Table of Contents    
ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 2  |  Page : 209-216  

Resveratrol nanoparticle pretreatment improved the oral bioavailability of bromocriptine: Involvement of liver and intestinal CYP3A enzyme inhibition


1 Department of Pharmacology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India
2 Department of Toxicology DMPK and Clinical Pharmacology Division, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India

Date of Web Publication18-Jul-2019

Correspondence Address:
Prasad Neerati
Department of Pharmacology, DMPK and Clinical Pharmacology Division, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_126_19

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   Abstract 


Background: Resveratrol (RSV) is a polyphenol belonging to phytoalexin family and has been reported to show inhibitory effects on CYP3A4 enzymes. However, there has been no report about the pharmacokinetic interaction of bromocriptine (BRO) with RSV and RSV nanoparticles (NRSV) in rats. Hence, the present study was undertaken in an attempt to enhance the oral bioavailability of BRO when BRO was pretreated with RSV and NRSV. Materials and Methods: Antisolvent precipitation method is used to prepare NRSV under temperature control. The following methods were used in this study, i.e., in vitro assessment of CYP3A activity in liver and intestinal microsomes and in vitro noneverted sac method. To confirm the in vitro findings, an in vivo pharmacokinetic study was also performed. Results: The results indicate that RSV significantly (P < 0.05) inhibited the CYP3A activity in intestinal and liver microsomes. In noneverted sac study, the intestinal transport and Papp of BRO were more significant (P < 0.05) in NRSV as compared to RSV group. Further, in vivo study revealed that the increased levels of Cmax and AUC were comparatively higher in NRSV-pretreated group than RSV group. In addition, pretreatment with RSV and NRSV significantly (P < 0.05) decreased the mean appararant clearance (CL/F) of BRO. Conclusion: NRSV pretreatment significantly increased the intestinal absorption and bioavailability of BRO probably by the inhibition of CYP3A-mediated metabolism in rats. However, further studies are needed to confirm these interactions in humans.

Keywords: Bromocriptine, CYP3A, high-performance liquid chromatography, oral bioavailability, resveratrol nanoparticles


How to cite this article:
Neerati P, Palle S. Resveratrol nanoparticle pretreatment improved the oral bioavailability of bromocriptine: Involvement of liver and intestinal CYP3A enzyme inhibition. J Nat Sc Biol Med 2019;10:209-16

How to cite this URL:
Neerati P, Palle S. Resveratrol nanoparticle pretreatment improved the oral bioavailability of bromocriptine: Involvement of liver and intestinal CYP3A enzyme inhibition. J Nat Sc Biol Med [serial online] 2019 [cited 2019 Oct 19];10:209-16. Available from: http://www.jnsbm.org/text.asp?2019/10/2/209/262943




   Introduction Top


Resveratrol (RSV) is a polyphenol belonging to the phytoalexin family, abundantly present in fruits, vegetables, grapes, peanuts, and blueberries.[1] RSV possesses diverse biochemical and physiological properties including antioxidative stress,[2] anti-inflammation, and anti-carcinogenic activities [3] as well as neuroprotective effects.[4] Previous in vitro and in vivo studies demonstrated that RSV is an inhibitor of cytochrome P450 (CYP) 3A4,[5],[6] and it inactivates the enzyme in the presence of nicotinamide adenine dinucleotide phosphate (NADPH).[7],[8] In addition, RSV and its metabolite RSV-3-sulfate have been reported as inhibitors of CYP3A.[9]

From the literature, it is evident that RSV is practically water insoluble and has low bioavailability.[10] Hence, clinical application of this drug greatly restricted. In this regard, to circumvent the problems of poor bioavailability and poor pharmacokinetics associated with RSV, several strategies such as nanoparticles, liposomes, and nanoemulsions have been developed by performing extensive studies.[11] According to the Noyes and Whitney equation, particle size reduction effectively increases the surface area of poorly water-soluble compounds and therefore enhances the solubility and dissolution rate.[12] Nanoparticle technology is an excellent drug delivery system developed to enhance the saturation solubility and bioavailability of many therapeutic drugs, especially highly hydrophobic agents like RSV.

