oral administration, respectively. The mean residence time of FEX was calculated using Napp. Data are expressed as the mean ± S.E. Statistical comparisons were conducted using the Student’s t-test. Significant difference was assumed when a p value is less than 0.05.

RESULTS Plasma Concentration of FEX After its Intravenous Administration to Rats at 3 h After the Oral Administration of CsA The plasma concentrations of FEX observed after its intravenous administration with or without CsA are shown in Figure 1a. Its plasma concentration–time profile exhibited a biphasic curve, and CsA administration increased the plasma concentration of FEX at all time points. The kinetic parameters of FEX are shown in Table 2. CsA significantly increased the initial concentration (C0 ) and AUCp of FEX to 205% and 213% of the control values, respectively. Accordingly, the CLtot,p of FEX significantly decreased to 44.4% of the control value. After the oral administration of FEX at 3 h after the oral administration of CsA or vehicle, the plasma concentration of FEX was increased by the CsA administration (Fig. 1b), that is, CsA increased the maximum concentration (Cmax ) and AUCp of FEX to 392% and 900% of the control values, JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 1. Plasma concentration of FEX after its intravenous (a) or oral (b) administration at 3 h after the oral administration of vehicle or CsA. The plasma concentration of FEX was examined after its intravenous (1 mg/kg) or oral (10 mg/kg) administration to rats at 3 h after the oral administration of 10 mg/kg CsA (䊉) or vehicle (). Each point represents the mean ± S.E. (n = 3–4).

oral administration at 24 h after the oral administration of CsA or vehicle was minimally affected by the administration of CsA (Fig. 2b), that is, no significant change was observed for Cmax and AUCp of FEX after the coadministration of CsA (Table 3). CsA slightly decreased the F of FEX to 78.7% of the control value.

respectively (Table 2). F of FEX was increased to 423% of the control value by administration of CsA (Table 2). Plasma Concentration of FEX After its Intravenous or Oral Administration to Rats at 24 h After the Oral Administration of CsA The plasma concentration of FEX after its intravenous administration at 24 h after the oral administration of CsA or vehicle was increased by the CsA administration, with the C0 and AUCp of FEX increasing to 226% and 188% of the control values, respectively (Fig. 2a and Table 3). Accordingly, the CLtot,p of FEX decreased to 54.3% of the control value. On the contrary, the plasma concentration of FEX after its

Blood Concentration of CsA Figure 3 shows the blood concentration–time profile of CsA after its oral administration. Three hours after the oral administration of CsA, its concentration in the systemic circulating blood was 0.669 ± 0.163 :M. Its concentration decreased with respect to time following first-order kinetics, and its blood

Table 2. Pharmacokinetic Parameters of FEX After its Administration to Rats at 3 h After the Oral Administration of Vehicle or CsA

Intravenous administration C0 (mg/L) AUCp,0–120 (mg·min/L) AUCp,0–inf (mg·min/L) CLtot,p (mL·min/kg) Mean residence time (MRT; min) Oral administration Cmax (mg/L) AUCp,0–240 (mg·min/L) AUCp,0–inf (mg·min/L) MRT (min) F (%)

CsA (−)

CsA (+)

4.20 ± 0.96 24.4 ± 4.0 27.2 ± 4.6 41.7 ± 7.5 39.1 ± 4.4

8.62 ± 1.39∗ 44.4 ± 6.7∗ 57.9 ± 10.6∗ 18.5 ± 5.7∗ 75.7 ± 13.7∗∗

0.0873 ± 0.0058 6.52 ± 0.94 8.10 ± 1.14 144 ± 32 2.98

0.342 ± 0.039∗∗ 51.8 ± 4.5∗∗∗ 72.9 ± 12.6∗∗ 189 ± 53 12.6

Data are shown as the mean ± S.E. (n = 3–5). ∗ p < 0.05 versus CsA(−). ∗∗ p < 0.01 versus CsA(−). ∗∗∗ p < 0.001 versus CsA(−).

