Resveratrol Enhances the Bioavailability of Fexofenadine in Healthy Human Male Volunteers: Involvement of P-Glycoprotein Inhibition

Resveratrol (RSV) (3,4’,5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin is naturally present in fruits, vegetables, grape skins and especially in red wine. RSV possessesdiverse biochemical and physiological properties including anti-inflammatory, immune modulatory activities as well as wide range of health benefits ranging from chemoprevention to cardio protection [1]. It is produced by the plants in response to stress, injury, ultraviolet irradiation and fungal infection as part of their defense mechanism. In addition, it is synthesized by grapes in response to fungal infections and foundin red wine at levels between 1 and 10 μM [2].


Introduction
Resveratrol (RSV) (3,4',5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin is naturally present in fruits, vegetables, grape skins and especially in red wine. RSV possessesdiverse biochemical and physiological properties including anti-inflammatory, immune modulatory activities as well as wide range of health benefits ranging from chemoprevention to cardio protection [1]. It is produced by the plants in response to stress, injury, ultraviolet irradiation and fungal infection as part of their defense mechanism. In addition, it is synthesized by grapes in response to fungal infections and foundin red wine at levels between 1 and 10 µM [2].
RSV modulates the synthesis of hepatic apolipoprotein, lipids, inhibits platelet aggregation and eicosanoid production in human platelets and neutrophils [3]. Moreover, RSV inhibits events associated with tumor initiation, promotion and progression [4]. It has been reported that RSV increased the accumulation of daunorubicin (a P-glycoprotein substrate) in KB-C2 cells in a concentration dependent manner by inhibiting P-gp (P-glycoprotein) [5]. Additionally, RSV has been found to reversethe multidrug resistance in KBv200 cells by inhibiting the multidrug resistant gene expression [6]. It has been previously reported that RSV enhanced the chemosensitivity of doxorubicin in multidrug resistant human breast cancer cells by increasing the cellular influx of doxorubicin [7]. In addition, RSV has been found to potentiate the cytotoxic activity P-gp substrates such as docetaxel and doxorubicin due to enhanced intracellular levels by inhibition of P-gp and down regulation of mdr1 gene [8]. Furthermore, in vivo studies in rats demonstrated that RSV was able to increase the bioavailability of P-gp substratesthrough the inhibition of P-gp mediated drug efflux [9,10]. Since RSV is a P-gp inhibitor and may potentially inhibit efflux transporter responsible for the poor absorption of P-gp substrates, the influence of RSV treatment on the pharmacokinetics of known P-gp substrate, fexofenadine hydrochloride (FEX) in healthy volunteers is the subject of current investigation.
intestinal permeability and almost no metabolism of FEX in vivo make it a suitable model substrate to evaluate the role of P-gp in drug efflux. The pharmacokinetic profile of FEX primarily depends on the activity of P-gp, an efflux transporter which is expressed in the small intestinemucosa, hepatocytes, kidney and blood-brain barrier [13,14].
In this study, we hypothesized that if RSV acts as aninhibitor of P-gp mediated efflux, possibly in the intestine,it will increase the bioavailability of FEX, a P-gpsubstrate. To our knowledge, there are no available data thatshow any inhibitory effect of RSV on P-gp mediateddisposition in humans. Therefore, we investigated thepossible effect of RSV on P-gp mediated efflux usingFEX as a P-gp substrate in healthy male volunteers.

Materials
FEX and Diphenhydramine (DPH) were obtained from Matrix Labs Limited (Hyderabad, India). RSV 500 mg capsules were procured from Zenith Nutritions (Banglore, India). FEX 120 mg tablets (Allegra) were purchased from Aventis Pharma Ltd (Mumbai, India). Solvents used for quantitative analysis (Merck, India) and all other chemicals, reagents which were used in the study are of analytical grade.

