The Effects of Jabara Juice on the Intestinal Permeation of Fexofenadine

Abstract

Jabara juice and its component narirutin inhibit the activity of organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1, which are considered to play significant roles in the intestinal absorption of fexofenadine. In this study, we investigated the effects of jabara juice on the intestinal absorption of fexofenadine in mice and the inhibitory effects of jabara juice and narirutin on the permeation of fexofenadine using Caco-2 cell monolayers and LLC-GA5-COL300 cell monolayers. In the in vivo study, the area under the plasma concentration–time curve (AUC) of fexofenadine in mice was increased 1.8-fold by jabara juice. In the permeation study, 5% jabara juice significantly decreased the efflux ratio (ER) of fexofenadine for Caco-2 monolayers. Furthermore, the ERs of fexofenadine and digoxin, which is a typical substrate of P-glycoprotein (P-gp), for LLC-GA5-COL300 cell monolayers were decreased in a concentration-dependent manner by jabara juice extract, suggesting that jabara juice may increase the intestinal absorption of fexofenadine by inhibiting P-gp, rather than by narirutin inhibiting OATPs. The present study showed that jabara juice increases the intestinal absorption of fexofenadine both in vivo and in vitro. The intestinal absorption of fexofenadine may be altered by the co-administration of jabara juice in the clinical setting.

INTRODUCTION

Jabara is a citrus fruit that grows in Japan. Recently, jabara has become increasingly popular as a dietary supplement for relieving pollen allergy symptoms because it contains a high amount of narirutin, which is considered to reduce inflammation by decreasing interleukin-4 and immunoglobulin E (IgE) levels.^1–3) Fexofenadine, a histamine receptor antagonist, is widely used for the treatment of allergies.⁴⁾ Therefore, jabara and fexofenadine may be taken simultaneously by patients with allergies. However, the influence of jabara juice on the intestinal absorption of fexofenadine has not been investigated.

Fexofenadine was reported to be a substrate of organic anion-transporting polypeptides (OATPs) 1A2⁵⁾ and 2B1,⁶⁾ which are involved in the intestinal absorption of various drugs.⁷⁾ Although controversy has existed over the expression of OATP1A2 in the human small intestine, Couto et al. have recently determined the expression level of OATP1A2 in the human jejunum to be 1.5 fmol/µg protein, which is comparable to that of OATP2B1 (2.65 fmol/µg protein).⁸⁾ Therefore, both transporters may contribute to the intestinal absorption of fexofenadine. Imanaga et al. reported that the area under the plasma concentration–time curve (AUC) of fexofenadine in subjects with the OATP2B1 c.1457 C > T variant was significantly decreased to 63% of that observed in subjects with wild-type OATP2B1, suggesting that OATP2B1 plays an essential role in the intestinal absorption of fexofenadine.⁹⁾ Moreover, intestinal OATPs are known to be the target molecules of food– and beverage–drug interactions.¹⁰⁾ Indeed, Dresser et al. reported that apple juice, orange juice, and grapefruit juice decreased the AUC of fexofenadine to 26.9, 30.6, and 36.7% of the control value, respectively.¹¹⁾ Phloridzin in apple juice,¹²⁾ hesperidin in orange juice,¹³⁾ and naringin in grapefruit juice¹⁴⁾ have been identified as OATP inhibitors. Regarding jabara, we have previously reported in an in vitro uptake study using HEK293 cells expressing OATP1A2 or 2B1 that jabara juice significantly inhibited the activities of both OATP1A2 and OATP2B1, and we identified narirutin as a novel OATP inhibitor that is found in jabara juice.¹⁵⁾ Therefore, the beverage–drug interaction between jabara juice and fexofenadine may be of clinical concern.

P-glycoprotein (P-gp) is an essential efflux transporter,¹⁶⁾ and hence, also affects the absorption of fexofenadine. Miyake et al. reported that the AUC of fexofenadine was 10.6-fold higher in P-gp knockout mice.¹⁷⁾ In addition, repetitive treatment with rifampin (600 mg once a day for 6 d), a potent inducer of P-gp, has been shown to increase the oral clearance of fexofenadine without affecting its elimination half-life in humans.¹⁸⁾ P-gp is also considered to be a target for food– and beverage–drug interactions. Indeed, Kampo, green tea, fruit juice, and their components have been reported to modify the activity of P-gp in in vivo or in vitro studies.^19–21) Moreover, jabara peel contains various P-gp inhibitors, such as 3,3,′4′5,6,7,8-heptamethoxyflavone (HMF) and 3,3′4′6,7,8-hexamethoxyflavone (nobiletin; NBL).^2,22–24) Therefore, jabara juice may affect the absorption of fexofenadine in different directions by inhibiting OATP1A2/2B1 and P-gp. However, the effects of jabara juice on the intestinal absorption of fexofenadine remain unclear.

In an in vivo study involving mice, we previously reported that cranberry juice, an OATP inhibitor, reduced the AUC of fexofenadine to 49% of the control value.²⁵⁾ In addition, Medwid et al. reported that the AUC of oral fexofenadine was 41% lower in Oatp2b1-knockout mice than in wild-type mice,²⁶⁾ suggesting that mice may be useful for assessing food–drug interactions between jabara juice and fexofenadine caused by the inhibition of OATPs. Other useful experimental models include cultured monolayers of intestinal epithelial cells, such as Caco-2 cells,²⁷⁾ and P-gp-overexpressing cell lines, such as LLC-GA5-COL300 cells. After being cultured for 21 to 29 d, Caco-2 cells differentiate into small intestinal epithelium-like cells with microvilli and express intestinal transporters, such as OATP1A2/2B1 and P-gp.²⁸⁾ In this study, we aimed to investigate the effects of jabara juice on the pharmacokinetics of fexofenadine in an in vivo study using mice. We also aimed to quantitatively assess the effects of jabara juice and narirutin on the permeation of fexofenadine across Caco-2 cell monolayers and the inhibitory effects of jabara juice extract on P-gp activity using LLC-GA5-COL300 cell monolayers that stably expressed P-gp.

