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Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition
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PHAREP 399 1–6 Pharmacological Reports xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2
Original research article
Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition
3 4 5
Q1 Satish
6 7
Kumar Bedada, Narsimha Reddy Yellu, Prasad Neerati *
Drug Metabolism and Clinical Pharmacokinetics Division, Department of Pharmacology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India
A R T I C L E I N F O
Article history: Received 13 June 2015 Received in revised form 29 August 2015 Accepted 31 August 2015 Available online xxx Keywords: Drug–phytochemical interactions Resveratrol Fexofenadine P-glycoprotein Pharmacokinetics
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A B S T R A C T
Background: Resveratrol (RSV) is a natural occurring antioxidant has been found to possess P-glycoprotein (P-gp) inhibition activity in vitro and in vivo, which may have the potential to cause drug–phytochemical interactions. The purpose of the present study was to evaluate the effect of RSV on the pharmacokinetics of fexofenadine (FEX), P-gp substrate in rats. Methods: A mechanistic evaluation was undertaken using in vitro non-everted sac and in situ intestinal perfusion studies to determine the FEX intestinal transport and permeability. These results were confirmed by an in vivo pharmacokinetic study of oral administered FEX (10 mg/kg) in rats. Results: The intestinal transport and apparent permeability (Papp) of FEX were increased significantly in duodenum, jejunum and ileum of RSV and verapamil (VER) pretreated groups when compared to FEX alone group. Similarly absorption rate constant (Ka), fraction absorbed (Fab) and effective permeability (Peff) of FEX were increased significantly in ileum of RSV and VER pretreated groups when compared to FEX alone group. In comparison with FEX alone, RSV pretreatment significantly increased maximum plasma concentration (Cmax) and area under the concentration–time curve (AUC), while there was no significant change was observed in T1/2 and Tmax of FEX. Conclusions: RSV significantly enhanced the exposure of FEX in rats likely by the inhibition of P-glycoprotein (P-gp) mediated efflux during the intestinal absorption, suggesting that there is a potential pharmacokinetic interaction between RSV and FEX. Therefore, further studies are recommended to evaluate the potential drug–phytochemical interactions in humans. ß 2015 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Introduction Q2
Resveratrol (RSV) (3,40 ,5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin is naturally present in fruits, vegetables, grape skins and especially in red wine. RSV possesses diverse biochemical and physiological properties including antiinflammatory, immune modulatory activities as well as wide range of health benefits ranging from chemoprevention to cardio protection [1]. It is produced by these plants in response to stress, injury, ultraviolet irradiation and fungal infection as part of their defense mechanism and it is synthesized by grapes in response to fungal infections and is found, therefore, in red wine at levels between 1 and 10 mM [2]. Resveratrol have recently been shown to exert genoprotective, cytotoxic, antiproliferative and proapoptotic actions in different tumoral cell lines [3].
* Corresponding author. E-mail address: [email protected] (P. Neerati).
RSV modulates the synthesis of hepatic apolipoprotein and lipids and inhibits platelet aggregation and eicosanoid production in human platelets and neutrophils [4]. Moreover, RSV inhibits events associated with tumor initiation, promotion, and progression [5]. 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) [6]. Additionally, RSV has been found to reverse the multidrug resistance in KBv200 cells by inhibiting the multidrug-resistant gene expression [7]. It has been previously reported that RSV enhanced the chemosensitivity of doxorubicin in multidrugresistant human breast cancer cells by increasing the cellular influx of doxorubicin [8]. In addition, RSV has been found to potentiate the cytotoxic activity of docetaxel and doxorubicin (P-gp substrates) due to enhanced intracellular levels by inhibition of P-gp and down regulation of mdr1 gene [9]. Furthermore, in vivo studies in rats demonstrated that RSV was able to increase the bioavailability of P-gp substrates through the inhibition of P-gpmediated drug efflux [10,11]. Since RSV is a P-gp inhibitor and may
http://dx.doi.org/10.1016/j.pharep.2015.08.018 1734-1140/ß 2015 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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potentially inhibit efflux transporter responsible for the poor absorption of P-gp substrates, the effect of RSV treatment on the pharmacokinetics of known P-gp substrate, FEX is the subject of current investigation. However, no information is available on the pharmacokinetic interaction between RSV and FEX. P-gp is a member of the adenosine triphosphate (ATP)-binding cassette family and is a 170-kDa plasma membrane-associated protein, present in small intestine, liver, kidney, and blood–brain barrier in both rodents and humans [12]. The presence of P-gp in animal and human organs and tissues alters the absorption of many P-gp substrate drugs [13]. FEX is a non-sedating antihistamine drug which is indicated for treatment of seasonal allergic rhinitis [14]. FEX has low oral bioavailability (approximately 33%) in humans due to P-gp-mediated drug efflux in intestine and it could be a suitable probe substrate drug to investigate the significance of P-gp-mediated drug interactions without interference of metabolic pathways [15]. The main objective of this study was to investigate the effect of RSV on the pharmacokinetics of FEX, P-gp probe substrate in rats. Moreover, it can be expected that concomitant use of RSV and drugs substrate for P-gp may increase the possibility of drug–phytochemical interactions. Therefore, the purpose of the present study was to investigate the effect of RSV on the pharmacokinetics of FEX in rats. The intestinal permeability characteristics of FEX were mechanistically investigated using the in vitro non-everted sac and in situ single pass intestinal perfusion methods. To confirm these findings, an in vivo pharmacokinetic study of oral administered FEX in rats with or without RSV pretreatment was performed.
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Materials and methods
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Chemicals and animals
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FEX was procured from Auro Labs Limited (Mumbai, India). Propranalol (PRP) and verapamil (VER) were obtained from Lupin Labs Limited (Pune, India). RSV and phenol red (PHR) were purchased from Sigma–Aldrich (Banglore, India) and Himedia (Mumbai, India) respectively. Solvents used for quantitative analysis were of HPLC grade (Merck, India) and all other chemicals, reagents which were used in the study are of analytical grade. Male Wistar rats weighing about 220–240 g were used for in vitro, in situ and in vivo pharmacokinetic studies were procured from Mahaveera Enterprises, Hyderabad, India. Rats housed in cages were kept in a room under controlled temperature (20–22 8C) and 12 h day–night cycle. Animals were used for studies after 1-week acclimatization with free access to water and feed. All animal study protocols were approved by Institutional Animal Ethics Committee of Kakatiya University (IAEC/04/UCPSC/KU/2014).
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In vitro non-everted intestinal sac study
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An in vitro non-everted intestinal sac study was performed according to the previously described methods [16,17]. Briefly, the rats were divided into three groups FEX alone (control), RSV pretreatment (test group) and VER pretreatment (positive control, standard P-gp inhibitor) groups each consisting of four animals. RSV and VER suspensions (prepared by suspending in 0.25% (w/v) of sodium carboxymethyl cellulose) were administered orally to pretreatment groups at a dose of 50 mg/kg and 25 mg/kg, respectively for 7 days and FEX alone group was kept as control. Both control and pretreatment group rats were sacrificed on 8th day by using anesthetic ether, the intestine was surgically removed and flushed with 50 mL of ice cold saline. The small intestine was cut into 3 segments, duodenum, jejunum and ileum of equal length (5 cm). The probe drug (FEX 500 mg/mL) was dissolved in pH 7.4 isotonic Dulbecco’s PBS (D-PBS) containing 25 mM glucose. The
probe drug solution (1 mL) was filled in the normal sac (mucosal side), and both ends of the sac were ligated tightly. The sac containing probe drug solution was placed in a beaker containing 40 mL of D-PBS, containing 25 mM glucose. The medium was prewarmed at 37 8C and pre-oxygenated with 5% CO2/95% O2 for 15 min., under bubbling with a CO2/O2 mixture gas, the transport of the FEX from mucosal to serosal direction across the intestinal sacs was measured by sampling the serosal medium at 0,15, 30, 45, 60 and 120 min. The samples of 1 mL were collected from FEX alone, RSV pretreated and VER pretreated groups at predetermined time points and stored at 80 8C until analysis. The drug transported from mucosal to serosal direction was measured by high performance liquid chromatography (HPLC).