Subjects
Twelve healthy male volunteers with an age of 28.17 ± 1.59 (range 26-31) years, weight of 68.3 ± 7.95 (range 52-82) kg, height of 173.2 ± 7.33 (range 165-188) cm and body mass index (BMI) of 22.76 ± 2.02 (range 17.99-24.95) were participated in the study ( Table 1). All participants gave their written informedconsent prior to the study. The participants were confirmed as healthy by their medical history, physical examination androutine laboratory tests before enrollment. All the subjectswere nonsmokers, ate a normal diet and were not taking anyherbal dietary supplements. The subjects were instructed toabstain from taking any medication for at least 2 weeks priorto and during the study period. All the subjects were asked toabstain from alcohol, caffeine-containing beverages, tea andfruit juices during the study period.

Study design
The study protocol was approved by the institutional human ethical committee (UCPSC/KU/BA/2012-08) of University College of pharmaceutical sciences, Kakatiya University, Warangal, India. A nonblinded, open-label, two-way crossover studywas performed. The study phases were divided into control andtreatment separated by a 2-week washoutperiod. The general study design was identical in control and treatment phases. The participants were administered with oral RSV 500 mg capsules oncedaily for duration of 10 days (Zenith Nutritions, Banglore, India). A single oral dose of FEX 120 mg (Allegra, Aventis Pharma Ltd, India) was administered during control phase and on eleventh day after RSV treatment with 240 mL water under fasting conditionsof more than 10 h. The doses of RSV and FEX were selected based on the marketed formulations. The drugs used in this study were given in the sitting position and the subjects remained seated for4 h after administration of FEX. Standardizedmeals were given at 4 and 10 h after dosing. Blood samples were drawn immediately before as well as 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36 and 48h after FEX administration. Blood samples were collected in heparinized vacutainers and centrifuged at 4,000 rpm for 15 min and theseparated plasma samples were stored at -70°C until the assaywas performed.

Determination of fexofenadine hydrochloride concentration inplasma
FEX concentration in plasma samples wasassayed using LC-MS/MS method [15] by employing protein precipitation extraction method. Aliquots of plasma samples (100 μL) were added to 200 μL acetonitrile with DPH (4 ng/mL) as an internal standard, then mixed in a vortex mixer for 3 min and centrifuged at 14,000 rpmfor 8 min. Next pipette out 150 μL of supernatant, then 5 μL aliquot was injectedinto the analytical column. The plasma concentration of FEX was analyzed by LC-MS/MS with Agilent 1200 quaternary pump, auto sampler with thermostat, column oven, online degasser and triple quadrupole mass spectrometer (Mass hunter software version B.03.01) with multimode source (Agilent Technologies, USA). A Symmetry C18 reverse phase column (75 mm × 4.6 mm, i.d. 3.5 µm) and the mobile phase (acetonitrile: 10 mM ammonium acetate: 0.1% formic acid = 70:30) at a flow rate of 0.2 mL/min were used. FEX was monitored in positive ion mode with the transition of m/z 502.3 to m/z 466.2 and DPH with the transition of m/z 256.2 to m/z 167.0 respectively. The lower limit of quantification for FEX was 0.5 ng/mL, and the assay range used was 0.5-1000 ng/mL. Correlation coefficient for FEX calibration curves was 0.998.The intra-day and inter-day coefficient of variations for the low andhigh quality control samples were less than 15%.

Pharmacokinetic analysis
The pharmacokinetics of FEXwas estimated usingnoncompartmental method using Phoenix WinNonlin 6.2.1 software (Certara, Pharsight Corporation, USA). Themaximum plasma concentration (C max ) and the time taken to reach C max is T max were estimated directly from the observedplasma concentration-time data. The elimination rate constant (K el ) was determined by linear regression analysis of thelog-linear part of the plasma concentration-time curve. Thetotal area under the plasma concentration-time curve (AUC)was calculated using the linear trapezoidal rule. The AUCfrom 0 to infinity (AUC inf ) was calculated as AUC inf =AUC +Ct/K el (where Ct was the last plasma concentration measured). The half-life (T 1/2 ) of FEX was calculated using T 1/2 =ln2/K el . The apparent oral clearance (CL/F) of FEX was calculated as CL/F=dose/AUC inf and the apparent volume of distribution (Vd/F) was calculated using Vd/F = CL/F/λz.

