Influence of hesperetin on the pharmacokinetics of diltiazem in rats

Cytochrome P-450 3A4 (CYP3A4) and P-gp substrates exhibit poor bioavailability. Diltiazem, is a benzothiazepine derivative used in various cardiovascular conditions. It is a heart muscle and blood vessel relaxant that improves blood pumping to the heart and from the heart. It has low bioavailability because it is a substrate of CYP3A4 and P-gp. Hesperetin (a flavanone) inhibited CYP3A4 and P-gp in previous investigations. The major goal of this research was to see how hesperetin affected diltiazem pharmacokinetics (PK) in rats and employing everted intestinal sac. Male Wistar rats were given diltiazem (15 mg/kg) alone or in combination with hesperetin (12.5, 25, and 50 mg/kg) once daily for 15 days to hesperetin-pretreated animals. Blood samples were collected on the 1st day in single-dose PK study and on 15th day in multiple dosage PK studies. In vitro , diltiazem was incubated with rat everted intestinal sacs in the presence and absence of hesperetin and conventional P-gp blockers . Diltiazem C max , area under the curve, and half-life ( T 1/2 ) rose two-fold in rats pretreated with Hesperetin compared to the diltiazem control group, although T max did not alter significantly. The value of Mean Residence Time increased by 37% to 47%. There has been a significant reduction in clearing and distribution rates. The results of an in vitro study showed that the transport of diltiazem was significantly increased in the presence of hesperetin and standard P-gp inhibitors. The current study found that hesperetin significantly increased diltiazem systemic absorption by inhibiting CYP3A4 and P-gp.


INTRODUCTION
Diltiazem is majorly prescribed to treat angina, supraventricular arrhythmias, and hypertension (Chaffman and Brogden, 1985;Pool, 1996;Weir, 1995). It undergoes an extensive presystemic metabolism because it is a substrate of cytochrome P-450 3A4 (CYP3A4) and P-glycoprotein (P-gp) (Lefebvre et al., 1996;Buckley et al., 1990). The bioavailability of diltiazem is approximately 40% and was found to be metabolized mainly into N-demethyldiltiazem in humans and dogs. The most common metabolites in rabbits and rats were desacetyldiltiazem and O-diacetyl-N-monomethyl diltiazem, correspondingly (Yeung et al., 1998). CYP3A4 is the major human variant of diltiazem N-demethylation in liver microsomes, and it's also located in the gut (Pichard et al., 1990). The metabolism of diltiazem can be more in the proximal segment of the small intestine than in the distal section (Kolars et al., 1992;Watkins et al., 1987). The P-gp and CYP34A might decrease the oral absorption of diltiazem. The calcium channel blockers verapamil, nicardipine, and diltiazem compete to suppress P-gp multidrug resistance, according to Yusa and Tsuruo (1989).
Diltiazem is not only an Multidrug Resistance modulator but also a precursor for CYP 3A4 and the P-gp efflux transporter Wacher et al. (2001) . Flavonoids are produced by a huge variety ). Hesperetin is a flavanone found naturally in citrus and grapefruits that have been shown to have anti-cancer, antioxidant, anti-inflammatory, anti-hypertensive, anti-atherogenic, hepatoprotective, and pass the blood-brain barrier. Furthermore, earlier research has shown that hesperetin and naringenin are CYP enzyme and P-gp *Corresponding Author Naveen Babu Kilaru, Department of Pharmaceutics and Pharmaceutical Biotechnology, KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, India. E-mail: naveenbabukvsr @ gmail.com inhibitors (Sridhar et al., 2014;Surya et al., 2014). The present study aimed to investigate the influence of hesperetin on the diltiazem pharmacokinetics (PK) in rats.

Drugs and chemicals
Throughout the investigation, analytical grade compounds were employed. Hesperetin was furnished by Sigma Aldrich. Diltiazem and ritonavir were provided as complimentary samples by Sipra Labs in Hyderabad, India. Finisar Chemicals Ltd. of Ahmadabad, India, provided acetonitrile for high-performance liquid chromatography (HPLC).

Study design
The current investigation involves two experiments, acute (single dose) and sub-acute (once daily for 15 days) administration of diltiazem and hesperetin, as previously reported (Challa et al., 2013).