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Calculation of apparent permeability coefficient (Papp)
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The apparent permeability coefficient (Papp) of FEX was calculated from the following equation [16]:
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P app ¼
dQ 1 dt AC 0
(1)
where dQ/dt is the transport rate of drug in the serosal medium, A is the surface area of the intestinal sacs and C0 is the initial concentration inside the sacs.
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In situ intestinal perfusion study
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The surgical procedure and the in situ single-pass intestinal perfusion (SPIP) study was performed according to the previously described methods [18–20]. Briefly, the rats were divided into three groups FEX alone (control), RSV pretreatment (test group) and VER pretreatment (positive control, standard P-gp inhibitor) groups each consisting of four animals. RSV and VER suspensions (prepared by suspending in 0.25%, w/v, of sodium carboxymethyl cellulose) were administered orally to pretreatment groups at a dose of 50 mg/kg and 25 mg/kg, respectively for 7 days and FEX alone group was kept as control. Both control and pretreatment group rats were subjected to surgery on 8th day. They were anaesthetized by thiopental sodium (50 mg/kg, ip) and placed on a hot pad to maintain normal body temperature. A midline incision was made on the abdomen and an ileal segment of approximately 8–12 cm was isolated using the ileo-caecal junction as a distal marker. Semi-circular incisions were made at each end and the lumen was rinsed with saline (37 8C) and the both ends were cannulated with PE tubing and ligated by using silk suture. Blank perfusion buffer (Phosphate buffer, pH 7.4) was first infused for 5 min at a flow rate of 1 mL/min by using Syringe pump (NE-1600, New Era Syringe Pumps, Inc. NY, USA), followed by perfusion of phosphate buffer saline (pH 7.4) containing FEX (50 mM), PRP (100 mM) and PHR (50 mg/mL) at a constant flow rate of 0.2 mL/min and the samples were collected at 10, 20, 30, 40, 50, 60, 70, 80 and 90 min. After cannulation the segment was covered with isotonic saline-wet gauze (37 8C). At the end of the perfusion the length of the segment was measured following the last sample collection. Perfusion samples were collected from FEX alone, RSV pretreated and VER pretreated groups at predetermined time points and stored at 80 8C until analysis. FEX concentrations in perfusion samples were measured by HPLC.
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Phenol red water flux correction
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Cout
(corr)
was calculated from the following equation [21]:
C outðcorrÞ ¼ C out
Concentration of phenol red in ðCPRin Þ Concentration of phenol red out ðCPRout Þ
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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where Cout (corr) is corrected outlet concentration of the drug, Cout is outlet concentration of the drug, CPRin is concentration of PHR entering the intestinal segment and CPRout is concentration of PHR exiting the intestinal segment.
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Calculation of effective permeability coefficient (Peff)
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It is calculated from the steady state concentration of compounds in the collected perfusate which is considered to be attainable when the concentration level of PHR (non-absorbable marker) is stable and it was reached at 30–40 min after the beginning of the experiment. The steady state effective permeability is calculated using the following equation [18]: P eff ¼
Q lnðC out =C in Þ 2prL
3
pH 4.4 adjusted with phosphoric acid) and the elution was monitored at 195 nm at a flow rate of 1.0 mL/min. In case of PRP the mobile phase was used a mixture of methanol: 0.05 mM KH2PO4 (55:45, v/v, pH 6.0 adjusted with potassium hydroxide) and the elution was monitored at 227 nm at a flow rate of 1.0 mL/ min [23]. All the in vitro, in situ perfusion and plasma samples were extracted using a simple protein precipitation method by adding acetonitrile (200 mL) to samples (100 mL). Samples were vortexed for 2 min and centrifuged at 13,000 rpm for 15 min. The resultant clean supernatant (20 mL) was injected and analyzed using HPLC method. The limit of detection was 0.01 mg/mL and the assay range used was 0.01–10 mg/mL. The average recovery of the drug was 88.98%. The intra-day and inter-day coefficients of variation for the low and high quality control samples were less than 15%.
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Spectrophotometric analysis of phenol red
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The PHR in phosphate buffer pH (7.4) has a characteristic red color which was measured spectrophotometrically at 560 nm [20].
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where Q is perfusion flow rate (0.2 mL/min), Cout is corrected outlet concentration of the drug, Cin is inlet drug concentration, r is radius of the rat small intestine (0.18 cm) and L is the length of the perfused intestinal segment (10 cm).
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Calculation of fraction of drug absorbed (Fab)
Pharmacokinetic analysis
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The fraction of drug absorbed is calculated using the following equation [22]:
Pharmacokinetic parameters were computed by non-compart- 230 mental model using Phoenix WinNonlin version 6.2 software 231 (Certara, Pharsight Corporation, USA). The plasma FEX concentra- Q3232 tion versus time plots were used to estimate the peak plasma 233 concentration (Cmax), time to reach peak plasma concentration 234 (Tmax), area under the concentration time curve to the last 235 sampling point (AUC0–t), area under the concentration time curve 236 to the infinity (AUC0–1), half-life (T1/2), apparent clearance (CL/F) 237 and apparent volume of distribution (Vd/F). 238
F ab ¼ 1
C out C in
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where Cout is corrected outlet concentration of the drug, Cin is inlet drug concentration.
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Calculation of absorption rate constant (Ka)
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The absorption rate constant is calculated using the following equation [22]: C out Q Ka ¼ 1 C in V
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where Q is perfusion flow rate (0.2 mL/min), Cout is corrected outlet concentration of the drug, Cin is inlet drug concentration and V is the volume of the perfused segment.
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Statistical analysis
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All mean values are presented as with their standard deviation (mean SD). Statistical analysis was performed by Student’s unpaired t test and analysis of variance (ANOVA) fallowed by Banferoni post-test using GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA, USA) at significance level of p < 0.05.
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Results
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In vivo pharmacokinetic study
Effect of RSV on intestinal transport and apparent permeability of FEX
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Rats were fasted overnight before oral dose administration and approximately 3 h post-dose. The rats were divided into two groups of control and pretreatment consisting of six animals each. RSV suspension at a dose of 50 mg/kg was administered orally to pretreatment group for 7 days. FEX was dissolved in ethanol and water (1:10) for oral administration. FEX at a dose of 10 mg/kg was administered to both control and pretreatment groups on 8th day. Under ether anesthesia, blood samples (approximately 0.25 mL) were collected from the retro-orbital plexus into heparinized micro centrifuge tubes prior to dosing and at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12 and 24 h post-dosing and plasma was obtained by centrifuging the blood samples at 13,000 rpm for 15 min and stored at 80 8C until HPLC analysis.