The CYP3A4 is one of the most important subfamily of CYP isoforms found in critical tissues such as the liver and intestine.[13],[14] CYP3A4 enzymes are responsible for first-pass metabolism and changes in absorption, distribution, and metabolism of orally administered many drugs.[15],[16] Therefore, inhibition of CYP3A4 activity greatly impacts the bioavailability of orally administered drugs which are substrates for this enzyme.

Bromocriptine (BRO) a semisynthetic derivative of ergot alkaloid, is an agonist of dopamine receptor, and directly and indirectly protects dopaminergic cells. BRO is widely used in the treatment of pituitary tumors, breast tumors, hyperprolactinemia, infertility, menstrual disorders, type 2 diabetes, and especially in Parkinson's disease.[17],[18],[19] BRO is available in tablet and capsule dosage form; it is easily absorbed through the gastrointestinal tract, but due to extensive first-pass metabolism, only 3%–5% of the ingested dose reaches the systemic circulation.[20],[21] BRO is a substrate of CYP3A4, extensively metabolized in the gastrointestinal tract and liver by the CYP3A4 cytochrome system.[22] Potent inhibitors or inducers of CYP3A4 may increase or reduce the circulating levels of BRO.[23] Therefore, oral administration of BRO with coadministered substances such as RSV and RSV nanoparticles (NRSV) could improve the efficacy and allow the optimal therapeutic dosing.

However, there has been no report about the pharmacokinetic interaction of BRO with RSV and NRSV in rats. Hence, the present study was undertaken in an attempt to enhance the oral bioavailability of BRO when BRO was pretreated with RSV and NRSV.

Rats were chosen as an animal model to investigate the pharmacokinetic interaction of BRO with RSV and NRSV although there should be some extent of difference in CYP enzyme activity between rat and human.[24] The influence of RSV and NRSV on the pharmacokinetics of BRO was evaluated in vivo by assessment of CYP3A activity in liver and intestinal microsomes. The intestinal permeability characteristics of BRO were mechanistically investigated using the in vivo noneverted sac method. To confirm these findings, an in vivo pharmacokinetic study of orally administered BRO in rats with RSV and NRSV pretreatments was performed.


   Materials and Methods Top


Materials

BRO was kindly donated by Inga laboratories Pvt. Ltd., Maharashtra, India. RSV, purchased from Sigma–Aldrich (USA), the absolute ethanol (99.5%–99.8%), and acetonitrile (high-performance liquid chromatography [HPLC] grade) were obtained from Merck, Mumbai, India. Water for analytical purpose is double-distilled, filtered using direct-Quv millipore, and sonicated for removing air bubbles. All other reagents used were also of analytical grade.

Preparation of resveratrol nanoparticles

The NRSV were prepared by temperature-controlled antisolvent precipitation method using syringe pump.[25] RSV was dissolved in the solvent ethanol and prepared a solution at a predetermined concentration of 60 mg/mL. The obtained organic solution was added to precooled (5°C) antisolvent (deionized water) in a drop-wise manner under rapid magnetic stirring at 1000 rpm. The formed NRSV was filtered and vacuum-dried.

Particle morphology

The particle size and morphology of samples were observed using a scanning electron microscope (SEM) Zeiss EVO 18-EDX special edition machine compatible with energy dispersive X-ray (EDX) machine. The powder samples were spread on a SEM stub and sputtered with gold before the SEM observations. The particle size and texture of nanoparticles can be analyzed using image magnification software compatible with SEM and helps in determining the presence and formation of NRSV. Five SEM pictures were used to find the average range of particle diameter.

In vitro dissolution testing

The in vitro dissolution of the prepared NRSV samples, as well as the original RSV, was determined using the paddle method (USP 29 type II) (Electro Lab, TDT 06P) in 100 mL of simulated intestinal fluid (SIF, pH 6.8). The rotation speed of paddle was set at 100 rpm, and the bath temperature was kept at 37°C ± 0.5°C. The original RSV and NRSV containing 5 mg of sample were tested for their dissolution in simulated intestinal fluid.[26] Samples of 1 mL volume were collected at 15, 30, 45, 60, 90, 120, and 180 min of dissolution time. The dissolution test for each sample was performed in triplicate, and the dissolution data were averaged. The absorbance was measured at 308 nm using ultraviolet (UV) spectrometer.

