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In vitro to in vivo evidence of the inhibitor characteristics of Schisandra lignans toward P-glycoprotein.

ARTICLE INFO

Keywords:

P-glycoprotein

Schisandra lignans extract

Herb-drug interaction

Inhibitor

ABSTRACT

Concomitant administration of herbal medicines with drugs that are P-glycoprotein (P-gp) substrates may produce significant herb-drug interactions. The purpose of this study was to evaluate the effects of Schisandra lignans extract (SLE) on P-gp thoroughly in vitro and in vivo, and to investigate the possible P-gp-based herb-drug interactions. In the in vitro experiments, the effect of SLE on the uptake and transport for P-gp substrates in Caco-2, LLC-PK 1 and L-MDR 1 cells were carefully investigated. Verapamil, a known P-gp inhibitor, was used as a positive control drug. Results shown that, 10 [micro]M verapamil and SLE (0.5, 2.0, and 10.0 [micro]g/ml) were observed to significantly enhance the uptake and inhibit the efflux ratio of P-gp substrates in Caco-2 and L-MDR1 cells. In vivo experiments showed that single-dose SLE at 500 mg/kg could increase the area under the plasma concentration time curve of digoxin and vincrisine significantly without affecting terminal elimination half-time. Long-term treatment with SLE for continuous 10 days could also increase the absorption of P-gp substrates with greatly clown regulation of P-gp expression in rat intestinal and brain tissues. In conclusion, SLE was a strong P-gp inhibitor, which indicated a potential herb-drug interaction when SLE was co-administered with P-gp substrate drugs.

[c] 2013 Published by Elsevier GmbH.

Introduction

In recent years, the use of herbal medicines (HMs) has increased dramatically in the Western world, and more than 80% of the population in developing countries relied on the use of herbal and other traditional medicines for their primary healthcare (Eisenberg et al. 1998; World Health Organization 2008). Herbal products contain multiple chemicals that are metabolized by phase I and phase II pathways and always serve as substrates for certain transporters. Importantly, almost 25% of all prescription drug users took HMs concomitantly with conventional medications, there was an associated possibility for herb-drug interactions to occur (Wold et al. 2005: Shahrokh et al. 2005). In accordance with the relevant European guidance documents, potential herb-drug interactions should be investigated critically for their clinical relevance, and a balanced assessment is required when regulatory documents are established (Steinhoff 2012).

In addition to cancer cells, P-gp is widely expressed in epithelial cells of normal tissues involved in drug disposition including the liver, intestine and kidney while providing a barrier to sites such as the brain and testes (Marzolini et al. 2004; Teft et al. 2011). Phytochemical-mediated alterations in P-gp activity may produce herb-drug interactions by altering drug absorption, distribution and elimination (Mills et al. 2005; Aller et al. 2009). Importantly, P-gp displayed broad substrate specificities toward many structurally and functionally unrelated compounds, such as flavone vinca alkaloids, anthracyclines, epipodophyllotoxins, taxans, and therefore, rationalized the MDR caused by P-gp (Dauchy et al. 2008; Gouaze et al. 2004; Loscher and Potschka 2005).

Schisandra was a common ingredient in prescriptions such as Shengmai-injection, Shenqi Wuweizi-Pian and Shengmai-Yin included in Chinese Pharmacopeia. It could be taken concomitantly with conventional medicine to have a protective effect against deficits of the lung, liver, and gall bladder, and to alleviate cough and satisfy thirst (Bae et al. 2012). In modern pharmaceuticals studies, Schisandra lignans, the major bioactive constituents, had been reported to enhance hepatic glutathione regeneration capacity (Ko et al. 1995) and reduce hepatotoxicity (Wang and Li 2006; Panossian and Wikman 2008). In our previous studies, a diagnostic fragment-ion-based extension strategy (DFIBES) was developed for the rapid detection and structural characterization of lignans in SLE based on liquid chromatography combined hybrid ion trap time-of-flight mass spectrometry (LC-IT-TOF/MS) analysis (Zheng et al. 2009; Liang et al. 2010). Besides, the pharmacokinetic characterizes of Schisandra lignans were obtained according to the relative quantitation assay and relative exposure approach. As the results, pharmacokinetic parameters of Schisandra lignans after a single intragastric dosage of SLE (500 mg/kg) including the area under the concentration-time curve (AUC), maximum plasma concentration ([C.sub.max]), and half-life time ([t.sub.1/2]) were calculated (Liang et al. 2010). The [C.sub.max] of Schizandrol A, Schizandrol B, Schisantherin A, Schisantherin B, Schizandrin B were 1159.33 [+ or -] 61.34, 78.64 [+ or -] 28.54, 80.02 [+ or -] 20.08, 43.37 [+ or -] 21.15, 44.80 [+ or -] 33.32 ng/ml, and tip of Schizandrol A, Schizandrol B, Schisantherin A, Schisantherin B, Schizandrin B were 2.23 [+ or -] 0.71, 7.30 [+ or -] 1.51, 12.60 [+ or -] 4.39, 5.77 [+ or -] 1.38, 6.16 [+ or -] 3.65h, respectively. In 2009, the effect of SLE on the oral pharmacokinetics of P-gp substrate talinolol in humans had been investigated. [AUC.sub.0-24] and [C.sub.max] of talinolol were found to be increased by 47% and 51% with SLE treatment (Fan et al. 2009). Xue et al. had reported that the extract of Schisandra sphenanthera could significantly increase the [AUC.sub.0-48h] and [C.sub.max] of Cyclosporin A (a substrate of CYP3A and P-gp) in rats by inhibiting the CYP3A-mediated metabolism and the P-gp-mediated efflux (Xue et al. in press). Besides, deoxyschizandrin in SLE had been proven effectively inhibit the P-gp-mediated efflux in Caco-2 cells (Yoo et al. 2007). Schisandrol A isolated from Fructus Schisandrae was found to be sensitive to HepG2-DR cell (Fong et al. 2007). Unfortunately, the current research about the inhibition effect of Schisandra on P-gp was still in its infancy and largely limited to the isolated components (deoxyschizandrin, Schisandrol A. etc.). As an integral part of the whole pipeline of herbal modernizations, potential P-gp mediated herb-drug interactions for SLE should be critically investigated due to their closely clinical relevance.

