FORMULATION AND EVALUATION OF DOLUTEGRAVIR SODIUM SOLID DISPERSIONS AND FAST DISSOLVING TABLETS USING POLOXAMER-188 AND JACK FRUIT SEED STARCH AS EXCIPIENTS

Objective: The current work mainly focuses on solubility enhancement of dolutegravir which is a biopharmaceutical classification system Class-II drug using jack fruit seed starch (JFS2) as excipient which improves the drug release. Materials and Methods: Starches were extracted using aqueous and alkali methods (sodium hydroxide at 0.1%, 0.25%, and 0.5% concentrations) from jack fruit seed powder. These starches were evaluated for phytochemical and physicochemical parameters. Fast dissolving tablets were prepared using dolutegravir sodium solid dispersion, JFS2, and croscarmellose sodium (CCS) in various concentrations using wet granulation technique. Various preand post-compression parameters were evaluated along with in vitro drug release studies; characterization studies such as Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), scanning electron microscopy, X-ray diffraction (XRD), and stability studies. Results: Phytochemical tests revealed the presence of only starch in all extracts. Starch prepared from 0.1% sodium hydroxide (JFS2) showed best physicochemical properties. From in vitro dissolution studies, it was observed that solid dispersion formulation DF3 containing dolutegravir sodium and poloxamer-188 in 1:1.5 ratios showed a better dissolution rate. From in vitro dissolution studies, tablet formulations DFT6 and DFT9 containing 12.5% w/w of JFS2 and 12.5% w/w of CCS showed enhanced dissolution rate compared with other formulations. FTIR and DSC studies revealed that there were 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 tablets prepared using jack fruit starch seed starch revealed the superdisintegrant property of starch.


INTRODUCTION
Solubility is an important physicochemical factor affecting the absorption of the drug and its therapeutic effectiveness. Nowadays, scientists are focusing on the development of oral dosage forms of poor aqueous solubility drugs through various solubility enhancing approaches. Among these approaches, preparation of solid dispersions is one of the widely used approaches. The term solid dispersion refers to a group of solid products consisting of at least two different components, a hydrophilic matrix and a hydrophobic drug [1,2].
The present study is aimed to develop dolutegravir sodium fast dissolving tablets using solid dispersion technique to improve the solubility and dissolution rate. Dolutegravir sodium is an antiretroviral agent which mainly acts by inhibiting the enzyme human immunodeficiency virus integrase which is needed for the viral replication process. According to the Biopharmaceutical Classification Scheme, dolutegravir can be considered as Class II drug, i.e., water-insoluble, lipophilic, and highly permeable compound. Therefore, it is possible to improve its bioavailability by increasing apparent solubility in water through solid dispersion technology [3][4][5]. Fast dissolving tablet is a solid dosage form containing a medicinal substance or active ingredient which disintegrates rapidly usually within a matter of seconds when placed on the tongue. It helps in faster drug release and better effect of formulation. Although several synthetic carriers have been developed, natural carrier development is gaining focus in recent days due to their easy availability [6].
A novel solid dispersion formulation of dolutegravir sodium was prepared by fusion method using poloxamer 188 as a carrier. Dolutegravir sodium tablets were prepared by wet granulation method with the best solid dispersion along with jack fruit seed starch (JFS2), employing a neoteric method which could prove the superdisintegrant property of JFS2.

Procurement of materials
Dolutegravir sodium was a gift sample from Dr. Reddy's Lab (Hyderabad, India). Poloxamer 188 was a gift sample from Pellets Pharma Ltd. (Hyderabad, India). Sodium hydroxide, magnesium stearate, and talc were procured form S. D Fine Chem. Ltd. (Mumbai, India). Croscarmellose sodium (CCS) was a gift sample from NATCO Pharma Ltd. (Hyderabad, India) and jackfruit seeds were procured from the local market (Guntur, Andhra Pradesh, India).
Extraction of jackfruit seed starch using alkali method JFS2 was isolated using aqueous and alkali extraction methods [7]. Jack fruit seed flour (5 gm) was added in water, 0.1%, 0.25%, and 0.5% of sodium hydroxide (100ml) and soaked (6 h or 8 h) at room temperature with occasional stirring. The slurry was filtered through 120 mesh stainless sieve. The filtrate solution was kept in a refrigerator for overnight and then removed, and the supernatant was discarded. The crude starch was washed with distilled water for 3 times, and the starch cake was dried at 40 o C for 24 h in tray dryer. The starch was grounded

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into a fine powder using mortar and pestle. The obtained starches were packed in a plastic bag and kept at room temperature until further use.

Phytochemical tests for jack fruit seed powder and extracted starches
The raw jackfruit powder and starch extracts were subjected to phytochemical tests for the identification of carbohydrates, proteins, alkaloids, glycosides, and steroids by various tests [8]. The results are given in Table 1.