MATERIALS AND METHODS

Materials

Fexofenadine hydrochloride, digoxin, and digitoxin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Fexofenadine-d₆ was purchased from MedChemExpress (Monmouth Junction, NJ, U.S.A.). Jabara juice was purchased from the Kitayamamura Jabaramura Center (Wakayama, Japan). Narirutin was purchased from Sigma-Aldrich (Dorset, U.K.). The unlabeled (standard) peptides and stable-isotope-labeled peptides (internal standard) used for the quantitative targeted absolute proteomics (QTAP) were purchased from SCRUM Inc. (Tokyo, Japan). Unless otherwise stated, all other reagents were purchased from Nacalai Tesque (Kyoto, Japan). Male ICR mice were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). Caco-2 cells and LLC-GA5-COL300 cells were acquired from the RIKEN Bioresource Research Center Cell Bank (Tsukuba, Japan).

QTAP Using LC-Tandem Mass Spectrometry (LC-MS/MS)

To determine the expression levels of OATP1A2, OATP2B1, P-gp, and multi-drug resistance protein 2 (MRP2) on the plasma membranes of Caco-2 cells using QTAP, plasma membrane samples were prepared using the sucrose cushion method and phase-transfer surfactant (PTS) method, as described previously.^29,30) Briefly, after being washed with Tris-sucrose buffer (10 mM Tris, 250 mM sucrose, and 1 mM ethylenediaminetetraacetic acid (EDTA); pH 7.4), the cells were subjected to nitrogen cavitation at 400 psi for 15 min on ice. After being centrifuged at 10000 × g for 10 min at 4 °C, the supernatant was gently layered on top of 38% (w/v) sucrose solution and centrifuged at 100000 × g for 40 min at 4 °C. The turbid layer at the interface was collected and centrifuged at 100000 × g for 40 min at 4 °C.²⁴⁾ Then, the pellet was dissolved in 150 µL of PTS solution (12 mM sodium dodecyl sulfate (SDS), 12 mM N-lauroylsarcosine sodium salt (SLS), and 100 mM Tris–HCl; pH 9.0), before being heated for 5 min at 95 °C. The protein concentration of the sample was then measured with a Pierce™ bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.), according to the manufacturer’s instructions.

Solution samples (containing 50 µg protein/sample) were reduced with 10 mM dithiothreitol at room temperature for 30 min, alkylated with 50 mM indole-3-acetic acid in the dark at room temperature for 30 min, and diluted four-fold with 50 mM ammonium bicarbonate. Then, 2.5 µL of 1% ProteaseMax solution (Promega, Madison, WI, U.S.A.) (final concentration: 0.01%), 1% lysyl endopeptidase (LysC), 1% of the total amount of protein (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 1% trypsin (Promega) were added to digest the peptide samples. The amounts of LysC and trypsin added to each sample accounted for 1% each of the total amount of protein present. The internal standards (IS) were then added to both the unknown and standard samples. The concentrations of the standard samples were set to 0, 0.3, 1, 3, 10, 30, and 100 nM. To remove the SDS and SLS, equal volumes of ethyl acetate and 0.5% trifluoroacetic acid were added, and the mixture was vortexed for 1 min. The mixture was centrifuged at 15000 × g for 2 min at 25 °C to separate the aqueous phase (liquid-liquid extraction), before the liquid-liquid extraction procedure was repeated. The aqueous phase was then subjected to desalting using graphite carbon (GC) and styrene-divinylbenzene (SDB) tips (GL Sciences Inc., Tokyo, Japan). An aliquot of 10 µg of protein/10 µL of the sample was injected into the LC-MS/MS system and separated using an XBridge BEH130 C18 column (3.5 µm, internal diameter: 1.0 × 100 mm; Waters). The mobile phase consisted of acetonitrile and 0.1% formic acid. It was pumped at a rate of 0.1 mL/min, and the gradient conditions were as follows (acetonitrile concentration): 5% (0–5 min)→5–80% (5–20 min)→95% (20–23 min)→5% (24–30 min). The MS/MS analysis was performed in positive-ion mode.^31,32) The transitions of the OATP1A2-, OATP2B1-, P-gp, and MRP2-specific peptides and the corresponding IS peptides detected by LC-MS/MS are listed in Supplementary Table 1.

Cell Culture

Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with glucose (4.5 g/L), 10% heat-inactivated fetal bovine serum (Biowest, Miami, FL, U.S.A.), 1% penicillin-streptomycin, and 1% non-essential amino acid mixed solution (Nacalai Tesque). The cells were seeded onto polycarbonate membrane inserts (pore size: 0.3 µm, growth area: 1.12 cm²) on 12-well plates (Transwell^®, Corning, NY, U.S.A.) at a density of 6.4 × 10⁴ cells/cm². Then, they were cultured at 37 °C, in an atmosphere containing 5% CO₂ and at 95% relative humidity, for 23–26 d to induce differentiation. Starting on day 7, the medium was changed every other day. To ensure the epithelial integrity of the Caco-2 cell monolayers, their transepithelial electrical resistance (TEER) was monitored using an ohmmeter (Millicell-ERS, Millipore, Billerica, MA, U.S.A.) during their incubation. TEER values were calculated using the following equation:

  

(1)

, where R_mono is the resistance in the presence of both the cell monolayer and filter, R_blank is the resistance of the filter itself, and A is the surface area of the filter (1.12 cm²).

LLC-GA5-COL300 cells were seeded onto a polystyrene membrane insert (pore size: 8.0 µm, growth area: 1.13 cm²; Thermo Fisher Scientific) coated with collagen I (Thermo Fisher Scientific) at a density of 5.0 × 10⁵ cells/cm² and grown in Medium 199 (Sigma-Aldrich), 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 300 ng/mL colchicine mixed solution at 37 °C, in 5% CO₂, and at 95% relative humidity for 4 d. From day 2 after the seeding, the medium was changed daily. It was replaced with colchicine-free medium 4 h before the experiment. To ensure the integrity of the LLC-GA5-COL300 cell monolayers, their TEER values were calculated according to the method described above.