The first step of this study was to investigate SLE as an inhibitor of P-gp using validated in vitro (Caco-2, LLC-PKI and LMDR1 cells). On the basis of inhibition predictions, the next step was to conduct an in vivo pharmacokinetic interaction studies in rats to investigate whether SLE could perpetrate P-gp-mediated herb-drug interactions with digoxin and vincrisine in the preclinical pharmacokinetics. In order to further confirm the SLE was a P-gp inhibitor, western blotting analysis was carried, and SLE was found significantly down-regulation of the P-gp expression in rat brain and intestinal. Thus, the inhibitor characteristics of SLE toward P-gp were systemically investigated in vitro and in vivo.

Materials and methods

Animals

Male healthy Sprague-Dawley rats (200 [+ or -] 20 g) were purchased from the Laboratory Animal Center of Peking University Health Science Center (Beijing, PR China) and kept in an environmentally controlled breeding room for at least 3 days before experimentation. The rats were fed with standard laboratory food and water and fasted overnight but with free access to water before the test. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals (National Research Council of USA, 1996) and the related ethical regulations of our university.

Materials

SLE was purchased from Nanjing Qingze Medical Technological Development Co. Ltd. (Nanjing, China). Digoxin, ginsenoside Rg1 (internal standard of digoxin), vincrisine, vinorelbine (internal standard of vincristine) and verapamil were purchased from Sigma-Aldrich (St. Louis, MO). [[.sup.14]C]Mannitol was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). HPLC-grade acetonitrile and methanol were purchased from Sigma-Aldrich. Deionized water was prepared by the Milli-Q system (Millipore Corporation, Billerica, MA) and was used throughout. Ethyl acetate and all other reagents were commercially available and of analytical grade.

Caco-2 cell culture

Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1 mM sodium pyruvate, and 100 U/ml penicillin and streptomycin (Invitrogen, Carlsbad, CA). The cells were grown in an atmosphere of 5% C[O.sub.2] at 37 [degrees]C, and cell medium was changed every other day. Cells were passaged upon reaching about 80% confluence.

Enhancing uptake of P-gp substrate by SLE in Caco-2 cell

To evaluate the action of SLE as a P-gp inhibitor, Caco-2 cells were incubated with SLE (0.5, 2.0 and 10.0 [micro]g/ml) or verapamil (10 [micro]M) for 1.0h, then were washed to discard SLE or verapamil before the addition of 5 [micro]M digoxin or 5 [micro]M vincrisine. The accumulation of 5 [micro]M digoxin or 5 [micro]M vincristine lasted for 2h, and the intracellular concentration of digoxin and vincrisine were measured by liquid chromatography combined mass spectrometry (LC/MS). All experiments were conducted in triplicate.

Inhibition of P-gp substrate transport by SLE in Caco-2 cell monolayers

Caco-2 cells with a density of 1.2 x [10.sup.5] cells/insert were seeded on a permeable polycarbonate insert (Millicell cell culture inserts, 0.4 [micro]m pore size, 12 mm diameter; Millipore Corporation) in 24-well tissue culture plates and were used for the experiment 18-21 days after seeding. [[.sup.14]C]Mannitol permeability and transepithelial electrical resistance measurements (Millicell-ERS epithelial volt-ohm meter; Millipore Corporation) were used to evaluate the integrity of Caco-2 cell monolayers. The monolayers used in transport studies had transepithelial electrical resistance values exceeding 600 [rho] [cm.sup.2], and the leakage of mannitol was less than 0.3% of the dose/h. Before initiation of transport studies, the cell monolayers were first washed with warm HBSS (pH 7.4) twice. HBSS containing SLE (0.5, 2.0 and 10.0 [micro]g/ml) or 1% ethanol (control) was then loaded into both apical and basolateral chambers. After incubation at 37 [degrees]C for 1.5h. 5 [micro]M digoxin was added to either the apical or basolateral side to evaluate the transport in absorptive and secretory directions, respectively, and the cell monolayers were incubated for another 2h. 10 [micro]M verapamil was used as a positive control inhibitor. At the designated time point, samples were taken from the receiving chamber for analysis. Digoxin and vincrisine concentrations were determined by LC/MS. All experiments were conducted in triplicate.