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Figure 2. Plasma concentration of FEX after its intravenous (a) or oral (b) administration at 24 h after the oral administration of vehicle or CsA. The plasma concentration of FEX was examined after its intravenous (1 mg/kg) or oral (10 mg/kg) administration to rats at 3 h after the oral administration of 10 mg/kg CsA (䊉) or vehicle (). Each point represents the mean ± S.E. (n = 3).

concentrations at 12 and 24 h after its oral administration were 0.384 ± 0.124 and 0.137 ± 0.045 :M, respectively. Expression Level of mRNA for Oatp2b1, Mdr1a, and Mdr1b in Rats We examined the mRNA expression levels of Oatp2b1, Mdr1a, and Mdr1b in the rat intestine at 24 h after the oral administration of CsA or vehicle (Fig. 4). No significant difference in the mRNA expression level of these transporters was observed between the groups.

DISCUSSION Previously, we reported that CsA has a long-lasting inhibitory effect on human OATP1B1 and OATP1B3,

and the hepatic uptake of BSP in rats, which is mediated by Oatps.26–27 In the present study, we examined the effect of the administration of CsA on the plasma concentration of FEX after its intravenous or oral administration to rats at 3 or 24 h after the oral administration of CsA. In particular, we focused on its long-lasting inhibitory effect on Oatps in the intestine. In the present study, FEX was used as a probe drug to examine the inhibitory effect of CsA on intestinal Oatps. In humans, in vitro analyses and in vivo studies on subjects carrying polymorphisms in OATP family transporters suggested that OATP1B1 and OATP1B3 are involved in the hepatic uptake of FEX and that OATP1A2 and OATP2B1 mediate its intestinal uptake.8,28–31 Thus, inhibition of intestinal

Table 3. Pharmacokinetic Parameters of FEX After its Administration to Rats at 1 Day After the Oral Administration of Vehicle or CsA CsA (−) Intravenous administration C0 (mg/L) AUCp,0–120 (mg·min/L) AUCp,0–inf (mg·min/L) CLtot,p (mL·min/kg) Mean residence time (MRT; min) Oral administration Cmax (mg/L) AUCp,0–240 (mg·min/L) AUCp,0–inf (mg·min/L) MRT (min) F (%)

2.89 ± 0.21 17.5 ± 0.9 21.8 ± 0.5 46.0 ± 0.1 68.9 ± 18.5 0.0418 ± 0.0080 3.20 ± 1.16 3.87 ± 1.26 165 ± 66 1.78

CsA (+) 6.53 ± 0.40∗∗∗ 33.6 ± 2.4∗∗ 41.0 ± 4.2∗∗ 25.0 ± 2.8∗∗ 60.9 ± 13.4 0.0685 ± 0.0131 3.96 ± 0.64 5.73 ± 1.19 205 ± 42 1.40

Data are shown as the mean ± S.E. (n = 3). ∗∗ p < 0.01 versus CsA(−). ∗∗∗ p < 0.001 versus CsA(−).

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Figure 3. Blood concentration of CsA after its oral administration. The blood concentration of CsA was examined after its oral administration (10 mg/kg). Each point represents the mean ± S.E. (n = 3).

OATPs by concomitant intake of fruit juices leads to reduction in the intestinal absorption and systemic exposure of FEX.12,32 On the contrary, GFJ increased mucosal to serosal transport of FEX in rat intestine because of inhibition of P-gp-mediated efflux.33 However, concomitant administration of apple or orange juice resulted in the reduced absorption of FEX in rats in vivo in a dose-dependent manner.34 It may suggest the involvement of intestinal Oatps in absorption of FEX in rats because these fruit juices inhibit OATP/Oatp family transporters in humans and

