INTRODUCTION:

Carvedilol (a β-receptor blocker) is used in the treatment of cardiovascular diseases like hypertension, ischemic heart disease, and congestive heart failure [1,2]. Carvedilol is an arylethanolamine that is used clinically as a racemic mixture of 2 enantiomers. The S-(–)- enantiomer has the β-adrenoceptor-blocking activity, while the racemate also has the α1-receptor-blocking activity due to the activity of the R-(+)-enantiomer [3, 4]. The drug is highly lipophilic and is also highly protein bound; it is rapidly absorbed and it undergoes extensive first-pass metabolism in the liver [3].

Lipophilic β-blockers, such as carvedilol, are metabolized via cytochrome P450 (CYP) enzymes. The drug is subject to an oxidative biotransformation and conjugation, being metabolized by CYP2D6 to 4’-hydroxyphenyl carvedilol and5’-hydroxyphenyl carvedilol, by CYP2C9 to O-desmethylcarvedilol, by CYP1A2 to 8-hydroxy carbazolylcarvedilol [5,6].

Resveratrol (RSV) (3,4′,5-trihydroxystilbene) is a naturally occurring phytoalexin present in grape skins, fruits, vegetables, and especially in red wine. RSV is known to have diverse biochemical and physiological properties including anti-inflammatory, immune modulatory activities as well as wide range of health benefits ranging from chemoprevention to cardio protection [7,8]. It is produced by the 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 Μm [9]. Resveratrol has recently been shown to exert genoprotective, cytotoxic, antiproliferative, and proapoptotic actions in different tumoral cell lines [10].

Resveratrol inhibits the metabolizing activity of phase-I enzymes (CYPs), and has an effect on P-gp in KB-2 cells [11]. The low bioavailability of oral carvedilol is mainly due to ﬁrst-pass metabolism and P-gp, an efﬂux transporter in the intestine. Thus, the pharmacokinetic interaction between carvedilol and resveratrol could be expected. Resveratrol and carvedilol could be prescribed for the prevention or treatment of cardiovascular diseases as a combination therapy. Therefore, the aim of this study was to investigate the effects of resveratrol on the intestinal transport of carvedilol in rats.

MATERIALS AND METHODS:

Carvedilol was gifted by Sun Pharmaceuticals, Simvasa, India. Resveratrol was gifted by Ranbaxy Laboratories India Ltd), Dulbeccos Phosphate Buffer pH 7.4 (Hi Media Mumbai, India Ltd), Methanol HPLC (E. Merck Ltd., India), Acetonitrile HPLC (E. Merck Ltd) were used. All other chemicals used were of AR grade.

NON-EVERTED INTESTINAL SAC STUDY: [12]

The animal study was conducted according to the protocol approved by animal ethics committee Anwarul Uloom College of Pharmacy, India. Male wistar rats weighing 200 ± 25 gm were selected for experiments. Resveratrol was administered to rats (n=3) at a dose of 10mL Kg-1 for seven days. Untreated rats (n=3) were used as control.

The rats were fasted overnight with free access to water before the experiments. Control rats and pretreated rats on seventh day were sacrificed 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 (10 cm). The probe drug (Carvedilol 10mg mL-1) was dissolved in pH 7.4 isotonic Dulbecco’s PBS (D-PBS) containing 25 mM glucose. The probe drug carvedilol solution (1mL) was filled in the normal sac (mucosal side), and both ends of the sac were ligated tightly. The sac containing probe drug solution was immersed in 40 mL of D-PBS, containing 25 mM glucose in the mucosal side. The medium was pre-warmed at 37°C and pre-oxygenated with 5% CO2/95% O2 for 15 minutes, under bubbling with a CO2/O2 mixture gas, the transport of the buspirone from mucosal to serosal (normal sac) surfaces across the intestine was measured by sampling the serosal medium periodically for 120 minutes. The samples of 1mL were collected at predetermined time intervals from the serosal medium and replenished with fresh buffer. The drug transported was measured using high performance liquid chromatography (HPLC) method.