Extraction of Jabara Juice Components

An ethyl acetate extract of jabara juice was used for the P-gp inhibition assay. After removing any sediment from the jabara juice by centrifuging it at 10000 × g for 20 min at 4 °C, the supernatant was filtered through a 0.2-µm filter. The filtrate was mixed with a double volume of ethyl acetate and shaken for 20 min. After being centrifuged at 2000 × g for 10 min at 4 °C, the organic layer was separated and evaporated under a nitrogen flow. The residue was dissolved in Hank’s balanced salt solution (HBSS; 125 mM NaCl, 4.8 mM KCl, 5.6 mM glucose, 1.2 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, and 25 mM 2-(N-morpholino) ethane sulfonic acid (pH 6.3) or 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4)) to produce an identical volume to that of the original juice.

Permeation Assay

After removing the growth media, the monolayers of Caco-2 or LLC-GA5-COL300 cells were washed with HBSS. They were then pre-incubated with 500 µL of pH 6.3 HBSS (apical side) and 1500 µL of pH 7.4 HBSS (basolateral side) at 37 °C under gentle shaking at 30 rpm for 30 min. After the pre-incubation procedure, the permeation assay was initiated by replacing the solution in the donor chamber with HBSS containing fluorescein isothiocyanate (FITC)-inulin (Sigma-Aldrich; molecular weight: 2000–5000) and fexofenadine (3 or 200 µM) or digoxin (1 µM). Then, the receiver chamber solution was replaced with 1.5 mL of fresh incubation solution. The plate was gently shaken at 37 °C and 30 rpm throughout the permeation assay. After the permeation assay had been initiated, a 25-µL aliquot of the sample solution was collected from the receiver chamber every 15 min until 75 min. Each sample was spiked with 225 µL of 50% methanol and stored at −20 °C until the analysis. An inhibitor (jabara juice, its extract, or narirutin) was added to the apical side during pre-incubation and incubation, while a positive control, 20 µM cyclosporine A, was added to both apical and basal sides during pre-incubation and incubation. To assess the integrity of the monolayers, 50 µL of receiver solution was collected at 75 min for FITC-inulin quantification. The TEER values of the monolayers were also measured after the experiment. The apparent permeability coefficient (P_app) was calculated using Eq. 2, and the efflux ratio (ER) was calculated using Eq. 3.

  

(2)

  

(3)

, where Q (τ), A, and C_o represent the amount of the substrate that had permeated through the monolayer at time τ (nmol), the surface area of the monolayer (cm²), and the initial concentration of the substrate in the donor chamber (µM), respectively. τ represents the incubation time used to calculate the P_app value, which was set at 75 min for this study. P_{app (B to A)} and P_{app (A to B)} represent the apparent permeability from the basolateral side to the apical side and that from the apical side to the basolateral side, respectively (cm/s).

Determination of Fexofenadine and Digoxin Concentrations Using LC-MS/MS

Sample Preparation

A 100-µL aliquot of the sample was spiked with 50 µL of the IS solution (1 ng/mL fexofenadine-d₆ or 10 ng/mL digitoxin), and then 500 µL of ethyl acetate was added. After being vortexed for 30 s, the mixture was centrifuged at 2000 × g for 10 min at 4 °C. Subsequently, 400 µL of the supernatant was collected and evaporated under a nitrogen flow. The resultant residue was redissolved with 50 µL of the mobile phase described below, and 10 µL of the solution was subjected to LC-MS/MS analysis.

The determination of digoxin and digitoxin levels by LC-MS/MS was conducted according to a method previously reported by our group.³³⁾ The concentrations of fexofenadine and digoxin were determined using an LC-MS/MS system consisting of a controller (CBM-20A, Shimadzu, Kyoto, Japan), a pump (LC-20AD, Shimadzu), a triple quadrupole mass spectrometer (LCMS-8050 or LCMS-8030, Shimadzu), and a column oven (CTO-20AC, Shimadzu). To determine the concentration of fexofenadine in a sample, the sample was separated using an octadecylsilane column (150×2.0 mm, 5C₁₈-MS-II, Cosmosil, Nacalai Tesque) equipped with a guard column (50 × 2.0 mm, 5C₁₈-MS-II, Cosmosil). The mobile phase consisted of 0.1% formic acid and ethanol (52 : 48, v/v) and was pumped at a flow rate of 0.2 mL/min. The MS/MS analysis was performed in positive-ion mode using electrospray ionization. Selected reaction monitoring was set to select mass-to-charge ratio (m/z) transitions of 502.3/466.4 m/z and 508.3/472.4 m/z for fexofenadine and fexofenadine-d₆, respectively, and the fexofenadine concentration was determined based on the peak area ratio of fexofenadine to fexofenadine-d₆.

In Vivo Study

Male ICR mice (body weight >30 g; 8 weeks of age) were maintained under standard conditions and given food and water ad libitum until the start of the experiment. Prior to the experiment, the mice were fasted overnight for at least 10 h with free access to water. Fexofenadine (5 mg/mL in 10% dimethyl sulfoxide) was mixed into jabara juice or the control solution (24 mg/mL sucrose and 11 mg/mL citric acid; pH adjusted to 3.5) and orally administered to the mice at a dose of 5 mg/kg (20 mL/kg). Blood samples (40 µL) were collected from the tail vein at 5, 10, and 30 min and 1, 2, 4, 6, 8, 12, and 24 h after the administration of the jabara juice or control solution. The blood samples were centrifuged at 2600 × g for 10 min at 4 °C to separate the plasma. The plasma samples were stored at −20 °C until the analysis. All of the animal experiments were conducted after approval had been obtained from the institutional animal care and use committee of Keio University, and were carried out in accordance with the International Guiding Principles for Biomedical Research Involving Animals (1985).