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HPLC analysis of FEX and PRP
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Shimadzu HPLC system equipped with a LC-20AD pump and SPD 20A UV visible detector and RP C18 column (Phenomenex Luna, 250 mm 4.6 mm ID, particle size 5 mm) was used for the analysis of samples. For FEX the mobile phase used was a mixture of 0.1 M triethylamine: acetonitrile: methanol (61:19.5:19.5, v/v,
The present investigation involves the determination of FEX intestinal transport and Papp in FEX alone, RSV pretreated and VER pretreated rats. Pretreatment with RSV (50 mg/kg) for 7 days resulted in a significant (p < 0.05) increase in mean cumulative concentrations (from mucosal to serosal direction) of FEX from 2.28 0.87 to 7.96 3.15 mg/mL in duodenum; 2.67 1.27 to 8.23 3.61 mg/mL in jejunum; 3.46 1.35 to 9.29 3.09 mg/mL in ileum (Fig. 1). In addition, pretreatment with positive control, VER (25 mg/kg) for 7 days resulted in a significant (p < 0.05) increase in mean cumulative concentrations (from mucosal to serosal direction) of FEX from 2.28 0.87 to 9.10 3.31 mg/mL in duodenum; 2.67 1.27 to 9.63 3.95 mg/mL in jejunum; 3.46 1.35 to 10.94 3.77 mg/mL in ileum (Fig. 1). The transport of FEX was found to be increased by 3.5-, 3.0-, and 2.7-fold in duodenum, jejunum and ileum, respectively in RSV pretreated group when compared with the FEX alone group. Further, the transport of FEX was found to be increased by 3.9-, 3.6-, and 3.2-fold in duodenum, jejunum and ileum, respectively in VER pretreated group when compared with the FEX alone group. The transport of FEX was increased from duodenum to ileum in RSV pretreated group which was comparable to positive control VER (standard P-gp inhibitor) pretreated group.
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Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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Fig. 1. Intestinal transport of FEX in duodenum, jejunum and ileum of Wistar rats. FEX alone group administered with FEX (10 mg/kg).
RSV pretreated
group administered with RSV (50 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th day.
VER pretreated group administered with VER (25 mg/kg) for 7 days
followed by FEX (10 mg/kg) on 8th day. Data are represented as mean S.D. (n = 4). *Significant difference (p < 0.05) in comparison with the control (two-way ANOVA followed by Bonferoni post-test).
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Pretreatment with RSV resulted in a significant (p < 0.05) increase in Papp of FEX from (0.091 0.003) 104 cm/s to (0.336 0.006) 104 cm/s in duodenum; (0.123 0.003) 104 cm/s to (0.368 0.007) 104 cm/s in jejunum; (0.144 0.014) 104 cm/s to (0.355 0.010) 104 cm/s in ileum (Fig. 2). In addition, pretreatment with positive control, VER resulted in a significant (p < 0.05) increase in Papp of FEX from (0.091 0.003) 104 cm/s to (0.364 0.023) 104 cm/s in duodenum; (0.123 0.003) 104 cm/s to (0.414 0.018) 104 cm/s in jejunum; (0.144 0.014) 104 cm/s to (0.424 0.023) 104 cm/s in ileum (Fig. 2). The Papp of FEX in duodenum, jejunum and ileum was found to be increased by 3.7-, 2.9-, and 2.5-fold, respectively in RSV pretreated group when compared with FEX alone group. Where as in case of VER pretreated group the Papp of FEX in duodenum, jejunum and ileum was found to be increased by 4.0-, 3.4-, and 2.9fold, respectively when compared with FEX alone group. The Papp values of FEX in RSV pretreated group were comparable to positive control VER (standard P-gp inhibitor) pretreated group.
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Effect of RSV on absorption rate constant, fraction absorbed and effective permeability of FEX
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FEX and PRP absorption rate constant (Ka), fraction absorbed (Fab) and intestinal effective permeability (Peff) were determined in rat ileum segment using SPIP technique. Ka, Fab and Peff values were
calculated from the steady-state concentrations in the perfusate collected from the outlet. Pretreatment with RSV for 7 days resulted in a significant (p < 0.05) increase in Ka of FEX from 0.028 0.002 min1 to 0.084 0.001 min1 and in case of pretreatment with VER resulted in a significant (p < 0.05) increase in Ka of FEX from 0.028 0.002 min1 to 0.092 0.001 min1 while there was no significant change was observed in Ka of PRP of both RSV and VER pretreated groups and it was found to be 0.044 0.002 min1, 0.045 0.001 min1 and 0.045 0.002 min1 in FEX alone, RSV pretreated and VER pretreated groups respectively (Fig. 3A). The increase in Ka of FEX in ileum was found to be 3.0- and 3.2-fold, respectively in RSV pretreated and VER pretreated groups when compared with the FEX alone group. Pretreatment with RSV for 7 days resulted in a significant (p < 0.05) increase in Fab of FEX from 0.142 0.009 to 0.429 0.004 where as in case of pretreatment with VER resulted in a significant (p < 0.05) increase in Fab of FEX from 0.142 0.009 to 0.467 0.005 while there was no significant change was observed in Fab of PRP of
Fig. 3. Absorption rate constant (Ka) (A), Fraction absorbed (Fab) (B) and Effective Fig. 2. Apparent permeability coefficient (Papp) of FEX in duodenum, jejunum, ileum
permeability (Peff) (C) of FEX and PRP in ileum of Wistar rats.
of Wistar rats.
group administered with FEX (10 mg/kg).
FEX alone group administered with FEX (10 mg/kg).
FEX alone
RSV pretreated group
RSV pretreated group administered with RSV (50 mg/kg) for 7 days followed by FEX
administered with RSV (50 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th
(10 mg/kg) on 8th day.
day.
VER pretreated group administered with VER
(25 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th day. Data are represented as mean S.D. (n = 4). *Significant difference (p < 0.05) in comparison with the control (two-way ANOVA followed by Bonferoni post-test).
VER pretreated group administered with VER (25 mg/kg) for 7 days
followed by FEX (10 mg/kg) on 8th day. Data are represented as mean S.D. (n = 4). *Significant difference (p < 0.05) in comparison with the control (two-way ANOVA followed by Bonferoni post-test).
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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both RSV and VER pretreated groups and it was found to be 0.223 0.014, 0.231 0.007 and 0.228 0.011 in FEX alone, RSV pretreated and VER pretreated groups respectively (Fig. 3B). The increase in Fab of FEX in ileum was found to be 3.0- and 3.3-fold, respectively in RSV pretreated and VER pretreated groups when compared with the FEX alone group. Pretreatment with RSV for 7 days resulted in a significant (p < 0.05) increase in Peff of FEX from (0.450 0.034) 104 cm/s to (1.655 0.024) 104 cm/s, where as in case of pretreatment with VER resulted in a significant (p < 0.05) increase in Peff of FEX from (0.450 0.033) 104 cm/s to (1.859 0.030) 104 cm/s while there was no significant change was observed in Peff of PRP of both RSV and VER pretreated groups and it was found to be (0.850 0.055) 104 cm/s, (0.882 0.027) 104 cm/s and 4 (0.870 0.043) 10 cm/s in FEX alone, RSV pretreated and VER pretreated groups respectively (Fig. 3C). The increase in Peff of FEX in ileum was found to be 3.7 and 4.1-fold, respectively in RSV pretreated and VER pretreated groups when compared with the FEX alone group. The Ka, Fab and Peff values obtained in RSV pretreated group were comparable to positive control VER (standard P-gp inhibitor) pretreated group.
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Effect of RSV on pharmacokinetics of FEX
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The plasma concentration-time plots of FEX after an oral administration of FEX (10 mg/kg) in control and pretreated (RSV 50 mg/kg) rats were characterized and depicted in Fig. 4. The mean pharmacokinetic parameters of FEX were summarized in Table 1. FEX oral pharmacokinetics were found to be significantly (p < 0.05) altered with RSV pretreatment for 7 days when compared with the control (FEX alone). The increase in Cmax, AUC0–t and AUC0–1 of FEX were found to be 3.6, 3.1 and 2.9- fold, respectively in RSV pretreated group. Further, the CL/F and Vd/F of FEX were significantly decreased while there was no significant change was observed in Tmax and T1/2 of FEX in RSV pretreated group when compared with FEX alone group.