LLC-PK1 and L-MDR1 cell culture

LLC-PK1 cells, a cell line lacking P-gp, and the derived cell line L-MDR1, which over-express human MDR1-encoded P-gp, were grown in minimum essential medium a with supplements at 37[degrees]C. 5% C[O.sub.2], and 95% humidity. LLC-PK1 and L-MDR1 cells were seeded at a density of 12 x [10.sup.6] cells per 150-[cm.sup.2] flask and grown for 7 days in medium 199 supplemented with 2 mM glutamine, 10% fetal bovine serum, 10 mg/500 ml of streptomycin, and 10,000 IU/500 ml of penicillin. To ensure a constant expression level of transport protein. L-MDR1 cells were grown in the presence of 640 nM vincristine. Cells were grown as monolayers on polycarbonate membrane filters (Transwell; Costar Corporation, Cambridge, MA) as outlined previously (Kim et al. 1998). Transepithelial resistance was measured in each well using a Millicell ERS ohmmeter (Millipore. Bedford. MA); wells registering a resistance of 200 [ohm] or greater, after correcting for the resistance obtained in control blank wells, were used in the transport experiments.

Inhibition of P-gp substrate transport by SLE in cultured LLC-PK1 and L-MDR1 cells

The LLC-PK1 and L-MDR1 cell monolayers were washed with warm Hank's balanced salt solution HBSS (pH 7.4) twice before initiation of transport studies. HBSS containing SLE (0.5, 2.0 and 10.0 [micro]g/ml), verapamil (10 [micro]M) or blank HBSS (control) was then loaded into both apical and basolateral chambers. After incubation at 37 [degrees]C for 1.5 h, 5 [micro]M digoxin or 5 [micro]M vincrisine was added to either the apical or basolateral side to evaluate the transport in absorptive and secretory directions, respectively, and the cell monolayers were incubated for another 2 h. Finally, the concentration of digoxin and vincrisine were determined by LC/MS. All experiments were conducted in triplicate.

Western blotting analysis

For Western blot analysis, crude membrane was prepared from rat brain and intestine as described previously (Ogihara et al. 1996). Three rats were given a dose of SLE intragastrically at 500 mg/kg suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) once a day for 10 continuous days, whereas the other three rats received the vehicle (0.5% sodium CMC-Na), serving as the control groups. On the 10th day, the rats were sacrificed by cutting femoral artery under deep isoflurane anesthesia. Intestinal or brain homogenates were lysed in ice-cold radioimmunoprecipitation assay lysis buffer with 0.02 mM phenylmethanesulfonyl fluoride for 30 min and then ultrasonicated for 60-s intervals in an ice bath. Samples were then centrifuged at 500 x g for 10 min at 4 [degrees]C. The supernatant was transferred to a new tube and centrifuged at 15,000 x g for 60 min at 4 [degrees]C. The supernatant (cytosol protein, such as 13-actin) and precipitant (membrane protein, such as P-gp) were both collected and stored at -80 [degrees]C until use.

Protein concentrations were measured using a BCA protein assay kit (Pierce Chemical, Rockford, IL) according to the manufacturer's instructions. Samples reconstituted in SDS-polyacrylamide gel electrophoresis sample loading buffer were boiled for 5 min for protein denaturation. Protein samples were separated on an 8% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Millipore Corporation). After blotting, the membrane was blocked with 10% bovine serum albumin in Tris-buffered saline-Tween 20 (TBS-T) for 1h at 37 [micro]C. Immunoblots were incubated with the primary monoclonal antibody to P-gp (1:800; clone3C3.2; Millipore Corporation) or [beta]-actin (1:800; Bioworld Technology, St. Louis Park, MN) for 24h at 4 [degrees]C. The membrane was washed (4x 10 min), incubated with the secondary antibody horseradish peroxidase-conjugated goat anti-mouse IgG (1:800; Boster Biological Technology, Wuhan, China) for 1h at 37[degrees]C and then washed three times with TBS-T. The signals were detected using an enhanced chemiluminescence kit (Pierce Chemical). The P-gp protein band intensity was normalized to that of [beta]-actin.

In vivo pharmacokinetic studies

To evaluate the effect of a single dose of SLE on the pharmacokinetics of each P-gp substrate, 24 rats were divided into four groups (group I-group IV) averagely. In group I: the rats were intragastrically administrated a single dose of SLE at 500 mg/kg. 2h later, digoxin (0.5 mg/kg), a P-gp substrate, was given to the rats by intragastric administration. In group II (control group): the rats received a single dose of vehicle (0.5% CMC-Na), and digoxin (0.5 mg/kg) was also given to the rats by intragastric administration. In group the rats received a single dose of SLE intragastrically at 500 mg/kg suspended in 0.5% CMC-Na. 2h later, vincrisine (1.0 mg/kg), one P-gp substrate, was given to the rats by intravenous injection. In group IV (control group): the rats received a single dose of vehicle (0.5% CMC-Na), and vincrisine (1.0 mg/kg) was also given to the rats intravenously.

To evaluate the effect of long-term treatment with SLE on the pharmacokinetics of each P-gp substrate, 24 rats were also divided into four groups (group I-group IV) with 6 animals each. In the group I and group III, rats were intragastrically administrated a dose of SLE at 500 mg/kg once a day for 10 continuous days, whereas the other two groups (group II and group IV) received the vehicle (0.5% CMC-Na), serving as the control groups. On the 11th day, digoxin was administered intragastrically to rats (group I and group II) at a single dose of 0.5 mg/kg, vincrisine was administered intravenously to rats (group and group IV) at a single dose of 1.0 mg/kg.