An aliquot of 5 µL of a plasma sample, 50 µL of fexofenadine-d₆, 5 µL of 50% methanol, and 500 µL of ethyl acetate were added to a glass microtube and vortexed for 1 min, before being centrifuged at 4 °C and 2000 × g for 10 min. An aliquot of 400 µL of the supernatant was collected and evaporated to dryness at 40 °C. The residue was redissolved in 50 µL of the LC-MS/MS mobile phase. LC-MS/MS-based analyses of the levels of fexofenadine and fexofenadine-d₆ were performed as described above.

The AUC (AUC_0–24 h) was calculated using the liner trapezoidal method. The mean residence time (MRT) was calculated by dividing the area under the first moment curve at 24 h by the AUC_0–24 h. The terminal (8–24 h) mono-exponential decline in plasma concentration after dosing was used to calculate the oral half-life (T_1/2) of fexofenadine. Data was included unless oral administration failed.

Statistical Analyses

Statistical comparisons between the jabara juice and control groups were performed using the independent samples t-test. The statistical significance of differences in P_app between the control and inhibitor-treated groups was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. p-Values of less than 0.05 were considered statistically significant.

RESULTS

The Effects of Jabara Juice on the Pharmacokinetics of Fexofenadine in Mice

The peak plasma concentration (C_max) and AUC values of fexofenadine in the jabara juice group were significantly increased to 241 and 185% of those seen in the control group. The t_1/2 value of fexofenadine was not affected by jabara juice (Fig. 1, Table 1).

Fig. 1. Plasma Concentration–Time Profiles of Fexofenadine after Its Oral Administration with/without Jabara Juice in Mice

Fexofenadine was spiked (5 mg/kg, 20 mL/kg) into pseudo-juice (pH adjusted to pH 3.5) (control; n = 6) or commercial jabara juice (n = 6), and the mixture was orally administered to mice. The open and closed circles represent the control and jabara juice groups, respectively. The inset shows a semi-logarithmic plot of the plasma concentration profiles. Each symbol and bar represent the mean ± S.D.

Table 1. Pharmacokinetics Parameters of Fexofenadine When Administration Alone (Control) or with Jabara Juice in Mice

Pharmacokinetics parameters	Control (n = 6)	Jabara juice (n = 6)
Cmax (ng/mL)	18.3 ± 6.84	44.1* ± 19.5
Tmax (h)	2.0 [2.0–4.0]	1.0* [1.0–1.0]
T1/2 (h)	6.45 ± 1.97	4.65 ± 0.94
AUC0–24h (h × ng/mL)	89.6 ± 26.7	165.5* ± 70.2
MRT0–24h (h)	4.65 (3.60–4.68)	2.94*(2.44–3.44)

The parameters were calculated from the data shown in Fig. 1. Data are presented as mean ± S.D. or geometric mean (−1 S.D.–+1 S.D.) T_max is shown as the median [Range]. * p < 0.05, independent samples t-test.

Detection and Quantification of Transporters in Caco-2 Cells

The absolute expression levels of OATP1A2, OATP2B1, P-gp, and MRP2 proteins in the plasma membrane samples were 3.54 ± 0.45, 4.40 ± 1.63, 3.59 ± 0.14, and 12.7 ± 1.55 fmol/µg protein, respectively, and that of Na⁺/K⁺-ATPase was 124.9 ± 13.1 fmol/µg protein (mean ± standard deviation (S.D.) of triplicate experiments).

The Effects of Jabara Juice or Narirutin on the Permeability of 3 or 200 µM Fexofenadine through Caco-2 Cell Monolayers

The P_{app (A to B)} of 3 µM fexofenadine was slightly increased by 1% jabara juice and significantly increased by 5% jabara juice. Cyclosporine A also slightly increased the P_{app (A to B)}. As for transport in the opposite direction, 5% jabara juice and cyclosporine A significantly decreased the P_{app (B to A)} to 67 and 15% of the control value, respectively. As a result, the ER of 3 µM fexofenadine was significantly decreased to 46% of the control value by 5% jabara juice (Fig. 2(a), Table 2).

Fig. 2. The Variations in the Apparent Permeability Coefficients (P_app) of 3 µM (a) and 200 µM (b) Fexofenadine in Caco-2 Cell Monolayers Treated with 1 or 5% Jabara Juice, or 20 µM Cyclosporine A

A to B and B to A represent permeation from the apical to the basolateral side and from the basolateral to the apical side, respectively. The actual P_{app (A to B)} and P_{app (B to A)} of 3 µM fexofenadine alone were (1.10 ± 0.08) × 10⁻⁷ cm/sec and (9.54 ± 0.75) × 10⁻⁷ cm/sec, respectively, and the actual P_{app (A to B)} and P_{app (B to A)} of 200 µM fexofenadine alone were (1.16 ± 0.08) × 10⁻⁷ cm/sec and (13.0 ± 1.30) × 10⁻⁷ cm/sec, respectively. Data are shown as the mean ± S.D., n = 5. * p < 0.05, according to Dunnett’s test (vs. fexofenadine alone).

Table 2. The Efflux Ratios of 3 or 200 µM Fexofenadine (FEX) for Caco-2 Cell Monolayers in the Presence of Diluted 1 or 5% Jabara Juice (JJ_diluted) or 20 µM Cyclosporine A (CsA)

		ER values
	FEX (µM)	3	200
FEX alone		8.74 ± 1.04	11.3 ± 1.6
with JJdiluted (%)
1		7.57 ± 1.47	10.2 ± 1.1
5		4.03* ± 0.66	7.15* ± 1.52
with 20 µM CsA		1.08* ± 0.27	1.09* ± 0.10
with 1% JJdiluted + 20 µM CsA		1.13* ± 0.27	1.07* ± 0.13

* ER: Efflux ratio (P_{app (B to A)}/P_{app (A to B)}), calculated from the data shown in Fig. 2; Data are shown as the mean ± S.D., n = 5. *: p < 0.05, according to Dunnett’s test (vs. fexofenadine alone).