Animals

Male albino Wistar rats (180–250 g) were used for the study. The rats were kept in polyacrylic cages and maintained under standard laboratory conditions (room temperature24°C–27°C and humidity 60%–65%). Each experimental group consisted of six animals that were chosen randomly from different cages. Handling and experimentation were conducted in accordance with the approved guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, and the experimental protocol was approved by Institutional Animal Ethical Committee (IAEC) Kakatiya University. Approval no: IAEC/06/UCPSC/KU/16.

In vitro assessment of CYP3A activity

An in vitro CYP3A activity was performed according to the previously described method.[27] This method is based on the principle of CYP3A converts erythromycin to N-demethyl erythromycin and formaldehyde, which produced yellow color with Nash reagent.[28]

Preparation of intestinal microsomes

The intestinal microsomes were prepared by slight modification of previously reported methods.[29],[30] The isolated intestine was cut into pieces, washed with ice-cold phosphate-buffered saline and then cut longitudinally to expose mucosa. The mucosal layer was scraped lightly from all pieces of intestine with the help of a cover slip. All scrapings were mixed together and centrifuged at 25 × g for 5 min. The pellet was suspended in 5.0 mL of ice-cold histidine–sucrose buffer (HSB) (histidine 5 mM, pH 7.0; sucrose 0.25 M; NaEDTA 0.5 mM; pH 7.4); homogenized and centrifuged at 15,000 × g for 10 min. The supernatant was carefully transferred to a clean tube. The pellet was resuspended in 5.0 mL of HSB and again centrifuged at 15,000 × g for 10 min. The supernatant after each centrifugation was taken together and mixed with 52 mM CaCl2 (0.2 mL per mL of the supernatant) to precipitate microsomes. After 15 min of standing, it was centrifuged at 20,000 × g for 15 min, the microsomal pellet was suspended in 0.5 mL of 0.1 M potassium phosphate buffer containing 20% glycerol, and stored at − 20°C until needed. The protein concentration of the microsomal fraction was determined by Biuret method using bovine serum albumin as the standard.

Preparation of liver microsomes

The liver microsomes were prepared by slight modification of previously reported method.[31] The liver isolated from rats was minced and homogenized in 5 mL of 0.25 M sucrose containing 10 mM Tris-HCl (pH 7.4) and then centrifuged at 600 × g for 5 min followed by 12,000 × g for 10 min. The postmitochondrial supernatant was separated, mixed with 52 mM CaCl2(0.2 mL per mL of the supernatant) to precipitate microsomes. After 15 min of standing, it was centrifuged at 20,000 × g for 15 min; the microsomal pellet was suspended in mixture of 150 mM KCl-10 mM Tris-HCl, and centrifuged at 20,000 × g for 20 min to obtain pinkish microsomal pellet, which was suspended in 0.5 mL of 0.1 M potassium phosphate buffer containing 20% glycerol, and stored at −20°C until needed.

Erythromycin-N-demethylation assay

The mixture of microsomal suspension (0.1 mL, 25%), erythromycin (0.1 mL, 10 mM), and potassium phosphate (0.6 mL, 100 mM, pH 7.4) was incubated at 37 °C along with RSV at a concentration of 0.1, 1, and 10 μM. The reaction between these agents was initiated by adding NADPH (0.1 mL, 10 mM), and terminated after 10 min, by adding ice-cold trichloroacetic acid (0.5 mL, 12.5% w/v) solution. It was centrifuged (2000 × g; 10 min) to remove proteins. To 1 mL of this supernatant, 1 mL of Nash Reagent (2 M ammonium acetate, 0.05 M glacial acetic acid, and 0.02 M acetyl acetone) was added and heated in a water bath at 50°C for 30 min. After cooling, the absorbance was read at 412 nm. The activity was calculated from standards (1–100 μM formaldehyde) prepared by substituting sample with the standard solution which was run in parallel. The CYP3A activity was expressed as μM of formaldehyde obtained per milligram of protein per hour.