In vivo pharmacokinetic studies
Male Wistar rats were used throughout the study and randomly assigned to four groups of six animals each per study. After overnight fasting, the animals in group 1 was treated with 15 mg/kg (oral) diltiazem in 1% Sodium Carboxymethylcellulose (SCMC) while the other groups viz., groups 2, 3, & 4 were administered with 12.5, 25, and 50 mg/kg hesperetin in 2% SCMC p.o, respectively, followed by diltiazem, 15 mg/kg p.o in singledose PK study (SDS). Similarly, in multiple dosing PK studies (MDS), the animals were given the same drugs once a day for 15 days.

Preparation of gut sac
The rat-everted gut sac model, a simple and effective in vitro model for evaluating drug absorption and processes by assessing drug content in the colon and conveying it via intestinal tissue, is used to assess diltiazem transport across the intestine (Barthe et al., 1999). Babu et al. (2013) presented a modified method for preparing everted sacs of rat ileum. The ileum of a rat was removed and flushed with ice-cold saline multiple times (0.9%). Under phenobarbitone (40 mg/kg) anesthesia, the distal part of the ileum from the rat intestines (approximately 15 cm each) was removed from an over day fasted male Wistar rat weighing around 180-220 g, everted after removal of fat and mesenteric connectors, and fused with a silk incision to make into sacs (Capraro et al., 2011).

Effect of hesperetin on diltiazem transport across gut sac
The everted sacs were supplied with a mixture containing diltiazem 50 µg/ml in the presence or absence of ritonavir 50 µg/ ml, conventional CYP3A4, and P-gp blocker (Kharasch et al., 2008;Kumar et al., 1996;Li et al., 2012), and hesperetin 50, 100, and 200 µg/ml. Diltiazem travel from the serosal to the mucosal side of the everted sac was measured by collecting 1 ml of the outer medium [replaced by 1 ml Krebs-Ringer bicarbonate buffer (KRB) buffer] at 10, 20, 30, and 60 minutes from an Erlenmeyer flask comprising 30mL of oxygenated (O 2 /CO 2 ; 95:5) KRB and incubating in a shaker bath at 37°C for 60 minutes. Each experiment was repeated three times.

Analytical methods
Diltiazem plasma concentrations were measured with changes using a technique published by Li et al., 2003). Briefly, a Shimadzu HPLC system with a pump (LC-20AT VP), a C 18 column (Kromasil 150 × 4.6 mm) with a particle size of 5 µm and a dual-wavelength ultraviolet (UV) visible detector (SPD-10A VP) was used. liquid chromatography solution software was used to gather and process the data. The mobile phase consisted of 0.2% formic acid solution in acetonitrile and water (80:20 v/v) that was ultrasonically degassed and filtered through a 0.45 µm membrane filter. The effluent was monitored at 235 nm with a UV detector at a flow rate of 1 ml/minute. The total run time was 5.0 minutes and the diltiazem eluted at 4.8 minutes (Fig. 1). The analysis was performed at room temperature.

Extraction of diltiazem from plasma
Diltiazem was extracted from rat plasma using the liquid-liquid extraction method (Kallem et al., 2013). 1.5 ml tertbutyl methyl ether was added to a 50 µl plasma aliquot, vortexed, and centrifuged at 6,000 rpm for 5 minutes in each step. The residue (1.2 ml) was dried in a moderate nitrogen stream at 40°C. The dried residue was reconstituted and used for chromatography ( Fig. 1).

Calculation of PK parameters
Thermo Kinetica was used to perform a noncompartmental PK analysis of each rat's plasma concentrations versus time data.

Statistical analysis
GraphPad Prism software was used for data analysis and p-value < 0.05 was considered significant.

Influence of hesperetin on the PK of diltiazem in SDS
Diltiazem plasma concentrations versus time curves after oral administration of diltiazem alone and pre-treatment with hesperetin 12.5, 25, and 50 mg/kg in SDS are shown in Figure 2. Except for T max , all PK parameters were logarithmically converted and compared using one-way ANOVA and Dunnett's multiple comparisons test. The mean plasma PK parameters are shown in Table 1. Hesperetin raised the C max , area under the curve (AUC) 0-24, AUC0-, t 1/2 , and Mean Residence Time (MRT) of diltiazem and lowered the clearance and volume of distribution of diltiazem considerably (p < 0.001 hour at a dose of hesperetin 12.5, 25, 50 mg/kg respectively. The clearance of diltiazem was decreased from 3.000 ± 0.341 to 0.009 ± 0.0001 and 3.000 ± 0.341 to 0.002 ± 0.0001 and 3.000 ± 0.341 to 0.001 ± 0.0001 l/hour/kg at a dose of hesperetin 12.5, 25, 50 mg/kg respectively. The volume of distribution of diltiazem was decreased from 0.111 ± 0.01 to 0.007 ± 0.0001 and 0.111 ± 0.01 to 0.083 ± 0.001 and 0.111 ± 0.01 to 0.040 ± 0.001 ml/kg at a dose of hesperetin 12.5, 25, 50 mg/kg respectively.