At a higher substrate concentration of 200 µM, the P_{app (A to B)} of 200 µM fexofenadine was not affected by jabara juice or cyclosporine A, while its P_{app (B to A)} value was decreased by both jabara juice and cyclosporine A in a similar manner to that of 3 µM fexofenadine, resulting in a decrease in its ER (Fig. 2(b), Table 2).

Although the change was not significant, narirutin reduced the P_{app (A to B)} of 3 µM fexofenadine to 78 and 67% of the control value at 10 µM and 100 µM, respectively, but did not affect its P_{app (B to A)}, resulting in an increase in the ER of 3 µM fexofenadine (Fig. 3, Table 3).

Fig. 3. The Variations in the Apparent Permeability Coefficients (P_app) of 3 µM Fexofenadine in Caco-2 Cell Monolayers Treated with 10 or 100 µM Narirutin, or 20 µM Cyclosporine A

A to B and B to A represent permeation from the apical to the basolateral side and from the basolateral to the apical side, respectively. The actual P_{app (A to B)} and P_{app (B to A)} of 3 µM fexofenadine alone were (1.18 ± 0.30) × 10⁻⁷ cm/s and (11.1 ± 1.90) × 10⁻⁷ cm/s, respectively. Data are shown as the mean ± S.D., n = 5. * p < 0.05, according to Dunnett’s test (vs. fexofenadine alone).

Table 3. The Efflux Ratios of 3 µM Fexofenadine (FEX) for Caco-2 Cell Monolayers after the Coadministration of 10 µM or 100 µM Narirutin (NAR) or 20 µM Cyclosporine A (CsA)

	ER values
3 µM FEX alone	9.87 ± 4.39
With NAR (µM)
10	14.7 ± 2.25
100	14.9 ± 4.99
With 20 µM CsA	0.895* ± 0.208
With 10 µM NAR +20 µM CsA	0.928* ± 0.265

* ER: Efflux ratio (P_{app (B to A)}/P_{app (A to B)}), calculated from the data shown in Fig. 3; Data are shown as the mean ± S.D., n = 5. *: p < 0.05, according to Dunnett’s test (vs. fexofenadine alone).

The Effects of Jabara Juice Extract on P-gp Activity

In LLC-GA5-COL 300 cells, the P_{app (B to A)} of fexofenadine was only slightly higher than its P_{app (A to B)}, while digoxin, a typical P-gp substrate, showed obvious directional transport across the monolayer, with its P_{app (B to A)} being significantly higher than its P_{app (A to B)}. Jabara juice extract reduced the ER values of both fexofenadine and digoxin in a concentration-dependent manner (Fig. 4, Table 4).

Fig. 4. The Variations in the Apparent Permeability Coefficients (P_app) of 3 µM Fexofenadine (a) and 1 µM Digoxin (b) in LLC-GA5-COL300 Cell Monolayers Treated with 1, 3, 5, or 10% Jabara Juice Extract or 20 µM Cyclosporine A

A to B and B to A represent permeation from the apical to the basolateral side and from the basolateral to the apical side, respectively. The actual P_{app (A to B)} and P_{app (B to A)} of 3 µM fexofenadine alone were (6.31 ± 1.56) × 10⁻⁷ cm/s and (8.55 ± 1.93) × 10⁻⁷ cm/s, respectively, and the actual P_{app (A to B)} and P_{app (B to A)} of 1 µM digoxin alone were (3.76 ± 1.76) × 10⁻⁶ cm/s and (17.4 ± 5.06) × 10⁻⁶ cm/s, respectively. Data are shown as the mean ± S.D., n = 4 (fexofenadine alone) or 5. * p < 0.05, according to Dunnett’s test (vs. substrate alone).

Table 4. The Efflux Ratios of 3 µM Fexofenadine (FEX) or 1 µM Digoxin (DX) for LLC-GA5-COL300 Cell Monolayers after the Coadministration of 1, 3, 5, or 10% Jabara Juice Extract (JJ_extract) or 20 µM Cyclosporine A (CsA)

	ER values
	FEX	DX
substrate alone	1.41a ± 0.37	5.00 ± 1.97
With JJextract (%)
1	1.45 ±0.23	4.23 ± 1.68
3	1.40 ± 0.21	4.32 ± 0.81
5	1.27 ± 0.17	3.00 ± 1.18
10	1.27 ± 0.34	2.41* ± 0.178
With 20 µM CsA	1.11 ± 0.07	1.46* ± 0.224

* ER: Efflux ratio (P_{app (B to A)}/P_{app (A to B)}), calculated from the data shown in Fig. 4; Data are shown as the mean ± S.D., n = 4^a or 5. *: p < 0.05, according to Dunnett’s test (vs. substrate alone).

DISCUSSION

In the present study, the effects of jabara juice on the absorption of fexofenadine were investigated through a pharmacokinetic study involving mice and a permeation study using Caco-2 monolayers. Both in vivo and in vitro, P-gp inhibition by jabara juice, which leads to increased absorption, was found to have stronger effects on fexofenadine absorption than OATP inhibition by jabara juice. Although we have previously reported that jabara juice and its component narirutin inhibit the activity of OATP1A2 and OATP2B1,¹⁵⁾ their effects on P-gp function remained to be investigated. This was also the first study to identify the inhibitory effects of jabara juice on P-gp function in a permeation assay using P-gp-overexpressing LLC-GA5-COL300 cell monolayers.

