FORMULATION AND EVALUATION OF FEBUXOSTAT FAST DISSOLVING TABLETS USING ENTADA SCANDENS SEED STARCH AND POLOXAMER-188 AS SUPERDISINTEGRANTS

Objective: The present study focused on solubility enhancement of the Biopharmaceutical Classification System (BCS) Class-II drug, Febuxostat using various super disintegrants. Methods: Starches were extracted from Entada scandens seed powder by alkali method i.e., sodium hydroxide at 0.1%, 0.25% and 0.5% concentrations and water. Starches were evaluated for various phytochemical and physicochemical tests. Solid dispersions of Febuxostat were prepared by fusion method using poloxamer-188 alone and by mixing poloxamer-188 with Entada scandens seed starch. Various physical parameters were evaluated for these solid dispersions. Tablets were prepared using Febuxostat solid dispersions and varying concentrations of Entada scandens seed starch by direct compression technique. Pre and post-compression parameters were evaluated along with in vitro drug release studies, characterization studies like Fourier Transform Infra-Red spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD) and stability studies. Results: Phytochemical tests revealed the presence of starch in all extracts. Starch prepared from 0.5% sodium hydroxide (ESS4) showed best physicochemical properties. In vitro dissolution studies revealed that solid dispersion F4 containing Febuxostat and poloxamer-188 in 1:3 ratios showed better drug release. Formulation FE5 containing F4 solid dispersion with 12.5% w/w of ESS4 showed enhanced drug release. FT2 tablets having 12.5% w/w of ESS4 showed better drug release when compared to others. FTIR and DSC studies revealed no major interactions between drug and excipients. XRD studies revealed the nature of formulations. Accelerated stability studies showed that all tablets were stable. Conclusion: The super disintegrant property of Entada scandens seed starch was evaluated.


INTRODUCTION
Solubility is an important physicochemical factor which affects drug absorption and therapeutic effectiveness.Nowadays, scientists are focusing on the development of oral dosage forms of poor aqueous solubility drugs through various solubility enhancing approaches [1].Among these approaches, preparation of solid dispersions is one of the widely used techniques.The term solid dispersion means a group of solid products with at least two different components, i.e., a hydrophilic matrix and a hydrophobic drug [2,3].
Though drug administration can be made in various routes, the oral route is widely preferred due to its ease.But it is difficult in geriatrics and in children to swallow the drug.To hinder such problems, fast dissolving tablets (FDTs) are developed.These tablets when comes in contact with saliva in the mouth, they rapidly disintegrate and releases drug [4].Fast dissolving tablets are widely accepted due to patient compliance, fast action, increased bioavailability and quick disintegration [5].These are prepared by using direct compression technique.Usage of certain super disintegrants like sodium starch glycolate, croscarmellose, polyvinyl pyrrolidine, etc. provides faster tablet disintegration even at low concentrations.They are mixed up with a tablet and some encapsulated formulations which enhance the breakup of tablet and capsules "slugs" into smaller fragments in an aqueous environment which increases the available surface area.This promotes rapid drug release.They promote moisture penetration and dispersion of the tablet matrix [6,7].
Nowadays, natural super disintegrants are preferred over synthetic forms, due to huge availability, economic, non-irritating and non-toxic properties [8].Several gums and mucilages have super disintegrant property.These are useful in enhancing the water solubility of BCS Class-II drugs.These are drugs with low solubility and high permeability properties.Past studies revealed the applications of components from plants like Ocimum tenuiflorum, mucilages of Plantago ovata, Aloe vera and Hibiscus rosasinesis as super disintegrants [9].
In the present study, an attempt was made to extract starch from of Entada scandens seed powder and to use it as super disintegrant for formulating fast-dissolving tablets.Entada scandens belonging to the family Fabaceae grows in evergreen tropical climates of India, Africa, and Australia.Their seeds have a thick and durable seed coat which is brownish in color.It contains a fleshy white cotyledon.These are rich in starch contents [10].Febuxostat which is an anti-urolithiatic agent is selected as a drug candidate for the present study.It mainly acts by non-competitively inhibiting the enzyme xanthine oxidase.This prevents the conversion of hypoxanthine and xanthine to uric acid, thereby preventing the formation of crystals in kidneys.The bioavailability of Febuxostat is approximately 49%.More than 99% of the drug is bound to plasma proteins.It has an approximate elimination half-life of 5-8 h [11].Based on pharmacokinetic and pharmacodynamic parameters, Febuxostat is selected as a drug of choice for the present study.

