Quercetin declines plasma exposure of metoprolol tartrate in the rat model.

The study was undertaken to evaluate the effect of quercetin on the pharmacokinetics of Metoprolol tartrate. A single dose in vivo pharmacokinetic study was carried out in rat models. In this study, rats were treated with quercetin (10 mg/kg) and metoprolol tartrate (20 mg/kg) orally and blood samples were collected 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12 h post treatment. Plasma concentration of metoprolol tartrate was estimated using reverse phase-high-performance liquid chromatography method. Area under the plasma concentration-time curve (AUC0-12) of metoprolol has significantly (P < 0.001) decreased by 9.8 times in the metoprolol and quercetin combination group (9434.65 ± 3525.02) when compared with AUC0-12 metoprolol of metoprolol-alone treated group (962.17 ± 242.81). AUC0-∞ of metoprolol has significantly (P < 0.001) decreased by 14.9 times in the combination group (16670.79 ± 12129.06) in comparison to AUC0-∞ of metoprolol of metoprolol-alone treated group (1113.68 ± 441.83). the results obtained herein indicate that quercetin remarkably declines the plasma exposure of metoprolol when concomitantly administered by oral route.

INTRODUCTION

Metoprolol, a selective β1-adrenergic receptor antagonist, is widely used for the treatment of hypertension, angina pectoris, myocardial infarction, and arrhythmia.[1] It is marketed as a racemate, but the pharmacologic effect resides in the (S)-enantiomer.[2] Metoprolol is extensively metabolized in the liver through O-demethylation, α-hydroxylation, and N-dealkylation. Metoprolol O-demethylation accounts for about 65% of the dose whereas α-hydroxylation and N-dealkylation each account for 10%.[3] In vitro studies with human liver, microsomes have indicated that metoprolol α-hydroxylation is almost completely mediated by CYP2D6 and that O-demethylation is partially mediated by CYP2D6.[4] About 70% of metoprolol metabolism is estimated to be mediated by CYP2D6 in vivo.[5] In extensive metabolizers for CYP2D6, the metabolism of metoprolol is stereoselective, with the area under the plasma concentration-time curve extrapolated to infinity (AUC) of the (S)-metoprolol being significantly higher than that of (R)-metoprolol.[6]

Flavonoids are believed to alter the expression and activity of enzymes and transporters implicated in drug metabolism and excretion.[789] Effects of flavonoids on the pharmacokinetics of drugs have already been described in humans.[101112] Quercetin is the most predominant flavonoid in plant foods, herbs, beverages and dietary supplements, e.g. onions, grapes, berries, apples, red wine, tea, St. John's wort and ginkgo. The daily dietary intake of quercetin is estimated to be in the range of 4-68 mg based on epidemiological studies in the US, Europe, and Asia,[13141516] but can be as high as several 100 mg in the dietary supplement and several grams in anticancer therapy.[17]

According recent studies, there is an increasing evidence that quercetin interacts with numerous xenobiotics. For instance, quercetin enhances the bioavailability of numerous drugs like rosiglitazone,[18] fexofenadine[19] in humans, paclitaxel,[20] valsartan,[21] tamoxifen[22] in rat models, digoxin[23] in pigs. In contrast, quercetin decreases the bioavailability of simvastatin[24] in pigs and cyclosporine[25] in pigs and rats.

Quercetin was found to be cardio-protective based on experimental[2627] and epidemiological[141516] studies. Since metoprolol is cardioprotective in case of cardiovascular disorders like myocardial infarction, it is quite logical to investigate the effect of quercetin on the pharmacokinetics of metoprolol. Hence, the study was designed to see if there is any effect of quercetin on the pharmacokinetics of metoprolol in animal models.

MATERIALS AND METHODS

Drugs and chemicals

Metoprolol tartrate was obtained as a gift sample from Matrix Laboratories; Hyderabad (India). Quercetin was purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. All high-performance liquid chromatography (HPLC) grade solvents (acetonitrile, Sodium Lauryl Sulfate, Ortho Phospharic Acid, and water) were procured from SD Fine chemicals, Mumbai, India. All other chemicals used were of analytical grade and purchased from local chemical agencies.