In LLC-GA5-COL300 cell monolayers, both jabara juice extract and cyclosporine A inhibited the peremeation of digoxin but not fexofenadine (Fig. 4; Table 4), conceivably because of the lower contribution of P-gp (and therefore the larger contribution of passive diffusion) to the permeation of fexofenadine compared to that of digoxin across the LLC-GA5-COL300 cell monolayers. The LLC-GA5-COL300 cell monolayer may not be suitable for assessing the contribution of P-gp for some substrates, such as fexofenadine. Moreover, in LLC-GA5-COL300 cell monolayers, both jabara juice and cyclosporine A increased the P_{app (A to B)} of digoxin without affecting P_{app (B to A)} instead of decreasing the latter. Although a similar effect of cyclosporine A has been previously reported on the permeation of darunavir across the ABCB1-transfected LLC-PK1 cells,³⁴⁾ the reason for this observation remains to be further investigated. On the other hand, HMF and NBL are primary candidates for the P-gp-inhibiting components of jabara juice. Honda et al. showed that 20 µM HMF and 20 µM NBL weakly and potently inhibited the efflux activity of P-gp, respectively, as assessed by the uptake potentiation of vinblastine into LLC-GA5-COL150 cells,²³⁾ although they did not assess certain important parameters, such as IC₅₀ or inhibition constant (K_i) values. We preliminarily quantified the levels of HMF and NBL in the jabara juice used in this study and detected 53 µM of HMF and 1.6 µM of NBL (in-house data). Therefore, the inhibition of P-gp by jabara juice may be attributable to HMF and NBL, although other inhibitors may also contribute to it.

Although P-gp is considered to be the major efflux transporter in Caco-2 cells,³⁵⁾ another likely cause of the increased permeability of fexofenadine through Caco-2 cell monolayers seen in the presence of jabara juice is the inhibition of MRP2, an efflux transporter that is also expressed on the apical side of Caco-2 cell monolayers and is known to transport fexofenadine.^36,37) We detected high MRP2 expression in Caco-2 cell-derived samples (12.7 fmol/µg protein). Honda et al. also identified HMF and NBL as weak and moderate MRP2 inhibitors, respectively.²³⁾ Although Pan et al. previously reported that P-gp and MRP2 are also expressed in the mouse small intestine,³⁸⁾ each transporter’s contribution to the in vivo intestinal fexofenadine absorption remains to be investigated. On the other hand, Tahara et al. reported that Mdr1a/1b knockout mice showed a 6-fold higher plasma concentration of fexofenadine after the drug’s oral administration than wild-type mice, suggesting that P-gp significantly contributes to limiting the intestinal absorption of fexofenadine.³⁹⁾ Ming et al. evaluated the contributions of P-gp and MRP2 to limiting the permeation of fexofenadine through Caco-2 cells and concluded that P-gp was the major rate-limiting transporter of fexofenadine permeation.³⁶⁾ Taken together, it is likely that the increased fexofenadine absorption observed in this study was primarily attributable to P-gp inhibition, although the minor contribution of MRP2 may need to be investigated to fully understand the overall route of fexofenadine absorption from the intestine.

Both OATP1A2 and 2B1 are known to transport fexofenadine.^5,6) Although the expression levels of OATP1A2 and 2B1 on Caco-2 cells are disputed, we detected both transporters at comparable levels in the present study, suggesting that they may play an important role in drug permeation across Caco-2 cells. Indeed, in our Caco-2 monolayer experiment, narirutin, which is known to inhibit OATP1A2 and 2B1,¹⁵⁾ increased the ER of fexofenadine by reducing the apical-to-basal transport of the drug by approximately 70% without affecting its basal-to-apical transport (Fig. 3; Table 3). Taking into consideration that the K_i values of narirutin for OATP1A2 and OATP2B1 were reported to be 82 and 16 µM, respectively,¹⁵⁾ the maximum narirutin concentration in this study (100 µM) or its estimated concentration in 5% jabara juice (22 µM; calculated from the narirutin concentration of 100% jabara juice; i.e., 439 µM¹⁵⁾) may not be high enough to induce potent inhibition. While cyclosporine A is reported to inhibit both OATP2B1 and P-gp with the IC₅₀ or K_i values of 2.2⁴⁰⁾ and 0.3 µM,⁴¹⁾ respectively, 20 µM cyclosporine A decreased the ER value of fexofenadine in Caco-2 cell monolayers as well as jabara juice (Table 2). Therefore, the inhibition of P-gp was predominantly observed in the permeation of fexofenadine across Caco-2 cell monolayers in the presence of dual inhibitors, such as cyclosporine A or jabara juice.

Food–drug interactions involving fexofenadine have been well reproduced in previous studies using mice. For example, Medwid et al. reported that the co-administration of grapefruit juice decreased the AUC of fexofenadine to 35% of the control value in wild-type mice, but not in Oatp2b1-knockout mice.²⁶⁾ This observation is consistent with the results of a clinical study in which grapefruit juice decreased the AUC of fexofenadine to 52% of the control value.⁷⁾ In the mouse experiment conducted in the present study, jabara juice had the opposite effect on fexofenadine absorption to that of grapefruit. This difference between grapefruit juice and jabara juice may be explained by differences in their inhibitory components. Grapefruit juice contains naringin at a concentration (approx. 1200 µM) that is much higher than its IC₅₀ values for OATP1A2 and OATP2B1 (3.6 and 4.6 µM, respectively^12,14)). On the contrary, the concentration of narirutin in jabara juice (approx. 440 µM¹⁵⁾) is not very high in comparison to its K_i values of 82 and 16 µM for OATP1A2 and OATP2B1, respectively,¹⁵⁾ especially when we consider the dilution of the juice in the gastrointestinal tract. Jabara juice is also known to not contain naringin.¹⁵⁾ Therefore, the lack of naringin, a potent inhibitor, in jabara juice may explain the difference in inhibitory potency between grapefruit and jabara juice, although narirutin certainly inhibits OATPs.

In conclusion, using mice and Caco-2 cell monolayers, the intestinal absorption of fexofenadine was shown to be unexpectedly increased by jabara juice, conceivably due to the inhibition of P-gp overcoming the inhibition of OATPs. Jabara juice may alter fexofenadine absorption in humans. A further clinical study would help us to understand the clinical relevance of drug–jabara juice interactions.