In the experiments above, 0.2 ml blood samples were collected at 5, 10, 20, 40, 60, 120, 240, 360, 480 and 720 min after digoxin dosing. After intravenous administration of vincrisine, 0.2 ml blood samples were collected at 5, 10, 20, 30, 45, 60, 120, 240, 360, 480, 600 and 720 min. Plasma was obtained by centrifugation at 5000 x g for 10 min and stored at -20 [degrees]C before analysis.

Analysis of digoxin and vincrisine in cell lysates, buffer samples and rat plasma

An aliquot of 100 [micro]l of sample with 10 [micro]l of internal standard (ginsenoside Rg1 for digoxin analysis, vinorelbine for vincrisine analysis) spiked was extracted by 1 ml of ethyl acetate. After centrifugation, 750 [micro]l of the extracted organic layer was evaporated to dryness with a Thermo Savant SPD 2010 Speed Vac system (Thermo Fisher Scientific, Waltham, MA). The residue was reconstituted in 200 pi of acetonitrile, followed by another centrifugation. Then 5111 of the supernatant was injected for LC/MS analysis.

LC experiments were conducted on a Shimadzu (Kyoto, Japan) HPLC system. Chromatographic separation was achieved on a SymmetryShieldTM RP8 column (3.5 [micro]m, 50 mm x 2.1 mm I.D., Waters) at 40 [degrees]C. For the digoxin analysis, the mobile phase (delivered at 0.2 ml/min) consisted of solvent A, [H.sub.2]0 (containing 1.0 [micro]M N[H.sub.4]Cl) and solvent B, C[H.sub.3]CN. A binary gradient elution was performed: initial 25% B for 0.2 min, linear gradient 25-85% B from 0.2 to 7.0 min and 85% to 25% B from 7.0 to 8.0 min, and maintained until 12 min for column balance. The mass spectrometer of LC/MS system consisted of a Shimadzu LCMS-2010A quadrupole mass spectrometer interfaced by an ESI probe. The [[M+CI].sup.-] ions for the targeted analytes (m/z 815 for digoxin, m/z 835 for ginsenoside Rg1) were monitored simultaneously. For the vincrisine analysis, the mobile phase (delivered at 0.2 ml/min) consisted of solvent A. [H.sub.2]0 (containing 0.1% HCOOH) and solvent B. C[H.sub.3]OH. A binary gradient elution was performed: initial 30% B for 0.03 min, linear gradient 30-70% B from 0.03 to 3.0 min and 70% to 30% B from 3.0 to 4.0 min, and maintained until 9 min for column balance. The LC/MS system was operated in the positive mode. The m/z of [[M+H].sup.+] for vincrisine and vinorelbine were 826.5 and 779.5, respectively. The MS operating conditions were optimized as follows: drying gas 1.5l/min, curved desolvation line (CDL) temperature 250[degrees]C, heat block temperature 200[degrees]C, and detector voltage 1.6 kV.

Identification of lignans in SLE by LC-IT-TOF/MS

LC experiments were conducted on a Shimadzu (Kyoto, Japan) HPLC system. Chromatographic separation was achieved on a SymmetryShield[TM] RP8 column (3.5 [micro]m, 50 mm x 2.1 mm Waters) at 40[degrees]C. The mobile phase (delivered at 0.2 ml/min) consisted of solvent A, C[H.sub.3]OH/[H.sub.2]0 (5:95, v/v, containing 1.0 [micro].M C[H.sub.3]COONa) and solvent B, C[H.sub.3]OH/[H.sub.2]O (95:5, v/v, containing 1.0 [micro]M C[H.sub.3]COONa). A binary gradient elution was performed: initial 55% B for 0.2 min, linear gradient 55-65% B from 0.2 to 25.0 min and 65% to 85% B from 25.0 to 40.0 min, then quickly returned to initial 55% B and maintained until 48 min for column balance. The mass spectrometer of LC-IT-TOF/MS (Shimadzu, Japan) equipped with an ESI source in positive ion mode. The optimized analytical conditions were as follows: detector voltage, 1.60 kV; nebulizing gas ([N.sub.2]) flow, 1.5l/min; dry gas ([N.sub.2]) flow, 50 kPa; vacumn of TOF region, 1.5 x [10.sup.-4] Pa; ion trap vacumn, 1.7 x [10.sup.-2] Pa; ion accumulated time, 30 ms; precursor ion selected width, 3.0 amu. Scan ranges were set al m/z 350-700 for M[S.sup.], 100-600 for M[S.sup.2] and 50-500 for M[S.sup.3].

Data analysis

For the transport assay, the apparent permeabilities ([P.sub.app], cm/s) in each direction (A [right arrow] B and B [right arrow]A ) were calculated as follows, where A is the surface area of the membrane inserts (0.0804 [cm.sup.2]), [C.sub.0] is the initial concentration of the compound applied in the donor compartment (2 [micro]M), and [DELTA]Q is the amount (micromoles) of compound transported over time [DELTA]t (5h = 18,000 s) (Zhou et al. 2005):

[P.sub.app] = 1/A x [C.sub.0] x [DELTA]Q/[DELTA]t

The efflux ratio (ER) was a dimensionless number calculated as the ratio of the apparent permeability in the B [right arrow] A direction divided by the apparent permeability in the A [right arrow] B direction (Feng et al. 2008).