Statistical analysis
The results are expressed as the mean ± SD. Thepharmacokinetics of FEX between the control and treatment phaseswere compared to each other using a paired t-test (twotailed) or the Wilcoxon signed

Results
The pharmacokinetic parameters and mean plasma concentrationtime profiles of FEX after a 10-day treatment with RSV are shown in Table 2 and Figure 1, respectively. During the study period, no serious adverse effects related to the drugs were reported. When we measured the plasma FEX concentrations after a single oral dose of 120 mg FEX, those of RSV phase were increased compared to those of the control phase ( Figure 1 versus 342.62 ± 108.56 L, P<0.05) respectively after RSV treatment as compared to control phase.The AUC andC max of FEX were increased where as CL/F and Vd/F were decreased in all individualsupon RSV treatment as compared to control phase ( Figure 2). However, there was no significant change was observed in T 1/2 , T max and K el of FEX between RSV treatment and control phases. The 90% CI for the ratio of geometric means (RSV treatment phase:control phase) for AUC, C max, CL/F and Vd/F were fell outside the interval (0.8-1.25) but in case T 1/2 , T max and K el fell within the interval ( Table 2) which indicates the interaction between RSV and FEX, consequently the pharmacokinetics of FEX were altered upon RSV treatment.

Discussion
P-gp is an adenosine triphosphate (ATP)-dependentmultidrug transporter expressed mainly on theapical membrane of the intestine. P-gp has been reported topromote the elimination of many drugs into the intestinallumen, thereby limiting their gastrointestinal absorption.Pgp can also reduce the intracellular concentrations of manycytotoxic anti-tumor drugs and is involved in multidrug resistancein tumor cells [16]. Recently inhibition of this transporter protein was implicated as a mechanism underlying certain drug-phytochemical interactions. In the clinical setting, familiarity with known inhibitors of P-gp, together with knowledge of the role of P-gp in determining substrate pharmacokinetics, should increase awareness of potential adverse or desired effects when phytochemicals that interact with P-gp substrates.  Pharmacokinetic data are expressed as mean ± SD. *P < 0.05, compared with the control phase. Table 2: Pharmacokinetic parameters of fexofenadine after a single-dose administration of 120 mg fexofenadine hydrochloride in 12 healthy subjects, after 500 mg resveratrol once daily for 10 days.
In this study, we assessed the effect of RSV on the pharmacokinetics of FEX, a substrate of P-gp in humans and found that RSV affected the pharmacokinetics of FEX and increased its bioavailability. To assess the inhibitory effect of RSV on P-gp in humans, we used FEX as a P-gp substrate [17]. RSV treatment significantly increased the C max and AUC of FEX to 65.2% and 76.6% respectively as compared to control phase. On the other hand, CL/F and Vd/F of FEX were significantly decreased to 42.6% and 42.1% respectively as compared to control phase. However,there was no significant change was observed in T 1/2, T max and K el of FEX upon RSV treatment when compared to control phase. The interaction between RSV and FEX was also assessed by geometric mean ratios and 90% CI. The 90% CI for the ratio of geometric means for AUC, C max , CL/F and Vd/F were fell outside the interval (0.8-1.25) which indicates the significant difference in mean values of AUC, C max, CL/F and Vd/F between control and treatment phases. On the other hand, 90% CI for the ratio of geometric means for T 1/2, T max and K el fell within the interval (0.8-1.25) which indicates the insignificant difference in mean values of T 1/2, T max and K el between control and treatment phases. These findings provide in vivo evidence that RSV might increase the bioavailability of FEX via the inhibition of P-gp mediateddrug efflux during the absorption phase in the intestine. Likewise, other P-gp inhibitors such as itraconazole [18][19][20], ritonavir [21] exhibited similar inhibitory effects on the pharmacokinetics of FEX, namely an increase in C max and AUC, decrease in CL/F and Vd/F. On the other hand, in the case of P-gp inducers such as St. John's wort [22], rifampin [23] and carbamazepine [24], the CL/F and Vd/F of FEX were reported to have been increased while the C max and AUC were decreased. Furthermore, it has been reported that P-gp was the main transport protein responsible for FEX transport and the in vitro FEX transport was inhibited by various P-gp inhibitors such as ketoconazole, verapamil, erythromycin and ritonavir in the Caco-2 cell model [25,26]. Considering that T 1/2 and T max were not changed significantly by RSV, the results of the presentstudy suggest that RSV affects P-gp expressed in thesmall intestine instead of the kidney and liver.