In vitro noneverted intestinal sac study

An in vitro noneverted intestinal sac study was performed according to the previously described methods.[32],[33] The rats were divided into three groups control, RSV and NRSV each consisting of six animals. RSV and NRSV were administered orally at a dose of 40 mg/kg for 10 days, and other group was kept as control without any drug treatment. All the animals were sacrificed on 11th day, and 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 (BRO 500 μg/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°C and preoxygenated with 5% CO2/95% O2 for 15 min, under bubbling with a CO2/O2 mixture gas; the transport of the BRO from mucosal to serosal direction across the intestinal sacs was measured by sampling the serosal medium periodically for 120 min. The samples of 1 mL were collected from intestinal sacs of control, RSV, and NRSV rats at predetermined time points and stored at −80°C until analysis. The drug transported from mucosal to serosal direction was measured by HPLC.

Calculation of apparent permeability coefficient

The apparent permeability coefficient (Papp) of BRO was calculated from the following equation:[34]



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.

In vivo pharmacokinetic study

Rats were fasted for at least 24 h before experiments and approximately 3-h postdose. The rats were divided into 3 groups consisting of six animals each. Group I was administered with BRO (10 mg/kg; p.o.) on the 11th day. Group II was pretreated with RSV (40 mg/kg; p.o.) for 10 days and on the 11th day with BRO (10 mg/kg) followed by RSV, and Group III was pretreated with NRSV (40 mg/kg; p.o.) for 10 days and on the 11th day with BRO (10 mg/kg) followed by NRSV. Blood samples (approximately 0.25 mL) were collected from subclavian vein at specified time intervals, then placed into heparinized tubes and separated immediately by centrifugation (15000 rpm for 15 min). The plasma samples obtained were stored at −80°C until the HPLC analysis.

High-performance liquid chromatography analysis of bromocriptine

The drug analysis of samples was carried out using Shimadzu HPLC system equipped with a LC-20AD pump, SPD 20A UV visible detector, Rheodyne injector port (20 μL loop) and RP C18 column (Phenomenex Luna, 250 mm × 4.6 mm ID, particle size 5 mm). The mobile phase comprised of acetonitrile water (0.2% triethylamine) (70:30 v/v), adjusted to pH 3 with HCL. Analyses were run at a flow rate of 1 mL/min, and the elution was monitored at 280 nm.[35]

In vitro and plasma samples were extracted using a simple protein precipitation method by adding acetonitrile (200 μL) to samples (100 μL). Samples vortexed for 2 min and centrifuged at 15,000 rpm for 15 min. The resultant clean supernatant (20 μL) was injected and analyzed using HPLC method. The limit of detection was 0.01 μg/mL, and the assay range used was 0.01–10 μg/mL. The average recovery of the drug was 91.7%. The intra- and inter-day coefficients of variation for the low and high quality control samples were <10%.

Pharmacokinetic analysis

Pharmacokinetic parameters were computed by the noncompartmental model using Phoenix WinNonlin version 6.2 software (Certara, Pharsight Corporation, USA). The maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (tmax) were determined by a visual inspection of the experimental data. The plasma BRO concentration versus time plots were used to estimate the area under the concentration–time curve to the last sampling point (AUC0-t), area under the concentration–time curve to the infinity (AUC0-∞), half-life (t1/2), elimination rate constant (kel), and clearance (CL/F).

Statistical analysis

All mean values are presented as with their standard deviation (SD) (mean ± SD). Significant difference between means was evaluated by one-way analysis of variance followed by Bonferroni multiple comparison tests, P < 0.05 was considered as statistically significant.