ER = [P.sub.app](BL [right arrow] AP)/[P.sub.app](AP [right arrow] BL)

Pharmacokinetic calculation

The plasma concentration-time curves of digoxin and vincrisine in rats were obtained by plotting the mean plasma concentrations versus time. Pharmacokinetic parameters were calculated using the WinNonlin program (Pharsight Inc., Mountain View, CA). The elimination half-life ([t.sub.1/2]) value was calculated as 0.693/[beta], where [beta] is the elimination rate constant calculated from the terminal linear portion of the log plasma concentration-time curve. The total areas under the plasma concentration-time curve from time 0 to the last quantifiable time point ([AUC.sub.0-t]) and from time 0 to infinity ([AUC.sub.0-[infinity]]) were calculated using the log trapezoidal rule.

[AUC.sub.0-[infinity]] = [AUC.sub.0-[infinity]] + [C.sub.r]/[beta]

The maximum plasma concentration ([C.sub.max]) for digoxin was obtained by visual inspection of the plasma concentration-time curve, whereas the initial drug concentration (the extrapolated concentration at zero time) of the drug after intravenous injection was calculated by back-extrapolation of the plasma concentration-time curve to the y-axis. The plasma clearance (CL) was estimated by dividing the total administered close by the [AUC.sub.0-[infinity]].

Results

Identification of lignans in SLE by LC-IT-TOF/MS

Schisandra lignans, composed of A, B and C rings, are the major and characteristic constitutes of Schisandraceae plants. 31 kinds of schisandra lignans had been identified based on LC-IT-TOF/MS analysis (Fig. 1). In this process, a diagnostic fragment-ion-based extension strategy (DFIBES) was used to classify the lignans into five subfamilies according to their skeleton structures.

Enhancing uptake of P-gp substrate by SLE in Caco-2 cell

To evaluate the inhibitory potency of SLE as a P-gp inhibitor, the effect of SLE on the uptake of P-gp substrates (digoxin and vincrisine) had been investigated and the results are shown in Fig. 2. The uptake extent of digoxin and vincrisine could be significantly enhanced by verapamil (the positive control inhibitor). At the same time, SLE concentration dependently increased intracellular concentration of cligoxin in Caco-2 cells by 1.18-, 1.43-, and 2.04-fold when 0.5, 2.0, and 10.0 [micro]g/ml of SLE were loaded (p < 0.01). Similarly, the intracellular concentration of vincrisine could also be increased by 1.84-, 2.79-, and 3.64-fold when the SLE at 0.5, 2.0, and 10.0 [micro]g/ml were added in the Caco-2 cells.

SLE decreased the efflux ratio of digoxin across Caco-2 monolayers

The ability of SLE to inhibit P-gp was further validated on the efflux ratio of P-gp substrate in Caco-2 cells. The P-gp-mediated transport of the probe digoxin (5[micro]M) across Caco-2 cell monolayers in the absorptive (AP [right arrow] BL) and secretory (BL [right arrow] AP) directions and the corresponding [P.sub.app] values are shown in Fig. 3. The classic P-gp substrate digoxin possessed an efflux ratio of 3.23, which was far greater than 2.0, and this high efflux ratio of digoxin decreased significantly to 1.25 in the presence of 10 [micro]M verapamil. Similarly, the presence of SLE could greatly decrease the transport capability of digoxin across Caco-2 monolayers in the BL-AP direction, which correspondingly led to the decrease in efflux ratio for digoxin. When the concentrations of SLE were at 0.5, 2.0, and 10.0 [micro]g/ml, the efflux ratios of digoxin in Caco-2 monolayers were 1.44, 1.29 and 1.15, respectively. Thus, SLE concentration dependently decreased the efflux ratio of digoxin in Caco-2 monolayers.

The effect of SLE on the uptake of digoxin in cultured LLC-PK1 and L-MDR1 cells

Herein, we examined the effect of SLE on the uptake of digoxin in LLC-PK1 cells (P-gp-negative control cells), a cell line lacking P-gp, and the derived cell line L-MDR1, which overexpresses human MDR1-encoded P-gp. As shown in Fig. 4, intracellular accumulation of digoxin on LLC-PK1 cells had no significant difference in the absence and presence of verapamil and SLE. In contrast, the intracellular concentration of digoxin on L-MDR1 cells was greatly increased (~2.3-fold) in the presence of P-gp inhibitor verapamil. At the same time, SLE concentration dependently increased intracellular concentration of digoxin in Caco-2 cells by 2.7-, 2.9-, and 3.6-fold in the presence of 0.5, 2.0, and 10.0[micro]g/ml SLE, respectively (p < 0.01).