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Discussion
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With the great interest in dietary supplements containing herbal constituents as alternative medicines, much effort is currently being expanded toward identifying natural compounds of plant origins that modulate P-gp; however, there is far less information on the pharmacokinetic interactions between dietary supplements containing phytochemicals and drugs. Therefore, more preclinical investigations on the drug–phytochemical interactions should be performed to prevent potential adverse reactions or utilize those interactions for a therapeutic benefit. Therefore, the present study evaluated the effects of RSV, an antioxidant, on the
Table 1 Pharmacokinetic parameters of FEX in control and RSV pretreated group rats after oral administration of 10 mg/kg. Data values are presented as mean SD. (n = 6). Pharmacokinetic parameters
Cmax (ng/mL) Tmax (h) T1/2 (h) AUC0–t (ng.h/mL) AUC0–1 (ng.h/mL) CL/F (L/h/kg) Vd/F (L/kg)
Fexofenadine, PO, 10 mg/kg Control group
RSV pretreated group
163.41 53.44 1.25 0.27 8.25 0.83 834.35 95.61 969.34 87.03 10.39 0.95 124.34 23.31
594.04 130.48* 0.91 0.13 8.49 0.75 2555.31 704.04* 2871.39 740.57* 3.67 0.91* 45.24 12.61*
* Significant difference (p < 0.05) in comparison with the control (unpaired Student’s t-test).
5
Fig. 4. Plasma drug concentration-time plots of FEX in Wistar rats. FEX alone group administered with FEX (10 mg/kg). RSV pretreated group administered with RSV (50 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th day. Data are represented as mean SD. (n = 6).
pharmacokinetics of FEX in rats to examine a potential pharmacokinetic interaction between RSV and FEX via the inhibition of P-gp. In this study, FEX was used as a model substrate for P-gp to explore the interaction with RSV in rats by using different absorption systems. FEX is a specific substrate for P-gp, it is well tolerated in vivo without interference of metabolic pathways [15]. The level of P-gp (mdr1a) mRNA is increased significantly from the top to bottom of the rat small intestine and found that the level of P-gp was higher in ileum than jejunum and duodenum [24]. The non-everted sac model was originally used to evaluate drug transport mechanisms [25]. Genty et al. [26] stated that the passive permeability of actively transported molecules can be determined through non-everted rat gut sacs. Hence, in the present investigation, non-everted intestinal sac study [16] was performed to determine the role of P-gp in the intestinal transport and apparent permeability of FEX. As shown in Fig. 1 the transport of FEX was significantly increased in duodenum, jejunum and ileum sacs of RSV pretreated and VER pretreated group rats when compared with the FEX alone group rats. Similarly, Papp of FEX (Fig. 2) was significantly increased in duodenum, jejunum and ileum sacs of RSV pretreated and VER pretreated group rats in comparison with that of FEX control. 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 [6]. In addition, an in vitro study has demonstrated that RSV enhanced the chemosensitivity of doxorubicin by increasing the cellular influx of doxorubicin [8]. Additionally, RSV has been found to potentiate the cytotoxic activity of docetaxel and doxorubicin due to enhanced intracellular levels by inhibition of P-gp [9]. Furthermore, it has been found to be FEX transport from basal to apical direction was inhibited by VER in Caco-2 cells [27]. Thus, the increase in intestinal transport and Papp of FEX across the duodenum, jejunum and ileum with RSV and VER pretreatment might be attributed to inhibition of P-gp in intestine of rats. The obtained intestinal transport and Papp values of FEX in RSV pretreated group were comparable to VER (standard P-gp inhibitor) pretreated group. Further, in order to support the role of P-gp inhibition involved in intestinal transport of FEX, in situ perfusion studies were performed in ileum of rats. These techniques maintain an intact blood supply to the intestine and can be used to estimate the impact of clearance pathways such as enzymes and transporters present in the gut [18]. In the present study, SPIP was performed in ileum segment of rats to investigate the functional role of P-gp intestinal permeability of FEX. Ka, Fab and Peff of FEX were significantly increased in RSV and VER pretreated group rats in
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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comparison with FEX alone group rats. The increase in Ka, Fab and Peff of FEX in pretreated rats is attributable to the P-gp inhibition by RSV and VER. The Ka, Fab and Peff values obtained in RSV pretreated group were comparable to VER (standard P-gp inhibitor) pretreated group. These results were indicated that P-gp limits the intestinal permeability of FEX by extruding it back to the intestine. PHR was used as a non-absorbable marker in in situ technique. PHR concentration in the outlet perfusate was used to assess the steady-state condition in in situ perfusion technique. It is also used to indicate the intestinal mucosal integrity during perfusion [18]. PRP was used as a passive, highly permeable marker and as an indicator of major changes in mesenteric blood flow [20]. The permeability values of PRP in the FEX alone, RSV pretreated and VER pretreated group rats were found to be statistically insignificant, indicating that changes in FEX permeability in the presence of RSV and VER are not due to compromise in membrane integrity. In this study, we also investigated the effect RSV on the pharmacokinetics of FEX. Pretreatment with RSV for 7 days enhanced the oral bioavailability of FEX in rats. The AUC and Cmax were significantly increased in RSV pretreated group when compared with FEX alone control group. Further, the CL/F and Vd/F of FEX were significantly decreased while there was no significant change was observed in Tmax and T1/2 of FEX in RSV pretreated group when compared to FEX alone control group. Likewise, RSV exhibited similar inhibitory effects on the pharmacokinetics of P-gp substrate drugs nicardipine [10] and diltiazem [11], namely an increase in Cmax, AUC and decrease in CL/F. These findings provide in vivo evidence that RSV might increase the bioavailability of FEX via the inhibition of P-gp mediated drug efflux during the absorption phase in the intestine. Moreover, the results obtained in our study were comparable to previous work where piperine [15] and diosmin [23] substantially enhanced the systemic exposure of FEX by inhibition of P-gp in intestine of rats. Thus, the enhanced AUC and Cmax of FEX in the presence of RSV might be caused, at least in part, by the increased intestinal absorption of FEX via the inhibition of P-gp-mediated drug efflux, which in this study has been substantiated with non-everted intestinal sac and in situ permeability data. Therefore, the present study results suggesting that RSV resulted in drug–phytochemical interaction via the gastrointestinal tract.
440
Conclusions
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RSV is a naturally occurring antioxidant significantly increased the intestinal absorption and exposure of FEX indicating the inhibitory effect on P-gp-mediated drug efflux in rats. In addition, the intake of dietary supplements containing RSV may increase the absorption or exposure of drugs able to serve as P-gp substrates in addition to FEX. Therefore, further studies are recommended to evaluate the potential drug–phytochemical interactions in humans.
448
Conflicts of interest
449 450
The authors declare that there are no conflicts of interest. Acknowledgements
451 Q4 The authors would like thank to University Grants Commission 452 (UGC), New Delhi, India for financial support and Mr. Anand Gupta 453 of Certara Inc., USA for providing the Phoenix WinNonLin 6.2 454 software academic license.
