Effect of hesperetin on the pharmacokinetics of metoprolol succinate in rats

Metoprolol is a substrate of CYP3A4, 2B6, CYP2D6, CYP2C9, and p-glycoprotein (p-gp). Hesperetin was reported as an inhibitor of cytochrome P-450 (CYP) enzymes and p-gp. The objective of this study was to investigate the effect of hesperetin on the pharmacokinetics of metoprolol in rats and in vitro models. In in vivo studies, male Wistar rats were treated with metoprolol (30 mg/kg) once a day for 15 consecutive days alone and in combination with hesperetin (25, 50, and 100 mg/kg). Blood samples were withdrawn from the tail vein on the 1st day in the single-dose pharmacokinetic study and on the 15th day in the repeated-dose pharmacokinetic study. In in vitro studies, metoprolol was incubated in the presence or absence of hesperetin and traditional p-gp inhibitors using rat-everted gut sacs. Reverse phase-high-performance liquid chromatography (RP-HPLC) was used to determine the amounts of metoprolol in the plasma and incubated samples (RP-HPLC). The Cmax, AUC, and half-life (t1/2) of metoprolol significantly increased by twofold compared to the metoprolol group in rats pre-treated with hesperetin. The clearance and volume of distribution both decreased significantly. Metoprolol transport was dramatically increased in the presence of hesperetin and quinidine (standard p-gp inhibitor) in in vitro study. The present study results revealed that hesperetin significantly increased the absorption of metoprolol in rats and everted gut sacs in vitro might be due to the inhibition of CYP and p-gp.


Drugs and chemicals
Metoprolol and quinidine are gifted by Orchid Health Care, Chennai, India. Hesperetin was purchased from Sigma Chemical Co. (St. Louis, MO). The required analytical grade solvents for this study were purchased from Finar chemical Ltd, India.

Laboratory animals
Animal experiments were carried out following CPCSEA guidelines at KVSR Siddhartha College of Pharmaceutical Sciences ((993/PO/Re/S/06/CPCSEA). Mahaveer Enterprises in Hyderabad, India, provided male Wistar rats weighing 180-220 g and housed in an animal house. For at least one week before the commencement of the studies, the animals were housed in conventional laboratory settings (12/12 h light/darkness, 22 °C, and 50-60% humidity).

Study protocol
The present investigation was divided into a single-dose pharmacokinetic (SDPK) study and a repeated-dose pharmacokinetic (RDPK) study as previously mentioned in rats [19].

Single-dose pharmacokinetic study
Wistar rats were randomly divided into four groups of six animals each in SDPK. Metoprolol and hesperetin were suspended in 1% SCMC for oral administration.

Repeated-dose pharmacokinetic study
In the RDPK study, rats were treated with the above drugs once a day for 15 days. On the 15th day, 100 μL of blood was drawn from the tail vein at various intervals (0.16, 0.33, 0.5, 1, 2, 4, 6, 8, 12, and 24 h). The plasma was separated and stored at − 20 °C until analysis.

Drug absorption study in vitro Gut sac preparation
The method that was described previously by [20] for the preparation of everted gut sacs of rat ileum was followed for this study also. Pentobarbital sodium 40 mg/kg was used to anesthetize the rats, and the small intestine was removed [21]. The intestinal digesta was removed and cleaned with ice-cold saline, and the distal ileum (about 15 cm each) was extracted and everted using a glass rod.

Influence of hesperetin on the intestinal absorption
Krebs-Ringer bicarbonate (KRB) buffer containing metoprolol 50 µg/mL was filled in everted sacs. Each sac was placed in a 50-mL Erlenmeyer flask containing 30 mL oxygenated (O 2 /CO 2 ; 95:5) KRB and incubated in a shaker bath at 37 °C for 60 min. At 10, 20, 30, and 60 min, 1 ml of sample was collected from the outer medium and the 1 ml KRB buffer was replaced. The movement of metoprolol from the serosal to the mucosal side was determined using RP-HPLC after centrifugation at 6000 rpm for 10 min. Each experiment was carried out three times. The same study was repeated with and without of quinidine 50 µg/ml and hesperetin 25, 50, and 100 µg/ml.

Analytical methods
The plasma concentrations of metoprolol were determined using a previously described method [22]. The data were collected and processed using LC solution software (Tokyo, Japan). 0.2% in acetonitrile and water (80:20 v/v) WAS the mobile phase. Before use, the prepared mobile phase was ultrasonically degassed and filtered through a 0.45 µm membrane filter. The injection volume was 20 µL, and the effluent was monitored at a flow rate of 1 ml/min at 222 nm with a UV detector. The chromatographic run duration was 5.0 min, while metoprolol was eluted at 3.35 min (Fig. 1). The experiment was conducted out at room temperature.

Extraction of metoprolol from plasma
The metoprolol was extracted from rat plasma using a liquid-liquid extraction technique [22]. 1 mL tert-butyl methyl ether was added to a 50 µL plasma sample, vortex mixed for 5 min, and then centrifuged at 6000 rpm for 5 min. The supernatant (500 µL) was dried at 40 °C under a moderate nitrogen stream. The dried residue was reconstituted in 50 µL of mobile phase (80:20, v/v), and 20 µL of this was used to run the HPLC.

Calculation of PK parameters
Thermo Kinetica (Version 5.1) was used to carry out a non-compartmental PK analysis.

