Formulation and Evaluation of Fast Dissolving Tablet Containing Vilazodone Nanocrystals for Solubility and Dissolution Enhancement Using Soluplus : In vitro-In vivo Study

© 2018 S. G. Gattani and R. S. Moon. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author R. S. Moon, School of Pharmacy, Swami Ramanand Teerth Marathwada University, Vishnupuri, Nanded, Maharashtra, 431606, India. E-mail: rajmun @ rediffmail.com Formulation and Evaluation of Fast Dissolving Tablet Containing Vilazodone Nanocrystals for Solubility and Dissolution Enhancement Using Soluplus: In vitro-In vivo Study


INTRODUCTION
Therapeutic efficacy depends upon bioavailability, solubility, and release of drug molecules from the formulation.The aqueous solubility of therapeutically active substance governs dissolution, absorption and has an essential role in bioavailability of oral drug formulations (Patel et al., 2011).About more than 40% of drug molecules recognized during combinatorial selection programs are poorly water soluble.Low aqueous solubility is main trouble in formulation development, clinical testing and during screening new compounds for therapeutic activity (Lipinski, 2002).The low water solubility of many active pharmaceutical ingredients (APIs) excludes their use in certain clinical applications in spite of their efficacy.However, small particle formation technologies for these APIs may increase their effectiveness for oral, injectable and inhalation delivery by increasing surface area, dissolution rates and overall bioavailability (Vaughn et al., 2004).Systematic formulation approaches may make poorly soluble drugs bioavailable.Nanoparticle engineering technologies in formulation development of low water-soluble drugs is a promising strategy to enhance dissolution rates and thus bioavailability (Patel et al., 2011).Vilazodone is an antidepressant agent with poor aqueous solubility use as an alternative for patients who cannot tolerate treatment with other antidepressants.

Materials
Vilazodone was gifted by Clearsynth labs Pvt Ltd Mumbai, Soluplus a graft copolymer was a gift sample from BASF chemicals, Mumbai, PVP K-30, and microcrystalline cellulose was from Himedia Lab.Pvt. Ltd., Mumbai, all other materials and chemicals were procure from S D Fine chemicals Mumbai.

Preparation of nanoparticles
Nanoparticles were prepared by evaporative precipitation into aqueous solution method.Vilazodone was dissolved in dimethyl sulfoxide with warm at 70-80°C under constant stirring at 1000 rpm.The prepared drug solution was injected dropwise by using syringe into water containing soluplus with stirring on a magnetic stirrer at 1000 rpm, particles precipitated and milk like suspension formed in which added solution of PVP K-30.Resultant was then filtered and dried.Nanoparticles were ground in mortar and pestle and pass through sieve no 170 to obtain desired uniform size.The Nanoparticles of the drug with polymer was denoted with symbol VLZNP.

Ratio optimization (drug: polymer) by solubility study
The solubility of VLZNP determined in distilled water.The solubility of NPs was determined by taking an excess amount of NPs and adding them to 10 ml of Millipore water and kept at equilibrium on a magnetic stirrer for a period of 48 hours (REMI Instruments, Mumbai) at room temperature.The resultant was filtered through 0.45 μm nylon syringe filter and analyzed by UV-Visible spectrophotometer (Shimadzu Corporation, Japan) at 224 nm wavelength.Ratio (drug: carrier) with higher solubility was said to be optimized and processed further.

Drug entrapment efficiency of NPs
The Vilazodone percentage entrapped within nanoparticles determined by measuring the concentration of free Vilazodone in the dispersion medium.
where M initial drug is the mass of initial drug used for the assay, M free drug is the mass of free drug detected in the supernatant after centrifugation of the aqueous dispersion.

In-vitro dissolution test for VLZ nanoparticles
Dissolution of optimized ratio performed using USP type II dissolution apparatus (Electrolab) in 900 ml of 0.1 N HCl.Powder containing 40 mg (or equivalent) of Vilazodone added in medium maintaining temperature 37 ± 0.5°C and 50 rpm paddle speed.About 5 ml of aliquot were withdrawn at 2.5, 5, 10, 15, 20, 25 and 30 minute and replaced with the same quantity of prewarmed fresh dissolution medium.Aliquots filtered through 0.45 μm nylon syringe filter and analyzed spectrophotometrically at a wavelength of 224 nm (Medina et al., 2013;Javadzadeh et al., 2007).