References
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Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
PHAREP 399 1–6 Pharmacological Reports xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2
Original research article
Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition
3 4 5
Q1 Satish
6 7
Kumar Bedada, Narsimha Reddy Yellu, Prasad Neerati *
Drug Metabolism and Clinical Pharmacokinetics Division, Department of Pharmacology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India
A R T I C L E I N F O
Article history: Received 13 June 2015 Received in revised form 29 August 2015 Accepted 31 August 2015 Available online xxx Keywords: Drug–phytochemical interactions Resveratrol Fexofenadine P-glycoprotein Pharmacokinetics
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
A B S T R A C T
Background: Resveratrol (RSV) is a natural occurring antioxidant has been found to possess P-glycoprotein (P-gp) inhibition activity in vitro and in vivo, which may have the potential to cause drug–phytochemical interactions. The purpose of the present study was to evaluate the effect of RSV on the pharmacokinetics of fexofenadine (FEX), P-gp substrate in rats. Methods: A mechanistic evaluation was undertaken using in vitro non-everted sac and in situ intestinal perfusion studies to determine the FEX intestinal transport and permeability. These results were confirmed by an in vivo pharmacokinetic study of oral administered FEX (10 mg/kg) in rats. Results: The intestinal transport and apparent permeability (Papp) of FEX were increased significantly in duodenum, jejunum and ileum of RSV and verapamil (VER) pretreated groups when compared to FEX alone group. Similarly absorption rate constant (Ka), fraction absorbed (Fab) and effective permeability (Peff) of FEX were increased significantly in ileum of RSV and VER pretreated groups when compared to FEX alone group. In comparison with FEX alone, RSV pretreatment significantly increased maximum plasma concentration (Cmax) and area under the concentration–time curve (AUC), while there was no significant change was observed in T1/2 and Tmax of FEX. Conclusions: RSV significantly enhanced the exposure of FEX in rats likely by the inhibition of P-glycoprotein (P-gp) mediated efflux during the intestinal absorption, suggesting that there is a potential pharmacokinetic interaction between RSV and FEX. Therefore, further studies are recommended to evaluate the potential drug–phytochemical interactions in humans. ß 2015 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Introduction Q2
Resveratrol (RSV) (3,40 ,5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin is naturally present in fruits, vegetables, grape skins and especially in red wine. RSV possesses diverse biochemical and physiological properties including antiinflammatory, immune modulatory activities as well as wide range of health benefits ranging from chemoprevention to cardio protection [1]. It is produced by these plants in response to stress, injury, ultraviolet irradiation and fungal infection as part of their defense mechanism and it is synthesized by grapes in response to fungal infections and is found, therefore, in red wine at levels between 1 and 10 mM [2]. Resveratrol have recently been shown to exert genoprotective, cytotoxic, antiproliferative and proapoptotic actions in different tumoral cell lines [3].
* Corresponding author. E-mail address: [email protected] (P. Neerati).
RSV modulates the synthesis of hepatic apolipoprotein and lipids and inhibits platelet aggregation and eicosanoid production in human platelets and neutrophils [4]. Moreover, RSV inhibits events associated with tumor initiation, promotion, and progression [5]. 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) [6]. Additionally, RSV has been found to reverse the multidrug resistance in KBv200 cells by inhibiting the multidrug-resistant gene expression [7]. It has been previously reported that RSV enhanced the chemosensitivity of doxorubicin in multidrugresistant human breast cancer cells by increasing the cellular influx of doxorubicin [8]. In addition, RSV has been found to potentiate the cytotoxic activity of docetaxel and doxorubicin (P-gp substrates) due to enhanced intracellular levels by inhibition of P-gp and down regulation of mdr1 gene [9]. Furthermore, in vivo studies in rats demonstrated that RSV was able to increase the bioavailability of P-gp substrates through the inhibition of P-gpmediated drug efflux [10,11]. Since RSV is a P-gp inhibitor and may
http://dx.doi.org/10.1016/j.pharep.2015.08.018 1734-1140/ß 2015 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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potentially inhibit efflux transporter responsible for the poor absorption of P-gp substrates, the effect of RSV treatment on the pharmacokinetics of known P-gp substrate, FEX is the subject of current investigation. However, no information is available on the pharmacokinetic interaction between RSV and FEX. P-gp is a member of the adenosine triphosphate (ATP)-binding cassette family and is a 170-kDa plasma membrane-associated protein, present in small intestine, liver, kidney, and blood–brain barrier in both rodents and humans [12]. The presence of P-gp in animal and human organs and tissues alters the absorption of many P-gp substrate drugs [13]. FEX is a non-sedating antihistamine drug which is indicated for treatment of seasonal allergic rhinitis [14]. FEX has low oral bioavailability (approximately 33%) in humans due to P-gp-mediated drug efflux in intestine and it could be a suitable probe substrate drug to investigate the significance of P-gp-mediated drug interactions without interference of metabolic pathways [15]. The main objective of this study was to investigate the effect of RSV on the pharmacokinetics of FEX, P-gp probe substrate in rats. Moreover, it can be expected that concomitant use of RSV and drugs substrate for P-gp may increase the possibility of drug–phytochemical interactions. Therefore, the purpose of the present study was to investigate the effect of RSV on the pharmacokinetics of FEX in rats. The intestinal permeability characteristics of FEX were mechanistically investigated using the in vitro non-everted sac and in situ single pass intestinal perfusion methods. To confirm these findings, an in vivo pharmacokinetic study of oral administered FEX in rats with or without RSV pretreatment was performed.
70
Materials and methods
71
Chemicals and animals
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FEX was procured from Auro Labs Limited (Mumbai, India). Propranalol (PRP) and verapamil (VER) were obtained from Lupin Labs Limited (Pune, India). RSV and phenol red (PHR) were purchased from Sigma–Aldrich (Banglore, India) and Himedia (Mumbai, India) respectively. Solvents used for quantitative analysis were of HPLC grade (Merck, India) and all other chemicals, reagents which were used in the study are of analytical grade. Male Wistar rats weighing about 220–240 g were used for in vitro, in situ and in vivo pharmacokinetic studies were procured from Mahaveera Enterprises, Hyderabad, India. Rats housed in cages were kept in a room under controlled temperature (20–22 8C) and 12 h day–night cycle. Animals were used for studies after 1-week acclimatization with free access to water and feed. All animal study protocols were approved by Institutional Animal Ethics Committee of Kakatiya University (IAEC/04/UCPSC/KU/2014).
87
In vitro non-everted intestinal sac study
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An in vitro non-everted intestinal sac study was performed according to the previously described methods [16,17]. Briefly, the rats were divided into three groups FEX alone (control), RSV pretreatment (test group) and VER pretreatment (positive control, standard P-gp inhibitor) groups each consisting of four animals. RSV and VER suspensions (prepared by suspending in 0.25% (w/v) of sodium carboxymethyl cellulose) were administered orally to pretreatment groups at a dose of 50 mg/kg and 25 mg/kg, respectively for 7 days and FEX alone group was kept as control. Both control and pretreatment group rats were sacrificed on 8th day by using anesthetic ether, the intestine was surgically removed and flushed with 50 mL of ice cold saline. The small intestine was cut into 3 segments, duodenum, jejunum and ileum of equal length (5 cm). The probe drug (FEX 500 mg/mL) was dissolved in pH 7.4 isotonic Dulbecco’s PBS (D-PBS) containing 25 mM glucose. The
probe drug solution (1 mL) was filled in the normal sac (mucosal side), and both ends of the sac were ligated tightly. The sac containing probe drug solution was placed in a beaker containing 40 mL of D-PBS, containing 25 mM glucose. The medium was prewarmed at 37 8C and pre-oxygenated with 5% CO2/95% O2 for 15 min., under bubbling with a CO2/O2 mixture gas, the transport of the FEX from mucosal to serosal direction across the intestinal sacs was measured by sampling the serosal medium at 0,15, 30, 45, 60 and 120 min. The samples of 1 mL were collected from FEX alone, RSV pretreated and VER pretreated groups at predetermined time points and stored at 80 8C until analysis. The drug transported from mucosal to serosal direction was measured by high performance liquid chromatography (HPLC).