In vivo bioavailability study in male Wistar rats:

The animal study protocol was reviewed and approved by the institutional animal ethical committee, Anwarul Uloom College of Pharmacy, Hyderabad, India. Male Wistar rats (n = 6) weighing 220 to 250g were selected for the study. The oral bioavailability of carvedilol after pretreatment with Resveratrol (dose of 10mg kg_1 for 7 days) was compared with an oral solution (carvedilol 3 mg kg_1). The rats were allowed free access to food and water, until night prior to dosing, and were fasted for 10 h. For the first group, oral carvedilol solution was administered through feeding needle; the second groups were pretreated with resveratrol (10mg/kg), for 7 days. Blood samples (0.5mL) were collected from retroorbital plexus at preset intervals of 0, 0.1, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h, respectively, after administration of oral drug solution and after pretreatment with resveratrol. All blood samples were allowed to clot and centrifuged for 10 min at 10000rpm. The serum was separated and transferred into clean eppendrof tubes and stored at 40°C until HPLC analysis. The concentration of diltiazem in the samples was estimated by using HPLC.

HPLC ANALYSIS:

The HPLC system (Shimadzu, Japan) consisted of a LC–10AT solvent module, and a model SPD–10A, UV-Visible Spectrophotometric detector with LC 10 soft ware. The column used was a Kromasil KR 100-5C8 (stainless steel column of 25cm length and internal diameter of 4.6mm, packed with porous silicon spheres of 5m diameter). The mobile phase consisted of acetonitrile, 15 mm orthophosphoric acid (37:63), and 0.25 v/ v% triethylamine mixture, and was adjusted to pH 2.5 with orthophosphoric acid. The elute was monitored at 238 nm with a flow rate of 1 mL/min.

SAMPLE PREPARATION:

To 200μL of intestinal sac and serum samples, 100μL of methanol was added and vortexed on a cyclo-mixer for two minutes and centrifuged at 5000rpm for 15 min using Biofuge Fresco Centrifuge (Heraeus, Germany). The supernatant (20μL) was injected into HPLC.

Analysis of pharmacokinetic parameters:

All the pharmacokinetic parameters were analyzed by using Phoenix Win Nonlin version 6.2 software (Certara, Pharsight Corporation, L.P, USA). The statistical analysis was performed by using one-way analysis of variance using Graph Pad Prism version (6.07) (Graph Pad Software Inc., San Diego, CA, USA) at significance level of p < 0.05.

RESULTS:

Effect of resveratrol on intestinal transport of carvedilol:

In the present study, the mean transport of carvedilol from mucosal to werosal (normal sac) was determined in duodenum, jejunum and ileum regions of rat intestine in the absence and presence of resveratrol. The time course of carvedilol transport at different concentrations across rat small intestine of duodenum, jejunum and ileum was shown in Table 1.

Table 1. Mean± SD (n=3) cumulative transport of carvedilol (10mcg/ml) in intestinal sacs in Wistar rats.

Region	Control	Carvedilol+ Resveratrol	Carvedilol+ Verapamil
Duodenum	734.16±24.61	3320.17±53.19	1496.36±36.48
Jejunum	1246.65±37.71	6692.91±43.60	4342.65±44.76
Ileum	1401.24±38.65	6517.51±24.87	4536.98±23.98

The resveratrol pre-treatment for 7 days increased the mean cumulative concentration of carvedilol from 734.16±24.61 to 3320.17±53.19 in duodenum, 1246.65±37.71 to 6692.91±43.60 in jejunum and 1401.24±38.65 to 6517.51±24.87 in ileum respectively. The transport of carvedilol increased 4.5, 5.3 and 4.6 times after pre-treatment with resveratrol compared to respective controls. There was a statistically significant (P<0.05) difference. The transport was increased from duodenum to ileum.

Effect of resveratrol on pharmacokinetics of carvedilol:

The mean plasma concentration vs time profile of carvedilol in groups with and without resveratrol pretreatment is depicted in Fig 4. Pharmacokinetic parameters of carvedilol in control and pretreated group is shown in Table 2.