Characterization of nanoparticles
Nanoparticles which show better results in solubility and in vitro dissolution were further characterized to access interaction if any between drug and polymer during a month.

Fourier-transform infrared spectroscopy
Spectra of drug Vilazodone alone and its Nanoparticles carried out to check compatibility of the drug with polymers.The nanoparticle of the drug with the polymer (VLZNPs) triturated with potassium bromide IR grade.The dispersion was scanned using FT-IR Spectrophotometer (Shimadzu, Japan).The spectra of nanoparticles compared with the Spectra of vilazodone.

Differential scanning calorimeters (DSC)
DSC thermograms of drug Vilazodone and its nanoparticles obtained (Diamond DSC, Perkin Elmer) by heating at a rate of 10°C/min from temperature 0-300°C.

X-ray diffraction studies (XRD)
XRD study of drug Vilazodone and Nanoparticles access the changes in crystallinity of drug alone and when combined with the polymer.XRD patterns recorded using (Bruker, D8) with Cu-kα radiation.The scanning angle ranged from 20° to 80° of 2θ.

Scanning electron microscopy (SEM)
The surface morphology of nanoparticle formulation was examined by Scanning Electron Microscopy with Gold ion coating for 5-6 min.

Particle size
Particle size was studied by Dynamic Light Scattering method (Hackley and Clogston, 2007).

Preparation of fast dissolving tablet
The ratio of NPs which shows best results in solubility and powder dissolution was selected for preparation of fast dissolving tablet, table 2. The tablets were compressed by direct compression method using 10 mm punch on rotary tablet Minipress (Karnavati Pvt.Ltd, Gujarat).Pre and post compression evaluation of tablet Precompression and Post compression evaluation fast dissolving tablet were performed according to USP 30 NF 25 (Rockvillle, 2007).

In vitro dissolution test for fast dissolving tablet
In vitro dissolution test was performed according to USP apparatus Type II (Electrolab).Tablet containing 40 mg (or equivalent) of Vilazodone was added in dissolution media 900 ml gastric fluid maintaining the temperature at 37 ± 0.5°C and rotation speed of paddle at 50 rpm.About 5 ml of the aliquot were withdrawn at 2. 5,5,10,15,20,25,30 and 35 minute and replaced with 5 ml of fresh prewarmed gastric fluid, filtered through 0.45 μm nylon syringe filter and analyzed at 224 nm wavelength.

Stability studies
Optimized tablet formulation containing nanoparticles subjected to stress conditions of 40°C/75% RH for six months in stability chamber (Photostability chamber S R Lab Instruments Pvt. Ltd.Mumbai).

In vivo antidepressant activity evaluation
The study was conducted in compliance the protocol approved by the institutional ethical committee.Immobility time in mice evaluated as per the method described by (Porsolt et al., 1977).Mice were forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm) containing 19 cm of water (depth) at 25 ± 1°C; and the immobility time recorded for 6 minutes.Individual mouse considered to be immobile when it ceased struggling and remained floating motionless in the water, showing only those movements essential to keep its head above water.An increase in immobility time was considered an indication of depressive behavior.

Drug: polymer ratio optimization
NPs of the drug with polymer were analyzed for solubility study and results are shown in table 3. Solubility study shows that soluplus have very good solubility enhancing property may because of its good surfactant property (Pandey et al., 2012;Hardung et al., 2010).From solubility studies, it is found that up to certain ratio i.e. 1:2.5 solubility goes increases constantly and further increase in polymer concentration leads to decrease in solubility.Prepared nanoparticle samples showed increased solubility in water more than seven-fold higher (9.36 µg/ml) than pure Vilazodone (1.48 µg/ml).This optimized ratio was then confirmed with powder dissolution and further used for formulation development.The increase solubility of of Vilazodone from prepared nanoparticles may due to the reduction in particle size.

Drug entrapment efficiency of Nanoparticles
Percentage entrapment efficiency of NP4 was found to be maximum that is 95.32%.