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Calculation of apparent permeability coefficient (Papp)
116
The apparent permeability coefficient (Papp) of FEX was calculated from the following equation [16]:
117 118
P app ¼
dQ 1 dt AC 0
(1)
where dQ/dt is the transport rate of drug in the serosal medium, A is the surface area of the intestinal sacs and C0 is the initial concentration inside the sacs.
120 119 121 122
In situ intestinal perfusion study
123
The surgical procedure and the in situ single-pass intestinal perfusion (SPIP) study was performed according to the previously described methods [18–20]. Briefly, the rats were divided into three groups FEX alone (control), RSV pretreatment (test group) and VER pretreatment (positive control, standard P-gp inhibitor) groups each consisting of four animals. RSV and VER suspensions (prepared by suspending in 0.25%, w/v, of sodium carboxymethyl cellulose) were administered orally to pretreatment groups at a dose of 50 mg/kg and 25 mg/kg, respectively for 7 days and FEX alone group was kept as control. Both control and pretreatment group rats were subjected to surgery on 8th day. They were anaesthetized by thiopental sodium (50 mg/kg, ip) and placed on a hot pad to maintain normal body temperature. A midline incision was made on the abdomen and an ileal segment of approximately 8–12 cm was isolated using the ileo-caecal junction as a distal marker. Semi-circular incisions were made at each end and the lumen was rinsed with saline (37 8C) and the both ends were cannulated with PE tubing and ligated by using silk suture. Blank perfusion buffer (Phosphate buffer, pH 7.4) was first infused for 5 min at a flow rate of 1 mL/min by using Syringe pump (NE-1600, New Era Syringe Pumps, Inc. NY, USA), followed by perfusion of phosphate buffer saline (pH 7.4) containing FEX (50 mM), PRP (100 mM) and PHR (50 mg/mL) at a constant flow rate of 0.2 mL/min and the samples were collected at 10, 20, 30, 40, 50, 60, 70, 80 and 90 min. After cannulation the segment was covered with isotonic saline-wet gauze (37 8C). At the end of the perfusion the length of the segment was measured following the last sample collection. Perfusion samples were collected from FEX alone, RSV pretreated and VER pretreated groups at predetermined time points and stored at 80 8C until analysis. FEX concentrations in perfusion samples were measured by HPLC.
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Phenol red water flux correction
155
Cout
(corr)
was calculated from the following equation [21]:
C outðcorrÞ ¼ C out
Concentration of phenol red in ðCPRin Þ Concentration of phenol red out ðCPRout Þ
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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where Cout (corr) is corrected outlet concentration of the drug, Cout is outlet concentration of the drug, CPRin is concentration of PHR entering the intestinal segment and CPRout is concentration of PHR exiting the intestinal segment.
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Calculation of effective permeability coefficient (Peff)
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It is calculated from the steady state concentration of compounds in the collected perfusate which is considered to be attainable when the concentration level of PHR (non-absorbable marker) is stable and it was reached at 30–40 min after the beginning of the experiment. The steady state effective permeability is calculated using the following equation [18]: P eff ¼
Q lnðC out =C in Þ 2prL
3
pH 4.4 adjusted with phosphoric acid) and the elution was monitored at 195 nm at a flow rate of 1.0 mL/min. In case of PRP the mobile phase was used a mixture of methanol: 0.05 mM KH2PO4 (55:45, v/v, pH 6.0 adjusted with potassium hydroxide) and the elution was monitored at 227 nm at a flow rate of 1.0 mL/ min [23]. All the in vitro, in situ perfusion and plasma samples were extracted using a simple protein precipitation method by adding acetonitrile (200 mL) to samples (100 mL). Samples were vortexed for 2 min and centrifuged at 13,000 rpm for 15 min. The resultant clean supernatant (20 mL) was injected and analyzed using HPLC method. The limit of detection was 0.01 mg/mL and the assay range used was 0.01–10 mg/mL. The average recovery of the drug was 88.98%. The intra-day and inter-day coefficients of variation for the low and high quality control samples were less than 15%.
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Spectrophotometric analysis of phenol red
226
The PHR in phosphate buffer pH (7.4) has a characteristic red color which was measured spectrophotometrically at 560 nm [20].
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where Q is perfusion flow rate (0.2 mL/min), Cout is corrected outlet concentration of the drug, Cin is inlet drug concentration, r is radius of the rat small intestine (0.18 cm) and L is the length of the perfused intestinal segment (10 cm).
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Calculation of fraction of drug absorbed (Fab)
Pharmacokinetic analysis
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The fraction of drug absorbed is calculated using the following equation [22]:
Pharmacokinetic parameters were computed by non-compart- 230 mental model using Phoenix WinNonlin version 6.2 software 231 (Certara, Pharsight Corporation, USA). The plasma FEX concentra- Q3232 tion versus time plots were used to estimate the peak plasma 233 concentration (Cmax), time to reach peak plasma concentration 234 (Tmax), area under the concentration time curve to the last 235 sampling point (AUC0–t), area under the concentration time curve 236 to the infinity (AUC0–1), half-life (T1/2), apparent clearance (CL/F) 237 and apparent volume of distribution (Vd/F). 238
F ab ¼ 1
C out C in
181 180 182
where Cout is corrected outlet concentration of the drug, Cin is inlet drug concentration.
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Calculation of absorption rate constant (Ka)
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The absorption rate constant is calculated using the following equation [22]: C out Q Ka ¼ 1 C in V
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where Q is perfusion flow rate (0.2 mL/min), Cout is corrected outlet concentration of the drug, Cin is inlet drug concentration and V is the volume of the perfused segment.
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Statistical analysis
239
All mean values are presented as with their standard deviation (mean SD). Statistical analysis was performed by Student’s unpaired t test and analysis of variance (ANOVA) fallowed by Banferoni post-test using GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA, USA) at significance level of p < 0.05.
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Results
245
In vivo pharmacokinetic study
Effect of RSV on intestinal transport and apparent permeability of FEX
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Rats were fasted overnight before oral dose administration and approximately 3 h post-dose. The rats were divided into two groups of control and pretreatment consisting of six animals each. RSV suspension at a dose of 50 mg/kg was administered orally to pretreatment group for 7 days. FEX was dissolved in ethanol and water (1:10) for oral administration. FEX at a dose of 10 mg/kg was administered to both control and pretreatment groups on 8th day. Under ether anesthesia, blood samples (approximately 0.25 mL) were collected from the retro-orbital plexus into heparinized micro centrifuge tubes prior to dosing and at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12 and 24 h post-dosing and plasma was obtained by centrifuging the blood samples at 13,000 rpm for 15 min and stored at 80 8C until HPLC analysis.