Data analysis
Graph Pad Prism 5.0 was used to calculate all statistics (San Diego, CA). For multiple comparisons, one-way analysis of variance (ANOVA) and two-way ANOVA were applied to compare the PK parameter values and plasma concentrations, respectively. Significant was defined as a p < 0.05. Figure 2 shows the plasma concentrations of metoprolol vs time after oral administration of metoprolol alone and pre-treatment with hesperetin 25, 50, and 100 mg/kg in SDPK. The mean PK parameters are summarized in Table 1. Hesperetin enhanced the C max , AUC 0-24 , AUC 0-∞ , t 1/2 , and MRT of metoprolol and lowered the clearance and volume of distribution of metoprolol in the present study (p < 0.001     Effect of hesperetin on the p-gp mediated transport of metoprolol using everted rat gut sacs p-gp in the intestine acts as a barrier, preventing xenobiotics and medicines from reaching the intraluminal space. This has a major impact on p-gp substrate bioavailability and, as a result, therapeutic potential. The intestinal absorption of metoprolol was measured from the mucosal compartment to the serosal compartment using everted gut sacs. The addition of hesperetin to metoprolol improved its absorption in a concentration-dependent manner (Table 3). At a concentration of 50 µg/mL, the amount of metoprolol transported alone was found to be 12.562 ± 1.725 µg/ mL at 60 min. The amount of metoprolol transport was increased from 12.562 ± 1.725 to 14.684 ± 1.853 and 12.562 ± 1.725 to 18.362 ± 2.515 and 12.562 ± 1.725 to 20.655 ± 3.687 µg/mL in the presence of hesperetin at concentrations of 25, 50, 100 μg/mL at 60 min. To further establish the p-gp role in metoprolol transport, similar assays were performed in the presence of 50 µg/ mL quinidine, a p-gp inhibitor. In the presence of quinidine, the amount of metoprolol transport was increased from 12.562 ± 1.725 to 19.514 ± 3.561 µg/mL at a concentration of 50 µg/mL at 60 min. These study results indicated that hesperetin and quinidine enhanced the absorption of metoprolol due to the inhibition of p-gp.

Discussion
CYP enzymes and p-gp play a significant role in the firstpass metabolism of several orally administered drugs. The oral bioavailability of metoprolol is 50% due to its extensive first-pass metabolism. In the present SDPK and RDPK study, hesperetin which is known to be a has inhibitory effects on CYP3A4 [16], CYP2C9 [17], and CYP2B6 [18] was co-administered with metoprolol.  Similarly, the intestinal absorption of metoprolol was measured from the mucosal compartment to the serosal compartment using everted gut sacs and the intestinal absorption increased by 1.16-, 1.46-, and 1.6-fold when pre-treated with hesperetin at concentrations of 25, 50, 100 μg/mL establishing that p-gp suppression by hesperetin improved the intestinal absorption of metoprolol. These results are consistent with previous study reports. In human liver microsomes, hesperetin inhibited the CYP2C9-mediated conversion of diclofenac to 4'-hydroxydiclofenac. The clinical relevance here is that CYP2C9 is responsible for the biotransformation of drugs with a narrow therapeutic index [17]. This indicates that the concomitant administration of hesperetin with diclofenac results in enhanced bioavailability due to the inhibition of CYP2C9 by hesperetin. In another study, hesperetin inhibited the CYP3A4-mediated metabolism of felodipine in rats, thereby increasing its systemic exposure and suggesting the role of hesperetin as a CYP3A4 inhibitor [16]. Another study has shown the role of hesperetin as a weak inhibitory activity on CYP2B6 [18].
Concomitant administration of duloxetine and metoprolol orally in rat models results in increased the systemic exposure and plasma concentration of metoprolol in rats due to inhibition of CYP2D6 and p-gp by duloxetine [23]. In another study, felodipine, which is a substrate of CYP3A4, significantly increased the C max and AUC 0-12 of metoprolol increased in healthy male volunteers [24]. Similarly, co-administration of pyronaridine and artesunate (PA) increased the peak concentration of metoprolol by 47.93% and the AUC 0-t by 25.60%, indicating that PA co-administration will likely increase exposure to CYP2D6 substrates [25].
Amiodarone (AM) is the most effective antiarrhythmic drug, and its principal metabolite desethylamiodarone (DEA) is similarly effective. Both AM and DEA inhibit CYP2D6, which converts metoprolol to alpha-hydroxymetoprolol. The combination of amiodarone/desethylamiodarone and metoprolol was investigated in another study. The results suggested that concentrations of amiodarone and desethylamiodarone were increased and the plasma concentration of metoprolol still increased even at the decreased dosage of metoprolol decreased due to inhibition of CYP2D6 [26]. Similarly, hesperetin significantly increased the plasma concentration and systemic exposure of rasagiline in rats due to inhibition of CYP and p-gp [27] in another study. The authors of previous studies are also followed encapsulated techniques to improve the bioavailability of encorafenib [28] and 5-fluorouracil [29]. These findings of the other studies further support that results of increased bioavailability of the metoprolol in the current study might be due to the inhibition of CYP2D6, CYP3A4, CYP2C9 by hesperetin and increased intestinal absorption might be due to the inhibition of p-gp by hesperetin.

Conclusion
The results of this study have shown that hesperetin significantly enhanced the C max , AUC 0-24 , AUC 0-∞ , t 1/2 , and MRT and decreased the clearance, and volume of distribution of metoprolol (p < 0.001) in a dose-dependent manner might be due to the inhibition of CYP-mediated metabolism according to the findings of the investigation. Similarly, hesperetin and quinidine significantly increased the absorption of metoprolol in in vitro intestinal absorption study due to p-gp inhibition.