Acknowledgments

This study was supported by the Japan Research Foundation through JSPS Kakenhi grants (Grant Numbers: 19K07173 [T.A.] and 18K06758 [H.O.]); the Over the Counter (OTC) Self-Medication Promotion Foundation [to T.A.]; and the Japan Science and Technology Agency (Grant Number: JPMJSP2123).

Author Contributions

All of the authors contributed to the study conception and design. The mouse and permeation studies were performed by Hongye Han, and Ayuko Imaoka. The quantitative targeted absolute proteomics analysis was conducted by Momoko Nabeta, Kodai Yajima and Toshiaki Tsuchitani. The first draft of the manuscript was written by Hongye Han, and all of the authors commented on previous versions of the manuscript. All of the authors have read and approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Funaguchi N, Ohno Y, La BL, Asai T, Yuhgetsu H, Sawada M, Takemura G, Minatoguchi S, Fujiwara T, Fujiwara H. Narirutin inhibits airway inflammation in an allergic mouse model. Clin. Exp. Pharmacol. Physiol., 34, 766–770 (2007).
• 2) Azuma S, Murakami Y, Azuma E, Baba K, Taniguchi M. Anti-inflammatory effects of flavonoids in Citrus jabara fruit peels. Trends Immunother., 4, 5–14 (2020).
• 3) Azuma S, Murakami Y, Taniguchi M, Baba K, Mima T, Nakamura K. Daily intake of Citrus jabara fruit peel powder (Japanese Patent No. 5, 323, 127) improves allergy-like symptoms: a randomized double-blind parallel-group comparative study. Trends Immunother, 5, 21–31 (2021).
• 4) Simpson K, Jarvis B. Fexofenadine: a review of its use in the management of seasonal allergic rhinitis and chronic idiopathic urticaria. Drugs, 59, 301–321 (2000).
• 5) Han H, Akiyoshi T, Morita T, Kataoka H, Katayama K, Yajima K, Imaoka A, Ohtani H. Comparison of the transport kinetics of fexofenadine and its pH dependency among OATP1A2 genetic variants. Drug Metab. Pharmacokinet., 47, 100470 (2022).
• 6) Shirasaka Y, Mori T, Murata Y, Nakanishi T, Tamai I. Substrate- and dose-dependent drug interactions with grapefruit juice caused by multiple binding sites on OATP2B1. Pharm. Res., 31, 2035–2043 (2014).
• 7) Yu J, Zhou Z, Tay-Sontheimer J, Levy RH, Ragueneau-Majlessi I. Intestinal drug interactions mediated by OATPs: a systematic review of preclinical and clinical findings. J. Pharm. Sci., 106, 2312–2325 (2017).
• 8) Couto N, Al-Majdoub ZM, Gibson S, Davies PJ, Achour B, Harwood MD, Carlson G, Barber J, Rostami-Hodjegan A, Warhurst G. Quantitative proteomics of clinically relevant drug-metabolizing enzymes and drug transporters and their intercorrelations in the human small intestine. Drug Metab. Dispos., 48, 245–254 (2020).
• 9) Imanaga J, Kotegawa T, Imai H, Tsutsumi K, Yoshizato T, Ohyama T, Shirasaka Y, Tamai I, Tateishi T, Ohashi K. The effects of the SLCO2B1 c.1457C > T polymorphism and apple juice on the pharmacokinetics of fexofenadine and midazolam in humans. Pharmacogenet. Genomics, 21, 84–93 (2011).
• 10) Tanaka S, Uchida S, Miyakawa S, Inui N, Takeuchi K, Watanabe H, Namiki N. Comparison of inhibitory duration of grapefruit juice on organic anion-transporting polypeptide and cytochrome P450 3A4. Biol. Pharm. Bull., 36, 1936–1941 (2013).
• 11) Dresser GK, Bailey DG, Leake BF, Schwarz UI, Dawson PA, Freeman DJ, Kim RB. Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine. Clin. Pharmacol. Ther., 71, 11–20 (2002).
• 12) Shirasaka Y, Shichiri M, Mori T, Nakanishi T, Tamai I. Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions. J. Pharm. Sci., 102, 280–288 (2013).
• 13) Bailey DG. Fruit juice inhibition of uptake transport: a new type of food–drug interaction. Br. J. Clin. Pharmacol., 70, 645–655 (2010).
• 14) Bailey DG, Dresser GK, Leake BF, Kim RB. Naringin is a major and selective clinical inhibitor of organic anion-transporting polypeptide 1A2 (OATP1A2) in grapefruit juice. Clin. Pharmacol. Ther., 81, 495–502 (2007).
• 15) Morita T, Akiyoshi T, Sato R, Uekusa Y, Katayama K, Yajima K, Imaoka A, Sugimoto Y, Kiuchi F, Ohtani H. Citrus fruit-derived flavanone glycoside narirutin is a novel potent inhibitor of organic anion-transporting polypeptides. J. Agric. Food Chem., 68, 14182–14191 (2020).
• 16) Sakaeda T, Nakamura T, Okumura K. MDR1 genotype-related pharmacokinetics and pharmacodynamics. Biol. Pharm. Bull., 25, 1391–1400 (2002).
• 17) Miyake T, Tsutsui H, Haraya K, Tachibana T, Morimoto K, Takehara S, Ayabe M, Kobayashi K, Kazuki Y. Quantitative prediction of P-glycoprotein-mediated drug–drug interactions and intestinal absorption using humanized mice. Br. J. Pharmacol., 178, 4335–4351 (2021).
• 18) Hamman MA, Bruce MA, Haehner-Daniels BD, Hall SD. The effect of rifampin administration on the disposition of fexofenadine. Clin. Pharmacol. Ther., 69, 114–121 (2001).