205
HPLC analysis of FEX and PRP
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Shimadzu HPLC system equipped with a LC-20AD pump and SPD 20A UV visible detector and RP C18 column (Phenomenex Luna, 250 mm 4.6 mm ID, particle size 5 mm) was used for the analysis of samples. For FEX the mobile phase used was a mixture of 0.1 M triethylamine: acetonitrile: methanol (61:19.5:19.5, v/v,
The present investigation involves the determination of FEX intestinal transport and Papp in FEX alone, RSV pretreated and VER pretreated rats. Pretreatment with RSV (50 mg/kg) for 7 days resulted in a significant (p < 0.05) increase in mean cumulative concentrations (from mucosal to serosal direction) of FEX from 2.28 0.87 to 7.96 3.15 mg/mL in duodenum; 2.67 1.27 to 8.23 3.61 mg/mL in jejunum; 3.46 1.35 to 9.29 3.09 mg/mL in ileum (Fig. 1). In addition, pretreatment with positive control, VER (25 mg/kg) for 7 days resulted in a significant (p < 0.05) increase in mean cumulative concentrations (from mucosal to serosal direction) of FEX from 2.28 0.87 to 9.10 3.31 mg/mL in duodenum; 2.67 1.27 to 9.63 3.95 mg/mL in jejunum; 3.46 1.35 to 10.94 3.77 mg/mL in ileum (Fig. 1). The transport of FEX was found to be increased by 3.5-, 3.0-, and 2.7-fold in duodenum, jejunum and ileum, respectively in RSV pretreated group when compared with the FEX alone group. Further, the transport of FEX was found to be increased by 3.9-, 3.6-, and 3.2-fold in duodenum, jejunum and ileum, respectively in VER pretreated group when compared with the FEX alone group. The transport of FEX was increased from duodenum to ileum in RSV pretreated group which was comparable to positive control VER (standard P-gp inhibitor) pretreated group.
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Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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S.K. Bedada et al. / Pharmacological Reports xxx (2015) xxx–xxx
Fig. 1. Intestinal transport of FEX in duodenum, jejunum and ileum of Wistar rats. FEX alone group administered with FEX (10 mg/kg).
RSV pretreated
group administered with RSV (50 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th day.
VER pretreated group administered with VER (25 mg/kg) for 7 days
followed by FEX (10 mg/kg) on 8th day. Data are represented as mean S.D. (n = 4). *Significant difference (p < 0.05) in comparison with the control (two-way ANOVA followed by Bonferoni post-test).
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Pretreatment with RSV resulted in a significant (p < 0.05) increase in Papp of FEX from (0.091 0.003) 104 cm/s to (0.336 0.006) 104 cm/s in duodenum; (0.123 0.003) 104 cm/s to (0.368 0.007) 104 cm/s in jejunum; (0.144 0.014) 104 cm/s to (0.355 0.010) 104 cm/s in ileum (Fig. 2). In addition, pretreatment with positive control, VER resulted in a significant (p < 0.05) increase in Papp of FEX from (0.091 0.003) 104 cm/s to (0.364 0.023) 104 cm/s in duodenum; (0.123 0.003) 104 cm/s to (0.414 0.018) 104 cm/s in jejunum; (0.144 0.014) 104 cm/s to (0.424 0.023) 104 cm/s in ileum (Fig. 2). The Papp of FEX in duodenum, jejunum and ileum was found to be increased by 3.7-, 2.9-, and 2.5-fold, respectively in RSV pretreated group when compared with FEX alone group. Where as in case of VER pretreated group the Papp of FEX in duodenum, jejunum and ileum was found to be increased by 4.0-, 3.4-, and 2.9fold, respectively when compared with FEX alone group. The Papp values of FEX in RSV pretreated group were comparable to positive control VER (standard P-gp inhibitor) pretreated group.
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Effect of RSV on absorption rate constant, fraction absorbed and effective permeability of FEX
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FEX and PRP absorption rate constant (Ka), fraction absorbed (Fab) and intestinal effective permeability (Peff) were determined in rat ileum segment using SPIP technique. Ka, Fab and Peff values were
calculated from the steady-state concentrations in the perfusate collected from the outlet. Pretreatment with RSV for 7 days resulted in a significant (p < 0.05) increase in Ka of FEX from 0.028 0.002 min1 to 0.084 0.001 min1 and in case of pretreatment with VER resulted in a significant (p < 0.05) increase in Ka of FEX from 0.028 0.002 min1 to 0.092 0.001 min1 while there was no significant change was observed in Ka of PRP of both RSV and VER pretreated groups and it was found to be 0.044 0.002 min1, 0.045 0.001 min1 and 0.045 0.002 min1 in FEX alone, RSV pretreated and VER pretreated groups respectively (Fig. 3A). The increase in Ka of FEX in ileum was found to be 3.0- and 3.2-fold, respectively in RSV pretreated and VER pretreated groups when compared with the FEX alone group. Pretreatment with RSV for 7 days resulted in a significant (p < 0.05) increase in Fab of FEX from 0.142 0.009 to 0.429 0.004 where as in case of pretreatment with VER resulted in a significant (p < 0.05) increase in Fab of FEX from 0.142 0.009 to 0.467 0.005 while there was no significant change was observed in Fab of PRP of
Fig. 3. Absorption rate constant (Ka) (A), Fraction absorbed (Fab) (B) and Effective Fig. 2. Apparent permeability coefficient (Papp) of FEX in duodenum, jejunum, ileum
permeability (Peff) (C) of FEX and PRP in ileum of Wistar rats.
of Wistar rats.
group administered with FEX (10 mg/kg).
FEX alone group administered with FEX (10 mg/kg).
FEX alone
RSV pretreated group
RSV pretreated group administered with RSV (50 mg/kg) for 7 days followed by FEX
administered with RSV (50 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th
(10 mg/kg) on 8th day.
day.
VER pretreated group administered with VER
(25 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th day. Data are represented as mean S.D. (n = 4). *Significant difference (p < 0.05) in comparison with the control (two-way ANOVA followed by Bonferoni post-test).
VER pretreated group administered with VER (25 mg/kg) for 7 days
followed by FEX (10 mg/kg) on 8th day. Data are represented as mean S.D. (n = 4). *Significant difference (p < 0.05) in comparison with the control (two-way ANOVA followed by Bonferoni post-test).
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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both RSV and VER pretreated groups and it was found to be 0.223 0.014, 0.231 0.007 and 0.228 0.011 in FEX alone, RSV pretreated and VER pretreated groups respectively (Fig. 3B). The increase in Fab of FEX in ileum was found to be 3.0- and 3.3-fold, respectively in RSV pretreated and VER pretreated groups when compared with the FEX alone group. Pretreatment with RSV for 7 days resulted in a significant (p < 0.05) increase in Peff of FEX from (0.450 0.034) 104 cm/s to (1.655 0.024) 104 cm/s, where as in case of pretreatment with VER resulted in a significant (p < 0.05) increase in Peff of FEX from (0.450 0.033) 104 cm/s to (1.859 0.030) 104 cm/s while there was no significant change was observed in Peff of PRP of both RSV and VER pretreated groups and it was found to be (0.850 0.055) 104 cm/s, (0.882 0.027) 104 cm/s and 4 (0.870 0.043) 10 cm/s in FEX alone, RSV pretreated and VER pretreated groups respectively (Fig. 3C). The increase in Peff of FEX in ileum was found to be 3.7 and 4.1-fold, respectively in RSV pretreated and VER pretreated groups when compared with the FEX alone group. The Ka, Fab and Peff values obtained in RSV pretreated group were comparable to positive control VER (standard P-gp inhibitor) pretreated group.
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Effect of RSV on pharmacokinetics of FEX
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The plasma concentration-time plots of FEX after an oral administration of FEX (10 mg/kg) in control and pretreated (RSV 50 mg/kg) rats were characterized and depicted in Fig. 4. The mean pharmacokinetic parameters of FEX were summarized in Table 1. FEX oral pharmacokinetics were found to be significantly (p < 0.05) altered with RSV pretreatment for 7 days when compared with the control (FEX alone). The increase in Cmax, AUC0–t and AUC0–1 of FEX were found to be 3.6, 3.1 and 2.9- fold, respectively in RSV pretreated group. Further, the CL/F and Vd/F of FEX were significantly decreased while there was no significant change was observed in Tmax and T1/2 of FEX in RSV pretreated group when compared with FEX alone group.