Table 2: Pharmacokinetic Parameters of carvedilol in control and pretreated group

Parameters	Control	Control+ Res
Cmax (ng/ml)	285.43±22.32	658.65±34.33*
Tmax (h)	2	2
Auc0-∞ (ng*h/ml)	1134.45±433.43	1856.87±342.77*
T1/2 (h)	4.32±0.65	15.45±0.65*
Kel (l/hr)	0.3±0.67	0.15±0.43
Cl (l/hr/kg)	3.54±2.43	2.34±3.44

Data represented in mean± SD, *p<0.05

Pharmacokinetic parameters of carvedilol were altered by resveratrol as shown in table 2. Cmax was increased significantly from 285.43 to 658.65 i.e 3 fold increase in resveratrol treated group when compared with control group. Tmax was unchanged. Area under the curve of carvedilol in pretreated group was increased significantly (p<0.05) when compared to control group.

DISCUSSION:

With the great interest in herbal products 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 herbal products and medicines. Therefore, more preclinical and clinical investigations on the herbal constituent–drug interaction should be performed to prevent potential adverse reactions or utilize those interactions for a therapeutic beneﬁt. Therefore, the present study evaluated the effects of resveratrol, an antioxidant, on the intestinal transport of carvedilol in rats to examine a potential drug interaction between resveratrol and diltiazem via the inhibition of P-gp. Based on the broad overlap in the substrate speciﬁcities as well as colocalization in the small intestine, the primary site of absorption for orally administered drugs, P-gp, has been recognized as a concerted barrier to drug absorption [13,14].

The non-everted intestinal sac model was originally used to evaluate drug transport mechanisms [15,16] compared the permeability values of some actively transported molecules and passively absorbed compounds through everted and non-everted sacs and found that the permeability was higher for actively transported molecules when the sacs were everted. The permeability of passively absorbed drug diazepam remained the same whether the sacs were everted or not. These results suggested that the passive permeability of actively transported molecules can be determined through non-everted rat gut sacs [17].

Resveratrol irreversibly inhibits CYP3A4 with a Ki of 20 Mm [18,19]. Moreover, resveratrol increases the accumulation of daunorubicin (a P-gp substrate) in KB-C2 cells in a concentration-dependent manner, indicating that resveratrol also. Resveratrol (100mM) increased rhodamine retention in P-gp over-expressing MCF-7/ADR cells by 140%, indicating inhibition of P-gp activity [20]. Thus, the increase in carvedilol transport induces by resveratrol could indicate decreased P-gp efflux in the intestine.

A previous study suggests the role of resveratrol in increasing oral bioavailability of nicardipine by inhibiting P-gp efflux [21]. Resveratrol (2.5 and 10 mg/kg) significantly increased the oral bioavailability of diltiazem suggested that resveratrol could inhibit P-gp, an efflux transporter, and the first-pass metabolism during intestinal absorption in a study conducted by [22]. In present study resveratrol pretreatment increased the carvedilol transported across different segments of rat intestine. As suggested by the previous studies carvedilol is efflux transported by p-glycoprotein which might inhibited by resveratrol after pretreatment. Increase levels of carvedilol might be due to inhibition of p-glycoprotein.

CONCLUSION:

From the results it can be concluded that resveratrol may increase the bioavailability of carvedilol when used concomitantly. These results of increased bioavailability of carvedilol may be due to the inhibit of p-glycoprotien by resveratrol, which leads to decrease efflux of carvedilol in intestinal lumen. The effect of p-glycoprotein inhibition can be confirmed by the invitro model of non-everted intestinal sac study and the in vivo model of oral bioavailability in rats. Hence when food or other herbal products which contains resveratrol if given along with carvedilol it may increase the concentration of carvedilol by inhibiting its transport by altering the function of p-glycoprotein. Further studies are recommended to prove their influence in human volunteers or animal in vivo.

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Received on 05.08.2019 Modified on 28.08.2019

Research J. Pharm. and Tech. 2020; 13(4):1660-1664.

DOI: 10.5958/0974-360X.2020.00301.7