• 19) Zhang W, Han Y, Lim SL, Lim YL. Dietary regulation of P-gp function and expression. Expert Opin. Drug Metab. Toxicol., 5, 789–801 (2009).
• 20) Takanaga H, Ohnishi A, Matsuo H, Sawada Y. Inhibition of vinblastine efflux mediated by P-glycoprotein by grapefruit juice components in caco-2 cells. Biol. Pharm. Bull., 21, 1062–1066 (1998).
• 21) Ito K, Satoh T, Watanabe Y, Ikarashi N, Asano T, Morita T, Sugiyama K. Effects of Kampo medicines on CYP and P-gp activity in vitro. Biol. Pharm. Bull., 31, 893–896 (2008).
• 22) Takanaga H, Ohnishi A, Yamada S, Matsuo H, Morimoto S, Shoyama Y, Ohtani H, Sawada Y. Polymethoxylated flavones in orange juice are inhibitors of P-glycoprotein but not cytochrome P450 3A4. J. Pharmacol. Exp. Ther., 293, 230–236 (2000).
• 23) Honda Y, Ushigome F, Koyabu N, Morimoto S, Shoyama Y, Uchiumi T, Kuwano M, Ohtani H, Sawada Y. Effects of grapefruit juice and orange juice components on P-glycoprotein- and MRP2-mediated drug efflux. Br. J. Pharmacol., 143, 856–864 (2004).
• 24) Ikegawa T, Ushigome F, Koyabu N, Morimoto S, Shoyama Y, Naito M, Tsuruo T, Ohtani H, Sawada Y. Inhibition of P-glycoprotein by orange juice components, polymethoxyflavones in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells. Cancer Lett., 160, 21–28 (2000).
• 25) Morita T, Akiyoshi T, Tsuchitani T, Kataoka H, Araki N, Yajima K, Katayama K, Imaoka A, Ohtani H. Inhibitory effects of cranberry juice and its components on intestinal OATP1A2 and OATP2B1: identification of avicularin as a novel inhibitor. J. Agric. Food Chem., 70, 3310–3320 (2022).
• 26) Medwid S, Li MMJ, Knauer MJ, Lin K, Mansell SE, Schmerk CL, Zhu C, Griffin KE, Yousif MD, Dresser GK, Schwarz UI, Kim RB, Tirona RG. Fexofenadine and rosuvastatin pharmacokinetics in mice with targeted disruption of organic anion transporting polypeptide 2B1. Drug Metab. Dispos., 47, 832–842 (2019).
• 27) Awortwe C, Fasinu PS, Rosenkranz B. Application of Caco-2 cell line in herb–drug interaction studies: current approaches and challenges. J. Pharm. Pharm. Sci., 17, 1–19 (2014).
• 28) Hubatsch I, Ragnarsson EG, Artursson P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc., 2, 2111–2119 (2007).
• 29) Uchida Y, Ohtsuki S, Kamiie J, Ohmine K, Iwase R, Terasaki T. Quantitative targeted absolute proteomics for 28 human transporters in plasma membrane of Caco-2 cell monolayer cultured for 2, 3, and 4 weeks. Drug Metab. Pharmacokinet., 30, 205–208 (2015).
• 30) Masuda T, Sugiyama N, Tomita M, Ohtsuki S, Ishihama Y. Mass spectrometry-compatible subcellular fractionation for proteomics. J. Proteome Res., 19, 75–84 (2020).
• 31) Uchida Y, Tachikawa M, Obuchi W, Hoshi Y, Tomioka Y, Ohtsuki S, Terasaki T. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS, 10, 21 (2013).
• 32) Uchida Y, Ohtsuki S, Katsukura Y, Ikeda C, Suzuki T, Kamiie J, Terasaki T. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J. Neurochem., 117, 333–345 (2011).
• 33) Tsuchitani T, Akiyoshi T, Imaoka A, Ohtani H. Digoxin absorption decreased independently of P-gp activity in rats with irinotecan-induced gastrointestinal damage. J. Pharm. Health Care Sci., 7, 24 (2021).
• 34) Fujimoto H, Higuchi M, Watanabe H, Koh Y, Ghosh AK, Mitsuya H, Tanoue N, Hamada A, Saito H. P-glycoprotein mediates efflux transport of darunavir in human intestinal Caco-2 and ABCB1 gene-transfected renal LLC-PK1 cell lines. Biol. Pharm. Bull., 32, 1588–1593 (2009).
• 35) Petri N, Tannergren C, Rungstad D, Lennernäs H. Transport characteristics of fexofenadine in the Caco-2 cell model. Pharm. Res., 21, 1398–1404 (2004).
• 36) Ming X, Knight BM, Thakker DR. Vectorial transport of fexofenadine across Caco-2 cells: involvement of apical uptake and basolateral efflux transporters. Mol. Pharm., 8, 1677–1686 (2011).
• 37) Sampson KE, Brinker A, Pratt J, Venkatraman N, Xiao YL, Blasberg J, Steiner T, Bourner M, Thompson DC. Zinc finger nuclease–mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells. Drug Metab. Dispos., 43, 199–207 (2015).
• 38) Pan Y, Omori K, Ali I, Tachikawa M, Terasaki T, Brouwer K, Nicolazzo J. Altered expression of small intestinal drug transporters and hepatic metabolic enzymes in a mouse model of familial Alzheimer’s disease. Mol. Pharm., 15, 4073–4083 (2018).
• 39) Tahara H, Kusuhara H, Fuse E, Sugiyama Y. P-glycoprotein plays a major role in the efflux of fexofenadine in the small intestine and blood-brain barrier, but only a limited role in its biliary excretion. Drug Metab. Dispos., 33, 963–968 (2005).
• 40) Varma MV, Rotter CJ, Chupka J, Whalen KM, Duignan DB, Feng B, Litchfield J, Goosen TC, El-Kattan AF. pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1. Mol. Pharm., 8, 1303–1313 (2011).
• 41) Parasrampuria DA, Lantz MV, Benet LZ. A human lymphocyte based ex vivo assay to study the effect of drugs on P-glycoprotein (p-Gp) function1. Pharm. Res., 18, 39–44 (2001).