In vitro dissolution studies of nanoparticles
VLZNP powder released 82.36% of the drug in solution compared to a pure drug which released only 53.75%.From the results fig. 1 it can be concluded that % drug release of VLZNP is faster than pure VLZ and hence nanoparticles used for the preparation of tablet formulation.

Fourier transformed infrared spectra
From the spectra of vilazodone and its nanoparticles, it can be concluded that principle peaks of the drug remained unchanged in soluplus treated nanoparticles demonstrating the compatibility between drug and polymer.

Differential scanning calorimetry
DSC analysis performed to observe the thermal behavior of components used in Nanoparticles formulation.The DSC thermograms of the VLZ and VLZNP fig. 4 and fig. 5 showed a sharp characteristic endothermic peak at 276.62°C and 277.31°C respectively indicating crystalline nature of materials and there is no appearance or disappearance of one or more peak.

X-ray powder diffraction (XRD)
Diffractogram of Vilazodone showed numerous sharp peaks between 24.56 to 28.85 at 2θ and VLZNP showed the presence of less intense peaks between 24.76 to 20.57 at 2θ.The intensity of the peaks reduces indicating that soluplus interferes with the Vilazodone by forming H-bonding and thus reduces the crystallinity of the drug and indicates amorphization of the drug.

Scanning electron microscopy (SEM)
Pure drug particles are tabular while optimized NPs were found to be irregular in shape and size below 1000 nm.The SEM images (fig.8) clearly indicate a complex form of Vilazodone in Soluplus.

Particle size
The Dynamic Light Scattering measurements as depicted in fig.9, show 90% of particles falls below 100 nm and 10% of particle found coarser.

Pre and post-compression evaluation of fast dissolving tablet
All the pre and post compression evaluation parameters are found to be within the limit as per USP 30 NF25 results depicted in table 4 and 5.

In vitro dissolution of VLZNPs fast dissolving tablet
Release of VLZNPs tablet formulation F 2 (89.386%) exhibited improved dissolution behavior than formulation F 1 (86.74%), F 3 (84.581)and VLZ (51.65%) at the end of 30 min, this may due to decreased particle size, increased surface area and micellar effect of soluplus (Gattani et al., 2018).Results of the in-vitro drug release depicted in fig.10.

Stability study of vilazodone nanoparticles tablet
Optimized formulation (F 2 ) subjected to stability conditions (40°C/75% RH) and parameters such as color, odor, assay, and release were studied during 180 days of exposure.No significant change was observed in the stability parameters during exposure and formulation said to be stable and not much affected by stress conditions.

In vivo antidepressant activity evaluation
Experimental data revealed that mice treated with VLZNP antidepressant agent attenuate significantly (P < 0.001) immobility time compared to VLZ and vehicle-treated group (Table 7 & fig 11).From the data, it seems that VLZ and VLZNP elicited significant antidepressant-like activity.This improvement in the effect of VLZNP may govern by the decreased particle size and increased surface area and also may due to the micellar action of Soluplus.Thus raised solubility and the in vitro dissolution are supported by in vivo data and also kinetics.

CONCLUSION
Vilazodone was successfully formulated in nanoparticles with desired characteristics of size, shape, with reasonable stability and high entrapment efficiency and significantly greater solubility with faster drug dissolution compared to pure drug.The characterization parameters DSC, XRD, IR, SEM, and particle size confirm that the particles are crystalline in nature with irregular shape and uniform size i.e. below 100 nm and compatible with other excipients.Dissolution studies showed the maximum release of optimized Vilazodone nanoparticles (NP4) and optimized Vilazodone tablet formulation (F 2 ).This may be due to the increased surface area due to decreased particle size and may due to micelle action of Soluplus.Also from in vivo study, it seems that VLZNP elicited significant antidepressant-like activity as compared to VLZ.Thus in vitro data supported by in vivo results.Evaporative precipitation into aqueous solution thus may be a useful approach to produce nanoparticles of poorly soluble drugs.

Table 1 :
Formulation design of nanoparticles.

Table 3 :
Solubility of pure VLZ and VLZ nanoparticles.

Table 6 :
Stability data of VLZNPs tablet formulation F 2 .

Table 7 :
Effects of the vehicle, or VLZ and VLZNP on immobility time in mice.