Materials
Febuxostat was a gift sample from Mylan Laboratories Ltd. (Hyderabad, India).Croscarmellose sodium was a gift sample from NATCO Pharma Ltd. (Hyderabad, India).Poloxamer-188 was a gift sample from Pellets Pharma Ltd (Hyderabad, India).Sodium hydroxide, saccharin sodium, magnesium stearate, and talc were procured from S.D Fine Chem.Ltd. (Mumbai, India).Isopropyl alcohol was obtained from High Pure Fine Chem.(Chennai, India).Entada scandens seeds were procured from the local market (Kurnool, Andhra Pradesh, India).The seeds were identified and authenticated by Dr. K. Ammani, Professor and Head, Department of Botany, Acharya Nagarjuna University, Guntur and voucher specimen (17/01) was preserved in Department of Pharmaceutics, Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Chowdavaram, Guntur, India.

Extraction of starch from Entada scandens seeds
Entada scandens seed starch was isolated using aqueous and alkali extraction methods [12].5g Entada scandens seed flour was added into 100 ml distilled water, 0.1%, 0.25%, and 0.5% sodium hydroxide solutions separately and soaked (6 h and 8 h) at room temperature then stirred constantly.The slurry was filtered through 212 mesh stainless sieve and remaining sediment was washed with distilled water three times.The filtrates were combined and precipitated overnight at 4 °C.The supernatant was discarded and the crude starch was cleaned with distilled water.This step was repeated three times and the starch cake was dried at 40 °C for 24 h in oven dryer.The starch was ground with a mortar and pestle.The starches were packed in a plastic bag and kept at room temperature until further use.

Phytochemical tests for Entada scandens seed powder and extracted starches
The raw Entada scandens seed powder and starches extracted were subjected to various phytochemical tests for identification of carbohydrates, proteins, alkaloids, glycosides, steroids, flavonoids and saponins [13].The results were indicated in table 1.

Evaluation of physicochemical properties of Entada scandens seed powder and extracted starches
Various physicochemical properties like gelatinization temperature, pH, viscosity, swelling index, and water absorption index were evaluated using suitable methods [14].

Total microbial load of isolated Entada scandens starch
The total microbial load is an important parameter which decides the suitability of a substance for use as an excipient in the pharmaceutical dosage form.The starch powders were subjected to dry heat sterilization at 180 °C for 30 min.Then the starches were inoculated on medium and were incubated for 24 h.Then the colonies were counted using a microbial colony counter.The results were indicated in table 2.

Preparation of febuxostat solid dispersions by fusion method
Solid dispersions of Febuxostat were prepared using Poloxamer-188 in different ratios by fusion method.Poloxamer-188 was placed in china dish and heated at 40 °C until it gets melted and then Febuxostat was added to it.After vigorous stirring in normal temperature, the mixture gets solidified.The solid mass was crushed, pulverized and sieved.The granules obtained were stored in a desiccator for further studies.The results were given in table 3.

Preparation of febuxostat solid dispersions by fusion method using Entada scandens seed starch
After performing dissolution studies for Solid dispersions of Febuxostat, the formulation showing best dissolution profile was selected.To that formulation, Entada scandens seed starch (ESS4) was added in increasing concentrations i.e., 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 by keeping the optimized formulation constant.The optimized formulation was placed in a china dish and heated at 40 °C until it gets melted and then ESS4 was added to it.After vigorous stirring in normal temperature, the mixture gets solidified.The solid mass was crushed, pulverized and sieved.The granules obtained were stored in a desiccator for further studies.The results were given in table 4.

Evaluation of physical parameters
The prepared solid dispersions were evaluated for various physical parameters such as the angle of repose, Carr's index, Hausner's ratio, particle size and drug content [11].The results were indicated in table 5.

In vitro dissolution studies
Dissolution studies for all solid dispersions were performed in a calibrated 8 station dissolution test apparatus (LABINDIA DS8000) equipped with paddles employing 900 ml of 0.05M phosphate buffer pH 6.0 as dissolution medium.The paddles were operated at 50 rpm and the temperature was maintained at 37±1° C throughout the experiment [15].The samples were withdrawn at 5, 10, 15, 20 and 30 min and replaced with an equal volume of same dissolution medium to maintain the sink conditions throughout the experiment.
Samples withdrawn were suitably diluted with same dissolution medium and the amount of the drug dissolved was estimated by Lab India double beam U. V spectrophotometer (UV 3000+) at 315 nm.The dissolution profiles were indicated in tables 6 and 7.