Inhibition of P-gp substrate transport by SLE in cultured LLC-PK1 and L-MDR1 cells

In order to further confirm the SLE is a P-gp inhibitor, we compared the effect of SLE on the transport ability of P-gp substrate in LLC-PK1 cells and the derived cell line L-MDR1. Fig. 5A illustrates the absorptive (AP [right arrow] BL) and secretory (BL [right arrow] AP) transport of digoxin on LLC-PK1 cells (lacking P-gp), in the absence and presence of 10 [micro]M verapamil, SLE (0.5, 2.0, and 10.0 [micro]g/ml) as P-gp inhibitor. No transport difference was observed when verapamil or SLE was added. It was mean that the efflux of digoxin (5 [micro]M) across LLC-PKI cell monolayers in the absorptive (AP [right arow] BL) and secretory (BL [right arrow] AP) directions and the corresponding [P.sub.app] values were not affected by verapamil or SLE. On the other hand, we also compared the transport of digoxin on L-MDR1 cells (LLC-PKI cells stably transfected with the human MDR1 gene), in the absence and presence of 10 [micro]M verapamil, SLE (0.5, 2.0, and 10.0 [micro]/ml) as P-gp inhibitor (shown in Fig. 5B). Obviously, verapamil and SLE could significantly increase the transport of digoxin in the AP [right arrow] BL direction, and greatly decrease the transport of digoxin across L-MDR1 cell monolayers in the BL [right arrow] AP direction. The efflux ratio of digoxin could be decreased from 44.44 to 11.38 by 10 [micro]M verapamil. When the concentration of SLE were at 0.5, 2.0, and 10.0 [micro]g/ml, efflux ratios of digoxin across L-MDR1 cells monolayers were at 3.03, 1.34 and 1.05, respectively.

Single-dose SLE increased plasma concentrations of P-gp substrates in rats

In this process, the effect of SLE on the plasma pharmacokinetics of digoxin and vincrisine in rats was investigated. The plasma concentration--time profiles when digoxin or vincrisine was single administered alone or in combination with SLE at 500 mg/kg are shown in Fig. 6 and the pharmacokinetic parameters are listed in Table 1. Obviously, coadministration of SLE could significantly increase the [AUC.sub.0-[tau]] of digoxin 1.25-fold, from 61.80 [+ or -] 13.37 ng h/ml in the vehicle treated group to 84.38 [+ or -] 7.25 ng h/ml in the SLE-treated group. The clearance (CL) was decreased from 7.66 [+ or -] 1.53 ng h/ml in the vehicle treated group to 5.53 [+ or -] 0.48 ng h/ml in the SLE-treated group. The maximum plasma concentration ([C.sub.max]) and half-life time ([t.sub.1/2]) was almost not changed after SLE treatment. At the same time, coadministration of SLE significantly increased the [AUC.sub.0-12] of vincrisine 1.35-fold, from 414 [+ or -] 94 ng h/ml in the vehicle treated group to 547 [+ or -] 31 ng h/ml in the SLE-treated group. CL was decreased from 1.74 [+ or -] 0.17 ng h/ml in the vehicle treated group to 2.23 [+ or -] 0.47 ng h/ml in the SLE-treated group. [C.sub.max] and [t.sub.1/2] was not significantly altered by SLE.

Table 1 Effect of single-dose SLE on the pharmacokinetics
of digoxin (DCX) and vincristine (VCR).

Croups   [C.sub.max]  [t.sub.1/2]  CL (l/kg  [AUC.sub.0-[tau]]
             (ng/ml)          (h)        h)          (ng h/ml)

DGX      15.04 [+ or   3.08 [+ or  7.66 [+     61.80 [+ or -]
             -] 5.07      -] 1.23    or -]              13.37
                                      1.53

DGX +    17.46 [+ or   2.98 [+ or  5.53 [+     84.38 [+ or -]
SLE          -] 2.26      -] 0.36    or -]               7.25
                                      0.48

p             0.3099       0.8559   0.0089             0.0046

VCR     352.70 [+ or   6.33 [+ or  2.23 [+    414.49 [+ or -]
            -] 92.75      -] 3.04    or -]              94.12
                                      0.47

VCR +   317.94 [+ or   5.47 [+ or  1.74 [+    547.96 [+ or -]
SLE        -] 127.75      -] 1.76    or -]              31.30
                                      0.17

P             0.6014       0.5616   0.0339             0.0081

Croups  [AUC.sub.0-[infinity]]
                     (ng h/ml)

DGX       67.51 [+ or -] 13.43

DGX +      90.98 [+ or -] 8.39
SLE

p                       0.0046

VCR      462.31 [+ or -] 85.34

VCR +    580.14 [+ or -] 54.25
SLE

P                       0.0171


Long-term treatment with SLE increased plasma concentrations of P-gp substrates in rats

As shown in Fig. 6C, 10 continuous days administration of SLE could increase the [AUC.sub.0-[tau]] of digoxin about 2-fold, from 68.30 [+ or -] 25.96 ng h/ml in the vehicle treated group to 123.24 [+ or -] 52.78 ng h/ml in the SLE-treated group (p< 0.05). CL was decreased from 7.48 [+ or -] 3.09 ng him, in the vehicle treated group to 4.49 [+ or -] 1.75 ng h/ml in the SLE-treated group (p < 0.05). [C.sub.max] and [t.sub.1/2] was not significantly changed by co-administrating of SLE at 500 mg/kg (p >0.05). Besides, long-term (10 days) treatment with SLE could increase the [AUC.sub.0-[tau] of vincrisine about 1.6-fold, from 448.08 [+ or -] 123.68 ng h/ml in the vehicle treated group to 718.22 [+ or -] 56.02 ng h/ml in the SLE-treated group (p < 0.05), as shown in Fig. 6D. CL was decreased from 2.14 [+ or -] 0.54 ng h/ml in the vehicle treated group to 1.21 [+ or -] 0.08 ng h/ml in the SLE-treated group (p <0.05). [C.sub.max] and [t.sub.1/2] was not significantly changed by co-administrating of SLE at 500 mg/kg (p > 0.05). The pharmacokinetic parameters of digoxin and vincristine in rats are listed in Table 2.