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Discussion
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With the great interest in dietary supplements containing herbal constituents as alternative medicines, much effort is currently being expanded toward identifying natural compounds of plant origins that modulate P-gp; however, there is far less information on the pharmacokinetic interactions between dietary supplements containing phytochemicals and drugs. Therefore, more preclinical investigations on the drug–phytochemical interactions should be performed to prevent potential adverse reactions or utilize those interactions for a therapeutic benefit. Therefore, the present study evaluated the effects of RSV, an antioxidant, on the
Table 1 Pharmacokinetic parameters of FEX in control and RSV pretreated group rats after oral administration of 10 mg/kg. Data values are presented as mean SD. (n = 6). Pharmacokinetic parameters
Cmax (ng/mL) Tmax (h) T1/2 (h) AUC0–t (ng.h/mL) AUC0–1 (ng.h/mL) CL/F (L/h/kg) Vd/F (L/kg)
Fexofenadine, PO, 10 mg/kg Control group
RSV pretreated group
163.41 53.44 1.25 0.27 8.25 0.83 834.35 95.61 969.34 87.03 10.39 0.95 124.34 23.31
594.04 130.48* 0.91 0.13 8.49 0.75 2555.31 704.04* 2871.39 740.57* 3.67 0.91* 45.24 12.61*
* Significant difference (p < 0.05) in comparison with the control (unpaired Student’s t-test).
5
Fig. 4. Plasma drug concentration-time plots of FEX in Wistar rats. FEX alone group administered with FEX (10 mg/kg). RSV pretreated group administered with RSV (50 mg/kg) for 7 days followed by FEX (10 mg/kg) on 8th day. Data are represented as mean SD. (n = 6).
pharmacokinetics of FEX in rats to examine a potential pharmacokinetic interaction between RSV and FEX via the inhibition of P-gp. In this study, FEX was used as a model substrate for P-gp to explore the interaction with RSV in rats by using different absorption systems. FEX is a specific substrate for P-gp, it is well tolerated in vivo without interference of metabolic pathways [15]. The level of P-gp (mdr1a) mRNA is increased significantly from the top to bottom of the rat small intestine and found that the level of P-gp was higher in ileum than jejunum and duodenum [24]. The non-everted sac model was originally used to evaluate drug transport mechanisms [25]. Genty et al. [26] stated that the passive permeability of actively transported molecules can be determined through non-everted rat gut sacs. Hence, in the present investigation, non-everted intestinal sac study [16] was performed to determine the role of P-gp in the intestinal transport and apparent permeability of FEX. As shown in Fig. 1 the transport of FEX was significantly increased in duodenum, jejunum and ileum sacs of RSV pretreated and VER pretreated group rats when compared with the FEX alone group rats. Similarly, Papp of FEX (Fig. 2) was significantly increased in duodenum, jejunum and ileum sacs of RSV pretreated and VER pretreated group rats in comparison with that of FEX control. 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 [6]. In addition, an in vitro study has demonstrated that RSV enhanced the chemosensitivity of doxorubicin by increasing the cellular influx of doxorubicin [8]. Additionally, RSV has been found to potentiate the cytotoxic activity of docetaxel and doxorubicin due to enhanced intracellular levels by inhibition of P-gp [9]. Furthermore, it has been found to be FEX transport from basal to apical direction was inhibited by VER in Caco-2 cells [27]. Thus, the increase in intestinal transport and Papp of FEX across the duodenum, jejunum and ileum with RSV and VER pretreatment might be attributed to inhibition of P-gp in intestine of rats. The obtained intestinal transport and Papp values of FEX in RSV pretreated group were comparable to VER (standard P-gp inhibitor) pretreated group. Further, in order to support the role of P-gp inhibition involved in intestinal transport of FEX, in situ perfusion studies were performed in ileum of rats. These techniques maintain an intact blood supply to the intestine and can be used to estimate the impact of clearance pathways such as enzymes and transporters present in the gut [18]. In the present study, SPIP was performed in ileum segment of rats to investigate the functional role of P-gp intestinal permeability of FEX. Ka, Fab and Peff of FEX were significantly increased in RSV and VER pretreated group rats in
Please cite this article in press as: Bedada SK, et al. Effect of resveratrol on the pharmacokinetics of fexofenadine in rats: Involvement of P-glycoprotein inhibition. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.08.018
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comparison with FEX alone group rats. The increase in Ka, Fab and Peff of FEX in pretreated rats is attributable to the P-gp inhibition by RSV and VER. The Ka, Fab and Peff values obtained in RSV pretreated group were comparable to VER (standard P-gp inhibitor) pretreated group. These results were indicated that P-gp limits the intestinal permeability of FEX by extruding it back to the intestine. PHR was used as a non-absorbable marker in in situ technique. PHR concentration in the outlet perfusate was used to assess the steady-state condition in in situ perfusion technique. It is also used to indicate the intestinal mucosal integrity during perfusion [18]. PRP was used as a passive, highly permeable marker and as an indicator of major changes in mesenteric blood flow [20]. The permeability values of PRP in the FEX alone, RSV pretreated and VER pretreated group rats were found to be statistically insignificant, indicating that changes in FEX permeability in the presence of RSV and VER are not due to compromise in membrane integrity. In this study, we also investigated the effect RSV on the pharmacokinetics of FEX. Pretreatment with RSV for 7 days enhanced the oral bioavailability of FEX in rats. The AUC and Cmax were significantly increased in RSV pretreated group when compared with FEX alone control group. Further, the CL/F and Vd/F of FEX were significantly decreased while there was no significant change was observed in Tmax and T1/2 of FEX in RSV pretreated group when compared to FEX alone control group. Likewise, RSV exhibited similar inhibitory effects on the pharmacokinetics of P-gp substrate drugs nicardipine [10] and diltiazem [11], namely an increase in Cmax, AUC and decrease in CL/F. These findings provide in vivo evidence that RSV might increase the bioavailability of FEX via the inhibition of P-gp mediated drug efflux during the absorption phase in the intestine. Moreover, the results obtained in our study were comparable to previous work where piperine [15] and diosmin [23] substantially enhanced the systemic exposure of FEX by inhibition of P-gp in intestine of rats. Thus, the enhanced AUC and Cmax of FEX in the presence of RSV might be caused, at least in part, by the increased intestinal absorption of FEX via the inhibition of P-gp-mediated drug efflux, which in this study has been substantiated with non-everted intestinal sac and in situ permeability data. Therefore, the present study results suggesting that RSV resulted in drug–phytochemical interaction via the gastrointestinal tract.
440
Conclusions
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RSV is a naturally occurring antioxidant significantly increased the intestinal absorption and exposure of FEX indicating the inhibitory effect on P-gp-mediated drug efflux in rats. In addition, the intake of dietary supplements containing RSV may increase the absorption or exposure of drugs able to serve as P-gp substrates in addition to FEX. Therefore, further studies are recommended to evaluate the potential drug–phytochemical interactions in humans.
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Conflicts of interest
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The authors declare that there are no conflicts of interest. Acknowledgements
451 Q4 The authors would like thank to University Grants Commission 452 (UGC), New Delhi, India for financial support and Mr. Anand Gupta 453 of Certara Inc., USA for providing the Phoenix WinNonLin 6.2 454 software academic license.
References
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