Effect of hesperetin on the PK of diltiazem in MDS
Diltiazem plasma concentrations versus time curves in MDS patients after an oral dose of Diltiazem alone and  pretreatment with hesperetin 12.5, 25, and 50 mg/kg are also shown in Figure 2. Except for T max , all PK parameters were logarithmically converted and compared using one-way ANOVA and Dunnett's multiple comparisons test. The mean plasma PK parameters are shown in Table 2. Hesperetin raised the C max , AUC0-24, AUC0-, t 1/2 , and MRT of Diltiazem and lowered the clearance and volume of distribution of Diltiazem in studies (p < 0.001 .625 hour at a dose of hesperetin 12.5, 25, 50 mg/kg respectively. The clearance of diltiazem was decreased from 1.000 ± 0.1 to 1.000 ± 0.1 and 1.000 ± 0.1 to 0.800 ± 0.2 and 1.000 ± 0.1 to 0.800 ± 0.2 l/hour/kg at a dose of hesperetin 12.5, 25, 50 mg/kg respectively. The volume of distribution of diltiazem was decreased from 0.1001 ± 0.1 to 0.052 ± 0.005 and 0.1001 ± 0.1 to 0.038 ± 0.005 and 0.1001 ± 0.1 to 0.033 ± 0.005 ml/kg at a dose of hesperetin 12.5, 25, 50 mg/kg respectively.

Hesperetin's effect on P-gp-mediated diltiazem transport
P-gp operates as a barrier in the gut, lowering net absorption of xenobiotics and medicines into the intraluminal space, which can have a substantial influence on P-gp substrate bioavailability and therapeutic uses. Diltiazem bowel absorption was assessed using everted gut sacs from the mucosal to the serosal compartments. Hesperetin increased diltiazem absorption in a concentration-dependent manner (Table 3). When To validate the role of P-gp in diltiazem transport, the studies were performed in the presence of 50 µg/ml ritonavir, a P-up blocker. When administered in the presence of ritonavir, the amount of Diltiazem was increased from 11.026 ± 1.811 to 18.362 ± 3.652 at a concentration of 50 μg/ml at a time interval of 60 minutes. The findings show that ritonavir increased absorption at the incubation time.