   Results Top


Resveratrol nanoparticles characterization by scanning electron microscope

SEM micrographs of the RSV and NRSV are shown in [Figure 1]a and [Figure 1]b. It is observed that the RSV powder [Figure 1]a exhibited particles lacking uniformity in size and was much larger than the NRSV [Figure 1]b. On the other hand, NRSV prepared by syringe pump exhibited particles uniformity in size, less crystallinity, and absence of larger particles [Figure 1]b. As depicted in the image, the particles possessed uniform shape. The size of all particles was found to be within the range of 100–250 nm.
Figure 1: Scanning electron microscope photographs of (a) resveratrol and (b) resveratrol nanoparticles. (c) Dissolution profile of commercial resveratrol and resveratrol nanoparticles. (d) Effect of resveratrol on in vitro CYP3A activity in intestinal microsomes. Data are represented as mean ± standard deviation (n = 6). *Significant difference (P < 0.05) in comparison with the vehicle. Statistical analysis was carried out using one-way analysis of variance followed by Bonferroni multiple comparisons test. (e) Effect of resveratrol on in vitro CYP3A4 activity in liver microsomes. Data are represented as mean ± standard deviation n = 6). *Significant difference (P < 0.05) in comparison with the vehicle. Statistical analysis was carried out using one-way analysis of variance followed by Bonferroni multiple comparisons test. (f). Intestinal transport of bromocriptine in duodenum, jejunum, and ileum of Wistar rats. Data are represented as mean ± standard deviation (n = 6). *Significant difference (P < 0.05) in comparison with the bromocriptine alone. Statistical analysis was carried out using two-way analysis of variance followed by Bonferroni multiple comparisons test. (g) Apparent permeability coefficient (Papp) of bromocriptine in duodenum, jejunum, and ileum of Wistar rats. Data are represented as mean ± standard deviation (n = 6). *Significant difference (P < 0.05) in comparison with the bromocriptine alone. Statistical analysis was carried out using two-way analysis of variance followed by Bonferroni multiple comparisons test. (h) Plasma drug concentration–time plots of bromocriptine (10 mg/kg) in Wistar rats. Control group administered with bromocriptine alone on 11th day and pretreatment groups administered with resveratrol and NRSV for 10 days and on the 11th day with bromocriptine followed by resveratrol and resveratrol nanoparticles. Data are represented as mean ± standard deviation (n = 6)

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In vitro dissolution

The dissolution profiles of the commercial RSV and the formulated NRSV in simulated intestinal fluid SIF at pH 6.8 shown in [Figure 1]c. NRSV were dissolved 90% at 180 min. On the other hand, 58% of the commercial RSV was dissolved in the same period. It was observed that NRSV reached 50% dissolution in 45 min whereas the commercial RSV reached 50% dissolution at 120 min.

Assessment of in vitro CYP3A activity in intestinal microsomes

The extent of erythromycin-N-demethylation (EMD) due to CYP3A activity in intestinal microsomes is shown in [Figure 1]d. The EMD levels in RSV-treated group at all concentrations 0.1, 1, and 10 μM were significantly reduced (P < 0.05) when compared with vehicle.

Assessment of in vitro CYP3A activity in liver microsomes

The extent of EMD due to CYP3A activity in liver microsomes is shown in [Figure 1]e. RSV treatment significantly decreased P < 0.05 EMD levels at 1 μM and 10 μM while it has no significant effect at 0.1 μM.

Effect of resveratrol and resveratrol nanoparticles on intestinal transport and apparent permeability of bromocriptine

The present investigation involves the determination of BRO intestinal transport and Papp in control, RSV, and RSVN treatment groups. Pretreatment with RSV (40 mg/kg) for 10 days resulted in a significant (P < 0.05) increase in mean cumulative concentrations from mucosal to serosal direction of BRO from 9.45 ± 0.87 to14.27 ± 0.74 μg/mL in duodenum; 7.29 ± 0.62–12.78 ± 0.86 μg/mL in jejunum; and 5.25 ± 0.36–11.72 ± 0.75 μg/mL in ileum [Figure 1]f. In addition, pretreatment with NRSV (40 mg/kg) for 10 days resulted in a significant (P < 0.05) increase in mean cumulative concentrations from mucosal to serosal direction of BRO from 9.45 ± 0.87–18.65 ± 0.54 μg/mL in duodenum; 7.29 ± 0.62–16.53 ± 0.69 μg/mL in jejunum; and 5.25 ± 0.36–14.81 ± 0.62 μg/mL in ileum.