Preparation of febuxostat tablets
Febuxostat tablets were prepared by direct compression technique.The drug concentration was maintained constant, while the optimized solid dispersion concentration was increased.The raw materials were individually weighed and were then converted into damp mass by using isopropyl alcohol.The damp mass was passed through sieve no 20 to obtain granules and they were kept for drying.The prepared granules were passed through sieve no.40.The granules were taken into a plastic bag and lubricated with 1% talc, magnesium stearate and half of the starch.Then they were compressed as tablets using CLIT 10 station mini-press.To minimize the processing variables all batches of tablets were compressed, under identical conditions.The compositions of various tablet formulations were given in table 8. To minimize the processing variables, all batches of tablets were compressed under identical conditions.

Evaluation of pre-compression parameters
The prepared granules were evaluated for pre-compression parameters such as the angle of repose, Carr's index and Hausner's ratio [16].The results were given in table 9.

Evaluation of post-compression parameters
The compressed tablets were further evaluated for postcompression parameters such as weight uniformity, hardness, friability, wetting time, dispersion test and drug content [17].The results were given in table 10.

In vitro dissolution studies of Febuxostat tablets
Dissolution studies for tablet formulations were performed in a calibrated 8 station dissolution test apparatus (LAB INDIA DS8000) equipped with paddles (USP apparatus II method) employing 900 ml of 0.05M phosphate buffer pH 6.0 as dissolution medium.The paddles were operated at 50 rpm and the temperature was maintained at 37±1° C throughout the experiment.The samples were withdrawn at 5, 10, 15, 20 and 30 min and replaced with an equal volume of same dissolution medium to maintain the constant volume throughout the experiment.Samples were suitably diluted with same dissolution medium and the amount of drug dissolved was estimated by Lab India double beam U. V spectrophotometer (UV 3000+) at 315 nm.The dissolution profiles were given in table 11.

Characterization studies
Based on dissolution studies, the optimized formulations were subjected to FTIR and DSC studies to observe the drug-polymer interactions.X-Ray Diffraction (XRD) studies were performed to detect the nature of formulations.Scanning electron microscopy (SEM) analysis was performed to know surface characteristics.The results were shown in fig. 1, 2, 3 and 4.

Accelerated stability studies
Accelerated stability studies were carried out on optimized formulations as per International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines.These studies were carried out to investigate the effect of temperature on the physical properties and drug release of the tablets.The results were given in table 12.

Extraction of starch from Entada scandens seeds
The starches extracted from Entada scandens seed powder were crisp, slightly granular, free-flowing and stable in nature.

Phytochemical screening of Entada scandens seed flour and starch extracts
The raw Entada scandens seed powder and starches extracted were screened for the presence of various phytochemical constituents.The results were indicated in table 1.

Preparation of febuxostat solid dispersions by fusion method
Solid dispersions of Febuxostat were prepared using poloxamer-188 as a carrier in different ratios by fusion method.The composition was given in table 3.

Evaluation of physical parameters
Various physical parameters of Febuxostat solid dispersions were evaluated.The obtained results were indicated in table 5.

In vitro dissolution studies of febuxostat solid dispersions
Formulation F4, prepared in 1:3 ratios of Febuxostat and Poloxamer-188 showed maximum drug release proving that solid dispersion technique enhances drug release as suggested by the earlier studies [18,19].The dissolution profiles of Febuxostat solid dispersions were given in table 6.  showed maximum drug release.The fusion technique used for the formulation of solid dispersions was also proved to be most advantageous as suggested earlier [20].The dissolution profiles of Febuxostat solid dispersions were given in table 7.

Preparation of febuxostat tablets
Febuxostat tablets with various concentrations of Entada scandens seed starch (ESS4) and croscarmellose sodium (CCS) were prepared by direct compression technique.Formulations FT1 to FT3 were prepared using 10.0 to 15% of alkali extracted starch (ESS4).Formulations FT4 to FT6 were prepared using 10.0 to 15% of CCS.Whereas the formulation FTP doesn't contain only the solid dispersion FE5.The compositions were given in table 8.

Evaluation of pre-compression parameters
The pre-compression parameter values obtained for various prepared granules were given in table 9.The angle of repose, Carr's index, and Hausner's ratio values for granules were within the range specified.Thus all the prepared granules were found to be stable and suitable for compression of tablets.