Equipments

High-performance liquid chromatography (Waters 2695 [isocratic system]) with Gelman science vacuum pump and Lab India Ultraviolet (UV 3000+) UV-visible Spectrophotometer. Zodiac C8, 150 × 4.6 mm, 5 μm was used. The system was equipped with Empower-2 Software. Sonicator (Hwashin Technology, Seoul, Korea), Biofuge (Hearus instrument, Hanau, Germany), micropipettes, tubes (Tarsons Products Pvt. Ltd, Kolkata, India) were used.

Animals

Albino Wistar rats (National Institute of Nutrition, Hyderabad, India), of either sex, weighing 200-250 g, were selected. Animals were maintained under standard laboratory conditions at 25°C ± 2°C, relative humidity 50% ± 15% and normal photoperiod (12 h dark/12 h light). Commercial pellet diet (Rayon's Biotechnology Pvt Ltd, India) and water were provided ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee of KVSR Siddhartha College of Pharmaceutical Sciences and studies were carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, India. IAEC Clearance number of the article was IAEC/11/03/2014/008.

Pharmacokinetic study in rats

Experimental procedure

Wistar rats were randomly distributed into two groups of six animals in each group. Before doing, all experimental animals were fasted for 18 h and but water was given ad libitum. Experimental design was as follows. Group I: Metoprolol tartrate (20 mg/kg; p.o.), Group II: Metoprolol tartrate (20 mg/kg; p.o.) + (quercetin (10 mg/kg; p.o.). Blood was collected from orbital sinuses using 2 ml Eppendorf tubes containing sodium citrate as an anticoagulant. Plasma was separated by centrifugation at 5000 RPM/10 min and stored at −20°C until further analysis. Plasma concentration of metoprolol was estimated by a sensitive reverse phase-HPLC (RP-HPLC) method.

Preparation of drugs

Metoprolol tartrate was dissolved in distilled water whereas quercetin (10 mg) was accurately weighed before being triturated in a dry clean mortar with the addition of 30 μL of tween 80 and then, required a volume of 0.9% sodium CMC was added and triturated again to suspend the drug in it. Then, suspension was transferred to plastic vials. Quercetin suspension was administered concomitantly with metoprolol tartrate solution.

Blood sample collection from rats

In this study, blood samples were collected at time points 0, 0.25,0.5, 0.75, 1, 1.5, 2, 4, 8, 12 h post treatment from the retro-orbital sinuses using fine capillary tubes into 2 ml Eppendorf tubes containing sodium citrate as an anticoagulant. Plasma was separated by centrifugation at 5000 RPM/10 min and stored at −20°C until further analysis. Plasma concentration of metoprolol was estimated by a sensitive RP-HPLC method.

Estimation of metoprolol by a sensitive reverse phase-high-performance liquid chromatography method

Chromatographic conditions

The mobile phase consisted of a buffer (1.3 g of Sodium Lauryl Sulfate and 1 ml Ortho Phosphoric acid transfer into 1litre water and it was made to dissolve and adjust pH-2.0 with orthophosphoric acid) and acetonitrile in the ratio of (50:50). The injection volume was 20 μL. The mobile phase was delivered at 1.0 ml/min. The mobile phase was filtered through 0.22 μm membrane filter. The flow rate was adjusted to 1.5 ml/min, and the effluent was monitored at 224 nm. The total run time of the method was set at 6 min. Retention time was obtained at 4-5 min.

Preparation for the calibration curve of metoprolol tartrate for in vivo samples

Preparation of linearity solution

Linearity solutions of various concentrations were prepared ranging from 0.2 μg to 1.2 μg/mL of Metoprolol tartrate. To 250 μL of sample, 250 μL of the mobile phase was added and was mixed well. Further, 500 μL of acetonitrile was added to precipitate all the proteins and mixed in vortex cyclomixture. Then, these were centrifuged at 4000 RPM for 15-20 min and supernatant solution was collected in HPLC vial and was injected into HPLC and chromatogram was recorded.