DISCUSSION
In the current investigation, Hesperetin dramatically changed the PK of Diltiazem in rats owing to an inhibition of CYP3A4 and P-gp. These results are consistent with previous study reports. Morin, a flavonoid, significantly increased diltiazem oral exposure. The C max and AUC have significantly increased from 173 ± 41.5 to 374 ± 55.2 ng/ml and 358 ± 56.9 to 642 ± 76.6 ng. hour/ml respectively at a dose of 7.5 mg/kg. the T 1/2 of Diltiazem was decreased from 13 ± 2.9 to 11 ± 3.2 hour and clearance was decreased from 710 ± 93.4 to 393 ± 38.5 ml/minute. Kg at a dose of morin 7.5 mg/kg. The increased oral absorption of Diltiazem is due to the inhibition of the CYP3A4-mediated metabolism of Diltiazem (Choi et al., 2005a). The bioavailability of diltiazem increased considerably in rabbits pretreated with quercetin compared to the control, but not in rabbits co-administered with quercetin. The Cmax and AUC significantly increased from 94.2 ± 23.5 to 99.3 ± 24.8 ng/ml and 232 ± 58 to 287 ± 71 ng/ml hour respectively at a dose of 2 mg/kg. The t 1/2 of the Diltiazem was increased from 11.3 ± 2.8 to 12.3 ± 2.9 hour at a dose of 2 mg/ kg. The increased bioavailability of Diltiazem in rabbits treated with quercetin could be attributed to quercetin's inhibition of the efflux pump P-gp and the first-pass metabolizing enzyme CYP 3A4 (Choi et al., 2005b).
The concomitant use of hesperetin significantly enhanced the oral exposure of Diltiazem in rats. The C max and AUC of Diltiazem were raised from 173 ± 41.5 to 375 ± 61.1 ng/ml and 358 ± 56.9 to 682 ± 54.8 ng/ml hour respectively at a dose of naringenin 15 mg/kg. There is no significant change in T 1/2 and T max (Choi et al., 2005c). The concurrent use of fluvastatin significantly enhanced the oral exposure of Diltiazem in rats. At a dosage of fluvastatin 2 mg/kg, the C max and AUC of Diltiazem rose from 174 ± 35.8 to 310 ± 62.1 ng/ml and 363 ± 63.9 to 628 ± 130 ng. hour/l, correspondingly, when fluvastatin was used concurrently. Diltiazem bioavailability increased considerably in rabbits pretreated with quercetin compared to the control, but not in rabbits co-administered with quercetin. Fluvastatin may inhibit presystemic metabolism during intestinal absorption, according to these studies. According to Lee et al. (1991) Diltiazem extraction ratios in the small intestine and liver were about 85% and 63%, significantly, following oral therapy of rats, suggesting that Diltiazem is widely extracted in both the small intestine and the liver . In conclusion, simultaneous fluvastatin medication may contribute, at least in part, to the increased oral exposure to Diltiazem by reducing both intestinal and hepatic extraction (Choi et al., 2006;Lee et al., 1991).
Simvastatin enhanced Diltiazem's oral absorption. At a dosage of 1 mg/kg, Diltiazem's C max and AUC were raised from 182 ± 33 to 246 ± 44 ng/ml and 270 ± 51 to 392 ± 74 ng. hour/ ml, accordingly. T max and t 1/2 were similarly elevated, although the differences were not important. Increased absorption in the small intestine due to P-gp inhibition and reduced first-pass metabolism of Diltiazem due to CYP3A subfamily inhibition in the small intestine and/or liver, rather than renal elimination of Diltiazem by simvastatin, could explain the rise in Diltiazem oral bioavailability . The resveratrol had increased the bioavailability of Diltiazem. At a dosage of resveratrol 10 mg/ kg, the C max and AUC of Diltiazem were considerably enhanced from 165 ± 37.8 to 259 ± 60.6 ng/ml and 342 ± 80.2 to 547 ± 131 ng/ml, correspondingly. Resveratrol did not alter T max . The resveratrol significantly increased the bioavailability of Diltiazem due to the inhibition of both the CYP3A4-mediated metabolism and the P-gp in the intestine and or liver (Hong et al., 2008).
The addition of lovastatin increased Diltiazem's systemic bioavailability. The AUC 0-∞ of Diltiazem was increased from 342 ± 69 to 508 ± 107 ng hour/ml at a 1 mg/kg dose of lovastatin. The C max of Diltiazem was increased from 165 ± 35 to 234 ± 53 ng/ml at a dosage of lovastatin 1 mg/kg. The T max of Diltiazem was decreased from 0.33 ± 0.13 to 0.29 ± 0.10 hour at a dosage of lovastatin 1 mg/kg. The volume of distribution of Diltiazem was reduced from 52.2 ± 14.9 to 42.4 ± 12.2 ml/kg at a dose of lovastatin 1 mg/kg. The clearance of Diltiazem was decreased from 45.2 ± 13.8 to 38.0 ± 9.9 ml/minute per kg at a dose of lovastatin 1 mg/kg. The increased bioavailability of Diltiazem in the presence of lovastatin may be due to lovastatin's suppression of the P-gp mediated efflux pump in the bowel and/or suppression of CYP3A4-mediated metabolic in the gut and/or liver (Hong et al., 2011). Rasagiline's concentration in the brain rose when it was coupled with hesperetin and significant naringenin. In the presence of hesperetin or naringenin, rasagiline transport from the mucosal to the serosal side did not change significantly ex vivo (rat-everted gut sacs used). Hesperetin and naringenin enhanced rasagiline systemic exposure via CYP1A2 inhibition but not P-gp suppression, according to our findings Ravindra et al., 2016). In vitro studies demonstrated that hesperetin enhanced felodipine intestinal absorption. Concurrent usage of hesperetin changed the PK of felodipine, resulting in increased systemic exposure (Sridhar et al., 2014).

CONCLUSION
Due to P-gp and CYP3A4 inhibition, hesperetin significantly increased the plasma concentration, AUC, t 1/2 , MRT, and greatly lowered the clearance, Vz/F, of diltiazem in rats. According to in vitro study results, diltiazem transport was significantly increased in the presence of hesperetin and ritonavir owing to P-gp and CYP3A4 suppression.