Evaluation of post-compression characteristics of febuxostat tablets
The direct compression method was found to be suitable for preparation of fast dissolving tablets.All the batches of tablets were compressed under identical conditions to minimize processing variables.They were evaluated for post-compression parameters such as weight uniformity, hardness, friability, wetting time, dispersion test and drug content.The results were given in table 10.Weight uniformity, hardness and friability loss of tablet formulations were within the specified limits and were found to be stable and suitable for further studies.

In vitro dissolution studies of febuxostat tablets
Dissolution studies were carried on Febuxostat tablets using U. S. P paddle method (apparatus II) with 0.05M phosphate buffer pH 6.0 as dissolution medium by maintaining the bath temperature at 37+1 °C and the paddles were operated at 50 rpm.The dissolution profiles of tablets were given in table 11.It was observed that the proportion of starch as super disintegrant has greatly influenced the dissolution parameters of various formulations.Formulation FT2 containing 12.5% w/w of ESS4 as super disintegrant exhibited closer dissolution profile with that of formulation FT5 prepared by 12.5% w/w of CCS.Several studies have been carried out previously indicating the advantages of super disintegrants in solubility enhancement [21].Those studies suggest the usage of a mixture of super disintegrants rather than single [22].Recent works focus on the natural starches application as super disintegrants [23,24].They have successfully proved the efficacy of natural starches as pharmaceutical excipients.

Accelerated stability studies of Febuxostat fast dissolving tablets
The fast-dissolving tablets FT2 and FT5 containing Febuxostat with FE5 solid dispersion mixed with ESS4 and CCS in 12.5% w/w respectively showed well in vitro dissolution and so was subjected to accelerated stability studies.The results were indicated in table 12.
The results showed that there were no visible and physical changes observed in the FDTs after storage.Weight uniformity, hardness, friability, wetting time, dispersion test and drug content were found to be uniform before and after storage at different conditions.It was also observed that there was no significant change in drug release from the FDTs.Thus the drug release characteristics of FDTs designed were found to be quite stable.

CONCLUSION
The current study shows that the proportion of starch as super disintegrant has greatly influenced the dissolution parameters of various formulations.Similar dissolution profiles were observed for formulations, FT2 containing 12.5%w/w of ESS4 and FT5 containing 12.5%w/w of CCS as super disintegrants.The super disintegrant effect of these starches might be due to the rapid uptake of water, followed by swelling that leads to an increase in hydrostatic pressure of tablet that ultimately disintegrates tablets faster.The optimized formulations showed no drug-excipient interactions when subjected to FTIR and DSC analysis.Similarly, crystallinity was observed using XRD studies.The formulations FT2 and FT5 were also found to be stable even when subjected to accelerated stability studies.
Based on the above studies, it was concluded that Febuxostat fast dissolving tablets prepared by Entada scandens seed starch extracted from 0.5% sodium hydroxide showed rapid drug release.

Physicochemical parameters of Entada scandens seed flour and starch extracts
Physicochemical parameters were evaluated for all the starches.The results were indicated in table 2. The starch ESS4 showed high swelling index and water absorption index which made it suitable for selection of solid dispersions and tablets.

Table 3 : Composition of febuxostat solid dispersions prepared by fusion method
One part is equal to 40 mg, similarly, solid dispersions were prepared by fusion method using Entada scandens seed starch (ESS4) as a carrier in different ratios with a constant amount of optimized formulation (F4).The composition was given in table4. *

Table 5 : Physical parameters of febuxostat solid dispersions
*FP indicates Febuxostat pure drug; n=3, SEM: standard error of mean

Table 11 : Dissolution profiles of febuxostat tablets prepared by direct compression method (FT1-FT6)
indicating the presence of C=C stretching,-C=O stretching,-O-H bending and-N=H wag.FT5, an FDT prepared with Febuxostat, FE5 and 12.5%w/w CCS exhibited peaks at 1638.13 cm -1 , 1413.59 cm -1 and 1059.33 cm -1 indicating the presence of-C=O stretching,-C≡N bending and-C≡N stretching.The detailed spectral elucidations were shown in fig.1.of Febuxostat with ESS4 clearly exhibited uniform dispersion of drug with spherical globular starch grains.The SEM image of Febuxostat with ESS4 and poloxamer-188 showed complete dispersion of drug with both the polymers.The SEM image of Febuxostat with CCS showed uniform dispersion of drug with blunt tubular crystals of CCS.The detailed SEM images were shown in fig. 3.