Evaluation of physicochemical properties of jack fruit seed powder and extracted starch
Various physicochemical properties such as gelatinization temperature, pH, viscosity, swelling index, and water absorption index were evaluated using suitable methods [9]. All the results are indicated in Table 2.

Total microbial load of isolated JFS2
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 agar medium was prepared and placed in an autoclave for 1 h. To this, bacterial culture like Escherichia coli was added, mixed well and poured into Petri plates and allowed to solidify for 10 min. After solidification, the starch powder was sprinkled and kept in an incubator for 24 h. According to many pharmacopeias, in case of excipients from natural origin, the total aerobic count should not be >1000 cfu/g and total fungal count should not exceed 100 cfu/g.

Acidity
About 1 g of starch was added to 100 ml of ethanol (70%) which was previously neutralized to phenolphthalein solution. This solution was shaken for 1 h, filtered and 50 ml of the filtrate was titrated with 0.1 M sodium hydroxide.

Fluorescence
About 500 mg of starch powder was dissolved in an organic solvent and placed on a glass slide. The slide was examined under ultraviolet (UV) cabinet for the presence of any fluorescent material.
Oxidizing substances To 5.0 g of sample, 10 ml of water and 1 ml of acetic acid were added and stirred until a homogeneous suspension was obtained. 0.5 ml of a freshly prepared saturated solution of potassium iodide was added, mixed and allowed to stand for 5 min.

Sulfated ash
About 1-2 g of the starch was placed in an accurately weighed crucible and ignited until thoroughly charred. Then, it is cooled and the residue was moistened with 1 ml of sulfuric acid. It was heated gently until white fumes are no longer evolved and ignited at 800 o C until black particles have disappeared. The crucible was cooled and few drops of sulfuric acid were added and heated. Then, it was weighed. This procedure was repeated until two successive weighing does not differ by >0.5 mg.

Loss on drying
Loss on drying is widely used to determine the moisture content of a sample, although occasionally it may refer to the loss of any volatile matter from the sample. Not >15% (for all starches except potato starch) and not >20% (for potato starch) of weight loss should be obtained. It was determined by drying 0.2 g of starch in an oven at 105°C.

Test for amylose content
About 100 mg of isolated starch sample was taken, and to it, 1 ml of ethanol and 9 ml of 1 N sodium hydroxide were added and kept aside for overnight. The suspension was thoroughly mixed. The dispersed sample was transferred to a 100 ml volumetric flask and diluted to the mark with distilled water. 5 ml of test starch solution was pipetted into a 100 ml volumetric flask and 1 ml of 1 N glacial acetic acid and 2 ml of iodine solution (0.02 N) were added. The volume was made up to 100 ml with distilled water and the absorbance was measured at 620 nm.

Preparation of dolutegravir sodium solid dispersions by fusion method
Solid dispersions of dolutegravir sodium were prepared using Poloxamer-188 in different ratios by fusion method. Poloxamer-188 was placed in a China dish and heated at 40°C until it gets melted, and then dolutegravir sodium 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 are given in Table 3.

Evaluation of pre-formulation parameters
The prepared solid dispersions were evaluated for various preformulation parameters such as angle of repose, Carr's index, Hausner's ratio, and particle size [10,11]. The results are indicated in Table 4.

Drug content uniformity for solid dispersions
Solid dispersions of dolutegravir sodium equivalent to 50 mg was weighed and transferred into a 100 ml volumetric flask. To this, a small quantity of methanol was added to dissolve. It was shaken occasionally for about 15 min, and the volume was made up to 100 ml by methanol. The solution was filtered using Whatman filter paper. The filtrate was subsequently diluted with 6.8 pH phosphate buffer, and the absorbance was measured at 258 nm using 6.8 pH phosphate buffer as blank.

In vitro dissolution studies
Dissolution studies for all solid dispersions were performed in a calibrated eight station dissolution test apparatus (LABINDIA DS8000) equipped with paddles employing 900 ml of 6.8 pH phosphate buffer as a dissolution medium. The paddles were operated at 50 rpm, and the temperature was maintained at 37±1°C throughout the experiment [12]. The samples were withdrawn at 5, 10, 15, 20, 30, 45, and 60 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 UV spectrophotometer (UV 3000+) at 258 nm. The dissolution profiles were indicated in Table 5 and the parameters in Table 6.

Preparation of dolutegravir sodium tablets
Dolutegravir sodium tablets were prepared from the optimized solid dispersions using wet granulation technique. The drug concentration was maintained constant, while jackfruit seed starch and CCS concentrations were increased. The raw materials were individually weighed, passed through sieve 80 and blended for 15 min using double cone blender. The powder mixture of lactose, half amount of jackfruit starch was then converted into damp mass using sucrose solution. The damp mass was passed through sieve 20 to obtain granules and they were kept in tray dryer for drying at 60°C for 1 h. The optimized solid dispersion was added to lactose granules and blended for 15 min using double cone blender. Flavoring agent was added to powdered mass and blended with 1% talc and magnesium stearate and compressed as    tablets using CLIT 10 station mini press. The compositions of various tablet formulations are given in Table 7.