Table 2 Effect of multi-dose SLE on the pharmacokinetics
of digoxin (DGX) and vincristine (VCR).

Groups   [C.sub.max]  [t.sub.1/2]  CL(l/kg  [AUC.sub.0-[tau]]
             (ng/ml)          (h)       h)          (ng h/ml)

DCX      11.75 [+ or   2.99 [+ or  7.48 [+     68.30 [+ or -]
             -] 4.22      -] 2.32    or -]              25.96
                                      3.09

DCX +    22.94 [+ or   2.17 [+ or  4.49 [+    123.24 [+ or -]
SLE         -] 13.67      -] 0.87    or -]              52.78
                                      1.75

P             0.0844       0.4359   0.0331             0.0452

VCR     455.13 [+ or   3.50 [+ or  2.14 [+    448.08 [+ or -]
           -] 101.52      -] 0.79    or -]             123.68
                                      0.54

VCR +   431.50 [+ or   4.44 [+ or  1.21 [+    718.22 [+ or -]
SLE         -] 51.13      -] 2.22    or -]              56.02
                                      0.08

p             0.6216       0.3555   0.0019             0.0006

Groups  [AUC.sub.0-[infinity]]
                     (ng h/ml)

DCX       76.14 [+ or -] 30.04

DCX +    126.79 [+ or -] 52.07
SLE

P                       0.0330

VCR     492.23 [+ or -] 120.99

VCR +    830.43 [+ or -] 53.51
SLE

P                       0.0001


Down-regulation of the P-Gp expression in rat brain and intestinal by SLE

The rats in group I were given a dose of SLE intragastrically at 500 mg/kg suspended in 0.5% sodium CMC once a day for 10 continuous days, and the other rats in group 11 received the vehicle (0.5% CMC-Na), serving as the control group. Intestinal or brain homogenates were used to investigate the effects of SLE on P-gp expression in vivo. As shown in Fig. 7A, significant difference on the P-gp expression in brain tissues was found between the two groups, and the P-gp expression in rat brain could be greatly inhibited by SLE. Similarly, the P-gp expression in rat intestinal could also be inhibited by administration of SLE (Fig. 7B). Significant difference was found on the protein expression between the control group and the SLE-treated group (p < 0.05), as shown in Fig. 7C.

Discussion

The widespread use of HMs had led to increasing concerns on potential herb-drug interactions through effects on enzyme pathways and P-gp regulation since HMs were often administered in combination with therapeutic drugs (Gouws et al. 2012). P-gp, one of the most well recognized efflux transporters, is present in many tissues including the intestine, brain, liver and kidney (Wakasugi et al. 1998). P-gp is vulnerable to inhibition, activation, or induction by herbal constituents (Aggarwal et al. 2006). The inhibition of P-gp by herbal constituents may provide a novel approach for reversing multidrug resistance in tumor cells, whereas the stimulation of P-gp expression or activity has implication for chemo-protective enhancement by HMs (Zhou et al. 2004). In conclusion, the modulation of P-gp activity and expression by HMs has shown to be responsible for several clinical herb-drug interactions.

Schisandra was always used as a popular Chinese traditional Chinese and Russian herbal formulae (Tang et al. 2012). In previous studies, several schisandra lignans were found to alter absorption and bioavailability of drugs that are P-gp substrates (Fan et al. 2009; Xue et al. in press; Yoo et al. 2007; Fong et al. 2007). However, it was worth mentioning that the inhibition effect of Schisandra on P-gp had not been thoroughly explored so far. The purpose of this study is to evaluate the effects of SLE on P-gp systematically and the possible P-gp-basal herb-drug interactions in vitro and in vivo.