The transport of BRO was found to be increased by 1.5-, 1.7- and 2.2-fold in duodenum, jejunum and ileum, respectively, in RSV-pretreated group when compared with the BRO alone group. Further, the transport of BRO was found to be increased by 1.9-, 2.2- and 2.8-fold in duodenum, jejunum, and ileum, respectively, in RSVN-pretreated group when compared with the BRO alone group. Pretreatment with RSV resulted in a significant P < 0.05) increase in Papp of BRO from 0.313 ± 0.063 × 10− 4 to 0.683 ± 0.056 × 10− 4 cm/s in duodenum; 0.269 ± 0.042 × 10− 4–0.594 ± 0.043 × 10− 4 cm/s in jejunum; and 0.203 ± 0.026 × 10− 4–0.539 ± 0.034 × 10− 4 cm/s in ileum [Figure 1]g. In addition, pretreatment with NRSV resulted in a significant (P < 0.05) increase in Papp of BRO from 0.203 ± 0.026 × 10− 4 to 0.649 ± 0.032 × 10− 4 cm/s in duodenum; 0.269 ± 0.042 × 10− 4–0.693 ± 0.042 × 10− 4 cm/s in jejunum; and 0.313 ± 0.063 × 10− 4–0.783 ± 0.053 × 10− 4 ileum [Figure 1]g. The Papp of BRO in duodenum, jejunum, and ileum was found to be increased by 2.1-, 2.2-, and-2.6-fold, respectively, in RSV-pretreated group when compared with BRO alone group. Whereas in case of RSVN-pretreated group, the Papp of BRO in duodenum, jejunum, and ileum was found to be increased by 2.5, 2.5, and 3.2 fold, respectively, when compared with BRO alone group. The increased intestinal transport and Papp of BRO were more prominent in RSVN-pretreated group than the RSV-pretreated group.

Effect of resveratrol and RSVN on pharmacokinetics of bromocriptine

The effects of RSV and NRSV (40 mg/kg, p.o.) on the plasma concentration–time plots of orally administered BRO (10 mg/kg, p.o.) were characterized and depicted in [Figure 1]h, and the pharmacokinetic parameters were summarized in [Table 1]. RSV and RSVN pretreatment for 10 days significantly (P < 0.05) altered the pharmacokinetics of oral BRO when compared with the control group BRO alone. The Cmax, AUC0-t, and AUC0-∞ of BRO were found to be increased by 1.6, 1.66, and 1.69 fold, respectively, in RSV-pretreated group when compared with the control group. The Cmax, AUC0-t and AUC0-∞ of BRO were found to be increased by 2.62, 2.18, and 2.21 fold, respectively, in NRSV-pretreated group when compared with the control group. Further, the CL/F of BRO was significantly decreased while there was no significant change observed in tmax of BRO in RSV and RSVN-pretreated groups when compared with control group. The changes in pharmacokinetic parameters of BRO were more prominent in RSVN-pretreated group than the RSV-pretreated group.
Table 1: Pharmacokinetic parameters of bromocriptine in control, resveratrol, and pretreated group rats after oral administration of 10 mg/kg postoperative bromocriptine

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Effect of resveratrol and resveratrol nanoparticles on pharmacokinetics of bromocriptine

The effects of RSV and NRSV (40 mg/kg, p. o.) on the plasma concentration–time plots of orally administered BRO (10 mg/kg, p. o.) were characterized and depicted in [Figure 1], and the pharmacokinetic parameters were summarized in [Table 1]. RSV and NRSV pretreatment for 10 days significantly (P < 0.05) altered the pharmacokinetics of oral BRO when compared with the control group (BRO alone). The Cmax, AUC0-t, and AUC0-∞ of BRO were found to be increased by 1.6, 1.66, and 1.69 fold, respectively, in RSV-pretreated group when compared with the control group. The Cmax, AUC0-t, and AUC0-∞ of BRO were found to be increased by 2.62, 2.18, and 2.21 fold, respectively, in NRSV-pretreated group when compared with the control group. Further, the CL/F of BRO was significantly decreased while there was no significant change observed in tmax of BRO in RSV and NRSV-pretreated groups when compared with control group. The changes in pharmacokinetic parameters of BRO were more prominent in NRSV-pretreated group than the RSV-pretreated group.


   Discussion Top


Herbal constituents have the greatest potential to modulate the expression and activity of drug-metabolizing enzymes, such as cytochromes P450, leading to clinically significant herb–drug interactions as they can affect the absorption and disposition of concomitantly administered drugs.[36],[37] Hence, it is best known that pharmacokinetic herb–drug interactions have attracted attention by the patients and healthcare professionals due to their improved therapeutic benefits.[38]

In the present study, the particle size of RSV was reduced by the temperature-controlled antisolvent precipitation method. Further, characterization of the NRSV was done by SEM and in vitro dissolution test to evaluate the size of the particles and dissolution rate. The SEM analysis of NRSV revealed the formation of nanoparticles, showed the spherical shape, and found to be within the range of 100–250 nm. In addition to that, in vitro dissolution study of NRSV has shown high dissolution rate as compared to the commercial RSV which may be attributed to the fact that the size reduction of a poorly water-soluble compound to nanoparticles results in increased surface specific dissolution rate.[39],[40] Here, we can conclude that the higher dissolution rate of NRSV resulted from the decreased particle size and could translate into increased bioavailability. Therefore, the increased bioavailability of RSV leads to enhancement of pharmacokinetic interaction with the concomitantly administered BRO which results in change in the total exposure of the BRO in the body and reflects in improved blood concentration.