Evaluation of post-compression parameters
The compressed tablets were further evaluated for post-compression parameters such as weight uniformity, hardness, friability, wetting time, dispersion test, and drug content [13]. The results are given in Table 8.

Drug content uniformity
Fast dissolving tablets of dolutegravir sodium from a batch were taken at random and were crushed to a fine powder. The powdered material was transferred into a 100 ml volumetric flask and few ml of methanol was added to it. It was shaken occasionally for about 30 min and the volume was made up to 100 ml by adding methanol. The resulting solution was set aside for few minutes and the supernatant solution was collected, filtered by Whatman filter paper. Then, the filtrate was subsequently diluted with phosphate buffer pH 6.8 and the absorbance was measured at 258 nm.

In vitro dissolution studies of dolutegravir sodium fast dissolving tablets
The dissolution test was carried out in United States Pharmacopoeia (USP) apparatus Type II (paddle) with 900ml of 6.8 pH phosphate buffer as a dissolution medium. The samples were drawn at 5, 10, 15, 20, 30, 45, and 60 min. The fresh volume of the medium was replaced with the withdrawn volume to maintain the sink conditions and constant volume throughout the experiment. Samples withdrawn were suitably diluted with same dissolution medium, and the amount of drug dissolved was estimated by ELICO SL UV -210 at 258 nm and subsequently analyzed for the cumulative percentage of drug released. The dissolution studies on each formulation were conducted in triplicate. The drug release profiles for all the formulations are given in Tables 9-11.

Reverse-phase high-performance liquid chromatography (RP-HPLC) studies
The dissolution studies for the optimized formulations were further analyzed by RP-HPLC method to achieve the concurrency [14]. The dissolution test was carried out in USP Apparatus Type II (paddle) with 900 ml of 6.8 pH phosphate buffer as the dissolution medium. The samples were drawn at 5, 10, 15, 20, 30, 45, and 60 min. The fresh volume of the medium was replaced with the withdrawn volume to maintain the sink conditions and constant volume throughout the experiment. Samples withdrawn were suitably diluted with the same dissolution medium, and the amount of drug dissolved was estimated by RP-HPLC. 40 µL of the respective concentration solution is injected to HPLC using a syringe. The sample was run for 10 min. The retention time and peak area were noted.

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The runs were continued for all concentrations. The dissolution profiles were drawn using method as described earlier. Peak area versus time chromatogram and data are given in Table 12 and shown in Fig. 1. The comparative drug release profile of optimized formulation DFT6 using RP-HPLC and UV spectrophotometric analysis is given in Table 13.

Statistical analysis
The results obtained were statistically evaluated. As the procedures performed and the results obtained were in triplicates, the mean along with their standard deviations was calculated for drug content, and standard error of mean was calculated for drug dissolution profiles.

Characterization studies
Based on the dissolution studies, the optimized formulations were selected, and Fourier transfer infrared (FTIR), and differential scanning calorimetry (DSC) studies were performed 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 on JFS2, dolutegravir sodium, poloxamer-188, CCS, optimized solid dispersion, and fast dissolving tablet formulations to know surface characteristics. The results are shown in Figs. 2-5.

Accelerated stability studies
Accelerated stability studies were carried out on optimized formulations as per International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines. After the stability studies, the formulations were evaluated for physical parameters, drug content, and drug release studies. The results are indicated in Tables 14-16.

Extraction of starch from jack fruit seeds
The starches extracted from jack fruit seeds were crisp, slightly granular, free-flowing and stable in nature.

Phytochemical screening of jack fruit seed flour and starch extracts
The raw jack fruit seed powder and starches extracted were screened for the presence of various phytochemical constituents.

Evaluation of physicochemical properties of jack fruit seed powder and extracted starch
All the parameters evaluated for jack fruit seed powder and extracted starches were within specified Indian Pharmacopoeial limits. Of all the starches, JFS2 with high swelling and water absorption index was selected for preparation of fast dissolving tablets.

Preparation of dolutegravir sodium solid dispersions by fusion method
Solid dispersions of dolutegravir sodium were prepared using Poloxamer-188 in different ratios by fusion method. The composition of dolutegravir sodium solid dispersions prepared by the fusion method is given in Table 3.

Evaluation of pre-formulation parameters
Various pre-formulation parameters of dolutegravir sodium solid dispersions were evaluated. The obtained results are indicated in Table 4.