In the in vitro experiment, the effect of SLE on the uptake and transport of P-gp substrates was firstly investigated in Caco-2 cell. As the results, SLE was found concentration-dependently increase intracellular concentrations of digoxin and vincrisine in Caco-2 cells, similar to the function of verapamil (the positive control inhibitor of P-gp). Besides, the presence of SLE could significantly decrease the transport capability of digoxin across Caco-2 monolayers in the BL-AP direction. When the concentrations of SLE were at 0.5, 2.0, and 10.0 the efflux ratios of digoxin in Caco-2 monolayers were decreased from 3.23 to 1.44, 1.29 and 1.15, respectively. According to the results above, SLE was speculated as a P-gp inhibitor. However, there was also an alternative possibility that SLE might be an inhibitor of other transporters or enzymes since digoxin and vincrisine were not the specific P-gp substrates. In order to further confirm the SLE as a P-gp inhibitor, the effect of SLE on the uptake and transport of P-gp substrates were subsequently investigated in LLC-PK1 and L-MDR1 cell. LLC-PK1, a porcine kidney-derived cell line, expresses very low level of P-gp, whereas L-MDR1 cells are LLC-PK1 cells stably transfected with the human MDR1 gene (Pastan et al. 1988). In this study, the observed efflux ratio of digoxin in L-MDR1 cells was about 1.0, and was not affected by verapamil and SLE. On the other hand, the efflux ratio of digoxin in L-MDR1 cells was found much higher than that in Caco-2 cells, which is in accordance with the higher expression levels of P-gp in L-MDR1 cells. In the presence of 10 p.M verapamil, a known P-gp inhibitor, the efflux ratio of digoxin across L-MDR1 cells was reduced from 44.44 to 11.38. Importantly, the transport capability of digoxin across L-MDR1 cells monolayers in the BL-AP direction were decreasing greatly with the increase in the concentration of SLE. When 0.5, 2.0, and 10.0 [micro]g/ml of SLE were added, the efflux ratio of digoxin across L-MDR1 cells monolayers was reduced from 44.44 to 3.03, 1.34 and 1.05, respectively. Besides, SLE and verapamil could greatly increase the uptake of cligoxin in L-MDR1 cell. As a control, no obvious effect on the uptake of digoxin was found on LLC-PK1 cells when SLE and verapamil were loaded. From the results above. SLE was definitely identified as a P-gp inhibitor.

In the in vivo experiment, the effects of SLE on the pharmacokinetics of P-gp substrates were investigated in rats with or without coadministration of SLE. Two P-gp substrates, digoxin and vincrisine, were chosen as the prototypical P-gp probes, and were given to rats via intravenous injection and intragastic administration, respectively. Oral coadministration of SLE led to a 1.25-fold increase in the [AUC.sub.0-12] of digoxin after single oral administration of a regular 500 mg/kg loading dose. At the same time, coadministration of SLE significantly increased the [AUC.sub.0-12] of intravenous vincrisine by 1.35-fold. Besides, 10 continuous days administration of SLE could increase the AUCO, of digoxin about 2-fold, from 68.30 [+ or -] 25.96 ng h/ml in the vehicle treated group to 123.24 [+ or -] 52.78 ng h/ml in the SLE-treated group (p < 0.05). Similarly, long-term (10 days) treatment with SLE could increase the [AUC.sub.[tau]] of vincrisine about 1.6-fold. In general, function and protein expression are often closely related. Many P-gp inhibitors can strongly decrease the expression of P-gp, in addition to the direct inhibitory effect on P-gp function (Hou et al. 2008). Herein, the effect of SLE on the P-gp expression in rat brain and intestinal was measured. Results from a Western blotting assay directly confirmed that P-gp expression in rat brain and intestinal could be greatly down-regulated by intragastic administration of SLE.

Overall, our results suggest that SLE is a strong P-gp inhibitor. It can not only increase the uptake and decrease the efflux ratios of P-gp substrate drugs on Caco-2 and L-MDR1 cells, but also increase the absorption/bioavailability of P-gp substrate drugs in rats. Moreover, long-term treatment with SLE could down-regulate the brain and intestinal expression of P-gp in rats.

Acknowledgments

Y.L. and Y.Z. contributed equally to this work. This study was supported by the National Nature Science Foundation (81102881), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGQ201109), and Jiangsu Province Key Lab of Drug Metabolism and Pharmacokinetics Projects (BM2012012).

Abbreviations: HMs, herbal medicines; P-gp, P-glycoprotein: SLE. Schisandra lignans extract; DDI, drug-drug interaction; TBS-T, Tris-buffered saline-Tween 20; AP, apical; BL, basolateral; HBSS. Hank's balanced salt solution; AUC, the area under the concentration-time curve; CL clearance; [C.sub.max], maximum plasma concentration; to, half-life time; HPLC, high-performance liquid chromatography: LC/MS, liquid chromatography combined mass spectrometry; LC-IT-TOF/MS, liquid chromatography combined hybrid ion trap time-of-flight mass spectrometry; DGX, digoxin: VCR, vincrisine; CMC-Na, sodium carboxymethyl cellulose.

* Corresponding author. Tel.: +8625 83271060; fax: +86 25 83271060.

** Corresponding author. Tel.: +86 25 83271128; fax: +86 25 83302827.

E-mail addresses: liangyan@cpu.edu.cn, liangyan0679@hotmail.com (Y. hang), jsxielin@sina.com.cn (L. Xie).

(1) These two authors contributed equally to this work.

0944-7113/$--see front matter [c] 2013 Published by Elsevier GmbH.

http://dx.doi.org/10.1016/j.phymed.2013.04.005

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Yan Lianga (a), (1), Yuanyuan Zhou (a), (1), Jingwei Zhang (a), Yanna Liu (a), Tianye Guan (a), Yu Wang (b), Lu Xing (a), Tai Rao (a), Lijun Zhou (a), Kun Hao (a), Lin Xie (a), *, Guang-ji Wang (a), **

(a) State Key Laboratory of Natural Medicines, Key Lab of Drug Metabolism & Pharmacokinetics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China

(b) State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
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Author:Liang, Yan; Zhou, Yuanyuan; Zhang, Jingwei; Liu, Yanna; Guan, Tianye; Wang, Yu; Xing, Lu; Rao, Tai;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Aug 15, 2013
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