In this study we assessed whether RSV could act as an inhibitor of P-gp mediateddrug efflux. Previously it has been reported that RSV exerted an inhibitory effect on P-gp mediated efflux in vitro resulted in increased intracellular accumulation of daunorubicin [5]. In addition, an in vitro study has demonstrated that RSV enhanced the chemosensitivity of doxorubicin by increasing the cellular influx of doxorubicin [7]. Additionally, RSV has been found to potentiate the cytotoxic activity of docetaxel and doxorubicin due to enhanced intracellular levels by inhibition of P-gp [8]. Further, in vivo studies in rats demonstrated that RSV was able to increase the bioavailability of P-gp substratesthrough the inhibition of P-gp mediated drug efflux [9,10]. It has been reported that RSV exertedan inhibitory effect on drugmetabolizingenzymeCYP3A4 in vitro [27], similarly RSV exhibited inhibitory effect on drugmetabolizingenzymes including CYP3A4, CYP2D6, and CYP2C9 in vivo in human volunteers [28]. Therefore, it is possible a priori to assume that the interaction between RSV and FEX might result from the inhibition of RSV on the metabolism of FEX. However, to our knowledge, FEX is not a substrate of these enzymes and it has been revealed that FEX itself is not further metabolized [29,12]. It has been previously reported that verapamil (a standard P-gp inhibitor) enhanced the bioavailability of FEX through P-gp inhibition [30]. Moreover, our results were comparable to previous work where quercetin substantially enhanced the bioavailability of FEX in healthy volunteers by inhibiting P-gpmediated drug efflux in the intestine [13]. Based on these findings, we suggest that RSV acts as an inhibitor of P-gpmediated efflux of FEX in humans.
Recently, it has been reported that in addition to P-gp,FEX is also a substrate of drug uptake transporters,such as organic anion transporter (OAT-3) and organicanion-transporting polypeptides (OATP)-A (OATP1A2) and OATP-B (OATP2B1) [12,17,31,32]. Dresser et al. [33] reported that grapefruit and apple juicesreduce FEX bioavailability through the inhibitionof OATP1A2 which resulted in delay in T max . However, the inhibition of uptaketransporters leads to a delay inT max , but no significant difference wasobserved in T max between the RSV treatment and control phases. Furthermore, eventhough the disposition of FEX may be affected by OATP, there were no reports available regarding the RSV having the inhibitory effect on OATPmediated efflux. Therefore, the role of OATP in the interaction between RSV and FEX may be minor compared to thatof P-gp.
RSV could be used as a P-gp inhibitor like verapamil, itraconazole and ritonavir and it is having the potential increase the bioavailability of P-gp substrates like digoxin, talinolol including FEX.In addition, the intake of dietary supplements containing RSV may increase the absorption or oral bioavailability of FEX and can be useful to reduce the dose of FEX and possibly helpful in reducing the side effects of FEX. However, further studies should be carried out in patients to explore the RSV mediated P-gp interactions and the potential clinical application of dietary phytochemicals as potent P-gp modulators.

Conclusions
The results of the present study showed that multiple use of RSV substantially enhanced thebioavailability of FEX, probably by the inhibition of P-gpmediated drug efflux in humans. The inhibition of P-gp by dietary phytochemicals containing RSV may provide a novel approach for improving the absorption and exposure of low bioavailable drugs like anti viral and anti cancer drugs.Accordingly, much effort is currently being expanded toward identifying natural compounds from plant origins that inhibitP-gp and developing as P-gp modulators.