In vitro dissolution studies of dolutegravir sodium solid dispersions
Formulation DF3, prepared in 1:3 ratios of dolutegravir sodium and Poloxamer-188 showed maximum drug release proving that solid dispersion technique enhances drug release as suggested by earlier studies [15]. The fusion technique used for the formulation of solid dispersions was also proved to be most advantageous as suggested earlier [16]. Employment of hydrophilic polymer was proved to be effective in dissolution enhancement [17]. The dissolution profiles of dolutegravir sodium solid dispersions are given in Table 5.

Formulation of dolutegravir sodium fast dissolving tablets
Dolutegravir sodium fast dissolving tablets with various concentrations of JFS2 and CCS were prepared by wet granulation technique. Formulations DFT2 to DFT7 were prepared using 2.5-15% of JFS2. Formulations DFT8 to DFT10 were prepared using 10-15% of CCS. Formulation DFT1 does not contain any superdisintegrant. The compositions are given in Table 7.

Evaluation of post-compression parameters
The wet granulation process was found to be suitable for compressing prepared granules as fast dissolving tablets [18,19]. All the batches of tablets were compressed under identical conditions to minimize processing variables. Then, the compressed tablets were further evaluated for post-compression parameters. The results are given in Table 8. Weight uniformity, hardness, and friability loss of all tablet formulations were within the specified limits. Thus, all the batches of tablet formulations were found to be stable and suitable for further studies.

In vitro dissolution studies of dolutegravir sodium fast dissolving tablets
Dissolution studies were carried on all fast dissolving tablet formulations using USP paddle method (Apparatus II) with phosphate buffer pH 6.8 as dissolution medium by maintaining the bath temperature at 37±1°C and the paddles were operated at 50 rpm. The dissolution profiles of all the fast dissolving tablets are given in Tables 9 and 10.
It was observed that the type of starch as superdisintegrant and its proportion in the formulation have greatly influenced the dissolution parameters of various formulations. Formulation DFT6 containing 12.5% w/w of JFS2 as superdisintegrant exhibited similar dissolution profile with that of the formulation DFT9 prepared by 12.5% w/w CCS. Several studies have been conducted earlier indicating the effect of superdisintegrants over solubility enhancement [20]. They suggest the usage of a mixture of superdisintegrants rather than single. Solid dispersions were already proved to be effective in dissolution rate enhancement [21]. Along with this, if starch was also incorporated, it could increase the dissolution rate more. Natural derivatives are nowadays playing an important role in solubility enhancement [22]. Recent studies suggest the application of natural starches as superdisintegrants. They also prove the equal efficacy of natural starches and already established superdisintegrants.

RP-HPLC studies
The dissolution studies for the optimized formulations were further analyzed by RP-HPLC method. The dissolution profiles obtained from RP-HPLC technique showed almost similar dissolution profile as that of UV spectrophotometric analysis which indicated the accuracy of data.

Characterization studies
Based on the dissolution studies performed on all the formulations, the optimized formulations were selected, and the following studies were done.

Accelerated stability studies of dolutegravir sodium fast dissolving tablets
The fast dissolving tablets DFT6 and DFT9 containing dolutegravir sodium which showed good in vitro performance were subjected to accelerated stability studies. These studies were carried out by investigating the effect of temperature on the physical properties and drug release from fast dissolving tablets. The results of these studies are given in Tables 14-16. The results thus indicated that there were no visible and physical changes observed in the frequency-discrimination thresholds (FDTs) even 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 starch JFS2 extracted from jack fruit seeds with 0.1% sodium hydroxide is found to be the best and is used as superdisintegrant for preparation of fast dissolving tablets. Dolutegravir sodium solid dispersions were prepared using various concentrations of JFS2 and poloxamer-188 and were subjected to in vitro dissolution studies. From these studies, it was observed that the proportion of starch as superdisintegrant has influenced the dissolution parameters of various formulations. Dolutegravir sodium fast dissolving tablets were prepared using various concentrations of JFS2 and CCS and were subjected to in vitro dissolution studies. From these studies, it was observed that the   proportion of starch as superdisintegrant has influenced the dissolution parameters of various formulations. Similar dissolution profiles were observed for formulations, DFT6 containing 12.5% w/w of JFS2 and DFT9 containing 12.5%w/w of CCS as superdisintegrants. The possible mechanism of superdisintegrant effect of these starches might be the rapid uptake of water, followed by swelling which causes the elevation of hydrostatic pressure in a tablet that leads to faster disintegration of tablets. The optimized formulations when subjected to FTIR and DSC analysis showed no drug-excipient interactions. Similarly, XRD studies were conducted to know the crystalline and amorphous nature of the samples. The optimized formulations DFT6 and DFT9 were also subjected to accelerated stability studies, which revealed that there were no significant changes in physical parameters and drug content even after the stability studies at various storage conditions which indicated that these formulations were stable.