Here, we explored the influence of RSV on EMD assay, which is an indicator of CYP3A activity. The results indicate that RSV inhibited the CYP3A activity in intestinal microsomes to a greater extent than the liver microsomes; it is due to the fact that intestinal microsomal preparations have a low overall concentration of CYPs in comparison to hepatic preparations.[24] These results are in accordance with the earlier reports, i.e., RSV enhanced the bioavailability of CYP3A4 substrates such as carbamazepine [16] and diltiazem [5] by CYP3A inhibition in rats. Considering that BRO is a substrate of CYP enzymes and inhibitory action on CYP may cause significant changes in the pharmacokinetic profile of BRO.

It has been clearly demonstrated that CYP3A4 is the predominant CYP form expressed in the human small intestine.[41],[42],[43] Orally administered xenobiotics, including therapeutic drugs initially metabolized in the enterocytes, epithelial cells of the human small intestine.[44] Toxicity and therapeutic efficacy of orally administered xenobiotics are substantially affected by the small intestinal CYP metabolism. Early studies reported that rifampin [41] induced the action of CYP3A4 by increasing human intestinal expression. In addition, constituents of grapefruit juice significantly increase the uptake of drugs such as felodipine,[45] saquinavir,[46] and ethinylestradiol [47] by decreasing the intestinal expression of CYP3A4.

In this study, the intestinal transport and Papp of BRO were significantly increased in duodenum, jejunum, and ileal sacs of rats pretreated with RSV and NRSV as compared to control group. The intestinal transport and Papp of BRO were more prominent in rats pretreated with NRSV than RSV-pretreated animals. Pretreatment with RSV and NRSV increased the Papp of BRO in rats, in the order duodenum > jejunum > ileum. We have found that our results are consistent with a study reported by Zhang et al.[48] Microsomal protein content decreased markedly along the small intestine from the duodenum to the ileum. Therefore, these studies further confirm the inhibitory action of RSV and NRSV on CYP3A activity.

Further, we also performed in vivo studies in rats to support in vitro liver and intestinal CYP3A activity and in vitro intestinal transport of BRO in noneverted sac model. Here, we evaluated the effect of RSV and NRSV on the metabolism and pharmacokinetics of BRO. RSV and NRSV pretreatment for 10 days increased the oral bioavailability of BRO in rats. RSV and NRSV groups have shown significant increase in the Cmax and AUC when compared to BRO group suggesting that pretreatment of RSV and NRSV for 10 days significantly altered pharmacokinetics and enhanced bioavailability of BRO. In addition, pretreatment with RSV and NRSV resulted in a significant decrease in mean CL/F of BRO while there was no significant change observed in tmax of BRO when compared to BRO group. Decreased CL/F, kel and increased t1/2 values indicating the inhibition of elimination of BRO upon RSV and NRSV pretreatment. Here, we observed that NRSV-pretreated group has shown the more prominent results than RSV pretreated group, which indicates improved therapeutic efficacy of NRSV.


   Conclusion Top


The results of the present study revealed that NRSV pretreatment significantly increased the intestinal absorption and bioavailability of BRO probably by the inhibition of CYP3A-mediated metabolism in rats. Therefore, the inhibition of CYP3A by dietary phytochemicals such as NRSV may provide a novel therapeutic approach for the clinical application which could improve the efficacy, reduce the incidence of side effects, and allow the optimal therapeutic dosing of BRO. However, further studies are necessary to conclude phytochemical-mediated CYP3A4 interactions with BRO in humans.

Acknowledgments

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. University College of pharmaceutical sciences, Kakatiya University, support for the routine reagents, and permission to animal holding for this research. Authors would like to thank Inga laboratories Pvt. Ltd., Maharashtra, for providing BRO as a gift sample.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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