Construction of calibration curve

A stock solution representing 100 μg/ml of metoprolol tartrate was prepared in a dilution (Water and acetonitrile were mixed in the ratio of 70:30) and this solution was stored at 2-8°C until use. Six different concentration levels (0.20, 0.30, 0.40, 0.80, 1.00 and 1.20 μg/ml) were prepared from each stock solution and diluted with above diluents. Each concentration solution was prepared in triplicate. A linear relationship was obtained between the peak area and the corresponding concentrations. The slope of the plot determined by the method of least-square regression analysis was used to calculate the metoprolol tartrate concentration in an unknown sample. A linear calibration curve in the range of 0.20 μg− 1 20 μg was established (r2 = 0.999). Retention time was obtained at 4-5 min.

Preparation of plasma sample

Plasma samples were labeled accordingly to their time intervals and then, centrifuged. To 250 μL of sample, 250 μL of the mobile phase was added and mixed well. Further, 500 μL of acetonitrile was added to precipitate all the proteins and mixed in vortex cyclomixture. Then, it was again centrifuged at 4000 RPM for 15-20 min and supernatant solution was collected in HPLC vial and was injected into HPLC and chromatogram was recorded.

Pharmacokinetic data analysis

The plasma concentrations versus time data obtained from each individual rat were submitted to a non-compartmental pharmacokinetic analysis using Kinetica Software (Version 5.1, Thermo Electron Corporation, and USA). The maximum plasma concentrations (Cmax) and times to achieve maximum plasma concentrations (Tmax) were obtained directly from the individual plasma concentration-time curves. AUC0-12 was calculated by the linear trapezoidal rule, and AUC0-∞ was determined by the following formula:

AUC0-∞ = Clast/Kel + AUC0-12

Where Clast is the last quantifiable concentration. The apparent total body clearance or oral clearance (CL/F) was calculated as follows:

CL/F = dose/AUC0-∞

The elimination rate constant (Kel) was obtained by linear regression of the terminal phase, and the elimination half-life (t1/2) was calculated as 0.693/Kel.

Statistical analysis

The results were expressed as mean ± standard deviation. Comparisons of plasma concentration versus time profiles of metoprolol tartrate-alone group and metoprolol tartrate with the quercetin combination group were analyzed using two-way ANOVA followed by Bonferroni post hoc test, whereas comparisons of pharmacokinetic parameters of these two groups were analyzed using unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001 were considered to be statistically significant.

RESULTS

Calibration curve

Linear relationship was obtained between the peak area and the corresponding concentrations. The equations of linear regression were performed using the least-square method. Retention time was obtained at 5 min. Chromatogram was shown in Figure 1 and Linearity graph was shown in Figure 2.

Figure 1

Metoprolol chromatogram in plasma spiked with metoprolol tartrate

Figure 2

Metoprolol linearity graph plotted with plasma spiked with Metoprolol tartrate

Effect of quercetin on plasma concentration time profiles of metoprolol

The plasma concentration versus time profiles of metoprolol in rats following oral treatment of metoprolol tartrate with and without quercetin was shown in Figure 3. From the comparison of plasma concentration profiles of metoprolol in the absence and presence of quercetin, it is clear that there was a significant reduction in the plasma drug exposure of metoprolol in the combination group at following time points 0.25 h (P < 0.001), 0.5 h (P < 0.001), 0.75 h (P < 0.001), 1st h (P < 0.001), 1.5th h (P < 0.001), 2nd h (P < 0.001) and 4th h (P < 0.05). It is quite interesting to find that the metoprolol concentrations in metoprolol and quercetin combination group are almost negligible at 4th h, whereas in the metoprolol alone group; metoprolol concentrations were even slightly present at 12th h.

Figure 3

Time versus plasma concentration profile of metoprolol in metoprolol alone treatment group and metoprolol and quercetin combination group

Effect of quercetin on pharmacokinetic parameters of metoprolol

The calculated pharmacokinetic parameters of metoprolol tartrate in rats were shown in Table 1. AUC0-12 of metoprolol has significantly (P < 0.001) decreased in the combination group (9434.65 ± 3525.02) than AUC0-12 of metoprolol of metoprolol-alone treated group (962.17 ± 242.81) [Figure 4]. This decrease is almost 9.8 times. AUC0-∞ metoprolol has significantly (P < 0.001) decreased in the combination group (16670.79 ± 12129.06) than AUC0-∞ of metoprolol of metoprolol alone treated group (1113.68 ± 441.83). This decrease is 14.9 times. In a similar manner, the Cmax of metoprolol has significantly (P < 0.001) decreased in the combination group (1411.03 ± 275.62) than Cmax of metoprolol of metoprolol alone treated group (962.17 ± 242.81). This decrease is almost 4 times. These parameters were altered without significant alteration in the tmax of metoprolol. Vd and CL of metoprolol has significantly (P < 0.01 and P < 0.001) decreased in the combination group, respectively.