rats.12 Actually, knocking out of Oatp1a/1b significantly reduced the intestinal accumulation of FEX in mice.35 In addition, Kikuchi et al.36 showed saturable transport of FEX in mucosal to serosal direction across upper small intestine tissue of rats using Ussing-type chamber. However, MacLean et al.11 reported that regional absorption of FEX in rat intestine did not correlate with the mRNA expression levels of Oatp1a5 and Oatp2b1. Thus, the contribution of uptake transporters to the intestinal uptake of FEX may be low, although uptake transporters including Oatps are partly involved in it. In the liver, Oatp1a1 and Oatp1a4 are reinvolved in the uptake of FEX.37 In addition to these transporters, Matsushima et al. reported that multidrug resistance-associated protein 2, bile salt export pump, and another unknown transporter(s) are involved in the biliary excretion of FEX, and Mrp3 is involved in its sinusoidal efflux in mouse liver.38 The transporters involved in the FEX transport in rodents are shown in Figure 5. The CLtot,p of FEX after its intravenous administration [41.7–46.0 mL/min/kg; Tables 2 and 3] was similar to the hepatic blood flow rate, and it was within the range of reported values [22–93 mL/min/kg].39–40 After the intravenous administration of FEX, its CLtot,p was decreased at both 3 and 24 h after the coadministration of CsA, possibly due to the inhibition of hepatic uptake transporters (Tables 2 and 3). Inhibition of hepatic Oatps by CsA should be discussed in relation to its concentration in the blood. CsA concentration in circulating blood was 0.67 and 0.14 :M at 3 and 24 h after its oral administration, respectively (Fig. 3). On the contrary, CsA concentration to produce 50% inhibition (IC50 ) on Oatp-mediated hepatic uptake is approximately 0.2 :M.2641–42 Considering that the unbound fraction of CsA in the blood is 10%43 ; the CsA concentration in the systemic circulation examined in

Figure 4. The mRNA expression levels of transporters in the rat ileal mucosa. The mRNA expression levels of Oatp2b1 (a), Mdr1a (b), and Mdr1b (c) were determined by real-time PCR. Their expression levels were normalized to the expression of villin mRNA. Data are shown as the mean ± S.E. [% of the CsA-untreated control]. JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 5. Schematic diagram representing the effect of CsA on the transport of FEX in rats. The transporters responsible for the transport of FEX are shown. Those affected by CsA administration are shown as black symbols. Three hours after the administration of CsA, it inhibits hepatic and intestinal Oatps and intestinal P-gp. In the intestine, CsA preferentially inhibits P-gp. On the contrary, 24 h after the administration of CsA, it inhibits hepatic and intestinal Oatps more strongly than intestinal P-gp.

the present study was too low to cause inhibition of hepatic Oatps in rats. Actually, addition of 90% rat plasma in incubation buffer increased the IC50 value of CsA for the uptake of cerivastatin to 2.3 :M.42 At 24 h after the oral administration of CsA, its total blood concentration was lower than its reported IC50 values for Oatp. Thus, it is less likely that CsA inhibits hepatic Oatps by competitive inhibition in the present study. This can be explained by the long-lasting inhibitory effect of CsA on Oatp-mediated hepatic uptake in rats, as shown in our previous report in which BSP was used as a substrate.26 Possibly because of the long-lasting inhibitory effect, the increases in the AUCp of FEX after its intravenous administration at 3 and 24 h after the administration of CsA were similar, although the blood concentration of CsA at 24 h after its administration was much lower than that observed at 3 h after its administration (Figs. 1–3 and Tables 2 and 3). On the contrary, different effects of CsA on the plasma concentration of orally administered FEX were observed at 3 and 24 h after its administration (Figs. 1 and 2). The plasma concentration of FEX was increased when it was orally administered at 3 h after the administration of CsA (Fig. 1), which might have been due to the inhibition of both its P-gp-mediated intestinal efflux and its hepatic uptake by CsA (Fig. 6a). However, when FEX was orally adminisDOI 10.1002/jps