Table 1

Comparison of pharmacokinetic parameters of metoprolol in rats before and after treatment with quercetin

Figure 4

Comparison of area under the plasma concentration-time curve from time 0 to 12 h of Metoprolol in Metoprolol alone treatment group and Metoprolol and quercetin combination group

DISCUSSION

The results of the study revealed that there is a significant decrease in the bioavailability of metoprolol administered concomitantly with quercetin, and that was evidenced clearly by significant (P < 0.001) decrease in the AUC0-12 and AUC0-∞. Mechanisms underlying the interaction between these two drugs are uncertain. However, the various possibilities of interaction are discussed. There was evidence that propranolol, beta-blocker, stimulated four to six-fold increase in MDR1 mRNA and P-glycoprotein (p-gp) protein expression measured by quantitative real-time polymerase chain reaction and immunoblotting, respectively. These changes were accompanied by an induction in transporter activity measured by rhodamine 123 efflux. In contrast, metoprolol, a compound with similar permeability but no affinity for PGP had no effect on PGP expression.[28] According to this study, it is clearly evident that metoprolol has no ability to increase the expression of p-gp, while other β-blocker like propranolol able to do so. Hence, the decreased bioavailability of metoprolol probably not attributed to the increased expression of p-gp.

Until date, there are no reports stating that metoprolol is a p-gp substrate though other beta-blocker, carvedilol is a known p-gp substrate.[29] Hence, it is very unlikely that metoprolol and quercetin interact at p-gp because metoprolol is not reportedly a p-gp substrate though quercetin is a known p-gp inhibitor.[30] This rule out the possibility of decreased bioavailability due to p-gp mediated interaction between these two drugs. It is presumed that these two drugs are likely to interact at other transporters.

Substantial evidence shows that these drugs interact at organic cation transporter (OCT-2) which is one of transporter accounts for the secretion of beta-blockers in the basolateral membrane of the intestine. In support of this idea, the previous study investigated the affect of selected flavonoids (quercetin, isoquercitrin, spiraeoside, rutin, kaempferol, naringenin, naringin and kempferol) on the transport of the P-gp substrate [3H] talinolol (beta-blocker) across Caco-2 cell monolayers. In addition, Researchers attempted to explore the mechanism behind the interaction observed in this system. The results revealed that six of the investigated flavonoids reduced the secretory flux of talinolol across Caco-2 cells. However, none of the selected flavonoids was able to replace [3H] talinolol from its binding to P-gp. However, the investigated flavonoids did show potency to inhibit OCT-mediated transport. Thus, previous in vitro results demonstrate that flavonoids bear the ability to interfere with secretory intestinal transport processes. This might be due to the interaction with P-gp, but apparently not via competition at the talinolol binding site of P-gp. Another mode of interaction may be the inhibition of members of the OCT-family, which is located at the basolateral membrane of intestinal epithelial cells.[9] Thus, metoprolol might be interacting at OCT-2 at intestine, and this interaction could be one possibility for the decreased bioavailability of metoprolol.

In addition, our results also revealed that there is significant (P < 0.001) decrease in the Cmax of metoprolol by quercetin co-administration. This reduction might be attributed to the significant enhancement of volume of distribution of metoprolol by quercetin. At the same time, clearance of the metoprolol was also significantly increased along with quercetin. It is unclear as to how and which mechanism is contributing to the increased clearance of the drug. Quercetin is believed to be a CYP3A4 inhibitor, whereas metoprolol is CYP2D6 substrate. Hence, it is very unlikely to have the interaction mediating CYP metabolic enzymes. It is imperative to identify the mechanisms to further clarify our result.

CONCLUSION

Our results reveal that quercetin reduced plasma exposure of metoprolol in rat models. However, mechanism of interaction is unclear. This interaction could be of clinical significance. However, further clinical studies are needed to confirm this interaction.