tered at 24 h after the administration of CsA, its plasma concentration was not significantly different from that observed in the vehicle-treated control (Fig. 2). Considering that the hepatic uptake of FEX was reduced even at 24 h after the administration of CsA, the absence of a significant change in the plasma concentration of FEX at 24 h after its oral administration can be explained by reductions in its intestinal absorption and hepatic uptake mediated by Oatps to the similar extent (Fig. 6b). Thus, the hepatic availability (Fh ) was increased and fraction absorbed in intestine as unchanged form (Fa ·Fg ) was decreased, resulting in slightly decreased or unchanged F (=Fa ·Fg ·Fh ). At 24 h after the administration of CsA, it inhibited the intestinal uptake of FEX more strongly than P-gp-mediated efflux. Actually, CsA concentration in the circulating blood (0.14 :M) was lower than its reported IC50 or inhibition constants on the human MDR1 (0.35–4.7 :M).44–48 Assuming the CsA concentration exposed to the intestinal uptake transporter(s), that is, its concentration in intestinal lumen is negligibly low at 24 h after its oral administration, the inhibitory effect of CsA on the intestinal Oatps should be the long-lasting one even after its removal. Inhibition of intestinal uptake of FEX by CsA should also be observed at 3 h after the oral administration of CsA. However, at that time point, P-gp inhibition JOURNAL OF PHARMACEUTICAL SCIENCES

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affected the plasma concentration of FEX to a higher extent than Oatp inhibition. In the present study, the mechanism of long-lasting inhibition of intestinal Oatps remains to be elucidated. We showed that the mRNA expressions of Oatp2b1 and Mdr1a/1b were not changed by CsA (Fig. 4). Previously, we proposed that long-lasting inhibition of CsA on the hepatic OATPs/Oatps may be related to the inhibitor concentration in the liver, but not the concentration of the inhibitor exposed to the transporter.26–27 However, P-gp inhibition should be related to the intracellular concentration of inhibitors because this transporter is an efflux transporter from inside the cells. Thus, the affinity of CsA as a transporter inhibitor may be higher for intestinal Oatps than for P-gp. It may be possible that different mechanisms are underlying for the Oatp and P-gp inhibitions. Although we could not detect altered localization of OATP1B1 in transporter-expressing cells by CsA treatment,27 human OATP2B1 was reported to be internalized after protein kinase C activation, resulting in changes in its transport activity.49 Recently, allosteric effect of nonsteroidal anti-inflammatory drugs was reported for OATP1B1 and OATP1B3.50 Thus, various mechanisms have been reported for altered transport activity of OATPs. Further studies are required to clarify the mechanism of long-lasting inhibition on intestinal Oatps. Uptake transporters for drug absorption in the intestine are important determinants of drug disposition. In humans, fruit juices alter the plasma concentration of FEX by inhibiting intestinal OATPs.51 Genetic polymorphisms in OATP2B1 also alter the intestinal absorption of various drugs including FEX, montelukast, and celiprolol, resulting in changes in their plasma concentrations.3152–53 Thus, long-lasting inhibition of intestinal OATP/Oatp by CsA might cause DDIs during intestinal absorption and reduce the plasma concentrations of drugs. However, CsA is a potent inhibitor of hepatic OATP/Oatp and may increase the plasma concentrations of the substrate drugs of these transporters in the liver due to reduced hepatic clearances even if they are substrates of intestinal OATP/Oatp.354–55 Among intestinal OATP/ Oatp substrates, levofloxacin is eliminated mainly by urinary excretion.56 Concomitant intake of fruit juice decreased its plasma concentration in humans, possibly due to the inhibition of intestinal OATPs.57 Accordingly, the long-lasting inhibition of intestinal OATP/Oatp by CsA might decrease the concentration of such drugs. In the present study, we first showed that CsA has long-lasting inhibitory effects on Oatps in the intestine as well as those in the liver. In rats, the longlasting inhibition of intestinal Oatps was observed at least 1 day after oral administration of CsA even after its elimination from the systemic circulation. Thus, JOURNAL OF PHARMACEUTICAL SCIENCES

also for humans, inhibition of intestinal OATPs by CsA may cause DDIs even after the concentration of CsA in circulating blood or intestine decreases below the concentration, which causes a significant inhibition on these transporters. The long-lasting inhibition of intestinal OATPs/Oatps by CsA should be cautious in clinical practices to avoid DDIs by the process of OATP/Oatp-mediated intestinal absorption.

ACKNOWLEDGMENTS This study was, in part, supported by the Grant-inAid for Young Scientists (B) provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan (#21790142). We are also grateful for The Uehara Memorial Foundation for supporting our research.

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