International Journal of Pharma and Bio Sciences

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1 Original Research Article Pharmaceutics International Journal of Pharma and Bio Sciences ISSN PREPARATION AND EVALUATION OF SELF EMULSIFYING DRUG DELIVERY SYSTEM OF ROSUVASTATIN CALCIUM AMIT VISHWAKARMA, MS NIMISHA*, RICHA SRIVASTAVA AND DIPTI SRIVASTAVA Amity Institute of Pharmacy, Amity University Uttar Pradesh Lucknow Campus. ABSTRACT The objective of present research work was to develop a self emulsifying drug delivery system of Rosuvastatin Calcium for increasing its solubility, and oral bioavailability. Solubility of Rosuvastatin Calcium was determined in various vehicles. Oil, Surfactant and Cosurfactant were selected based on the solubility and HLB (Hydrophilic lipophilic balance) value.to identify the microemulsion existing zone Pseudo-ternary phase diagrams were constructed. Solubility analysis was also done for optimization of formulation. The optimized microemulsion formulation was evaluated for Powder X-Ray Diffraction (XRD), SEM (Scanning electron microscopy) analysis, globule size, zeta potential and phase separation study. Average globule size and zeta potential of the optimized microemulsion formulation were found to be 30 nm, and mv, respectively. The viscosity and conductivity data indicated that the microemulsion was of the o/w type. Solubility of Rosuvastatin Calcium was successfully enhanced upto 82.32%. Percent drug release at the end of 60 mins was found to be %. The solubility of Rosuvastatin Calcium was found to be significantly enhanced after formulating into self emulsifying microemulsion. KEY WORDS: Pseudo ternary phase diagram, HLB, Nanoemulsion. MS NIMISHA Amity Institute of Pharmacy, Amity University Uttar Pradesh Lucknow Campus. *Corresponding author P - 117

2 INTRODUCTION Oral route has been the most preferable route of drug delivery due to the ease of administration and good patient compliance. The most common problem faced by drugs given through this route is due to the undesirable physicochemical properties of drug molecules namely high lipophilicity, low solubility etc 1,2.Most of new drug candidates show low solubility in water, which leads to poor oral bioavailability, high intra-and inter-subject variability and lack of dose proportionality. Thus, poor bioavailability remains a major hurdle in the way of drug development process and thus enhancement of bioavailability of poorly water soluble drugs becomes farthest challenge for a keen pharmaceutical scientist 3-5. Various approaches are used to improve the dissolution rate of the drug to enhance its solubility. In recent years, the focus has been on improving oral bioavailability of poorly water soluble drugs by increasing their solubility in lipids 6, 7. Among them, self micro emulsify drug delivery systems (SMEDDS) have shown reliable action for enhancing bioavailability of poorly soluble compounds.generally, SMEDDS are prepared in a liquid dosage form that can be administered in soft gelatin capsules, which have some disadvantages particularly in the manufacturing process and incompatibility problems with the shells of soft gelatin 8, 9. Many techniques are offered to convert conventional liquid SMEDDS to solid such as adsorptions to solid carriers, spray drying, spray cooling, melt extrusion, nanoparticles technology, supercritical fluid based methods, etc. But among these the adsorption technique is simple and just involves addition of liquid formulation onto carriers by mixing in a blender. The resulting powder may then be filled directly into capsules or, mixed with suitable excipients before compression into tablets. An important advantage of the adsorption technique is good content uniformity Rosuvastatin Calcium an HMG-CoA reductase inhibitor, a drug belongs to class of statins, used in combination with exercise, diet, and weight-loss to treat high cholesterol and related conditions, and to prevent cardiovascular disease was selected. It increases highdensity lipoprotein (HDL) ("good") cholesterol levels. It is used to slow atherosclerosis (narrowing of the arteries) in patients with high blood cholesterol levels. It is used in certain patients to reduce the risk of heart attack or stroke along with an appropriate diet. Rosuvastatin Calcium being unstable to acidic, oxidative and photolytic condition also having poor water solubility and low permeation results in a very low bioavailability and short shelf-life. Present research work concentrates on formulating Self- micro emulsifying drug delivery system (SMEDDS) of Rosuvastatin to enhance solubility and dissolution properties. Liquid SMEDDS possesses disadvantages like storage, portability and stability. Present research work aims to solidify SMEDDS by various methods using specific excipient which impart stability to drug during shelf-life as this technique offers an interesting and potentially powerful alternative carrier system for drug delivery due to its high solubilization capacity, transparency, thermodynamic stability, ease of preparation and high diffusion and absorption rates as compared to other oral dosage forms MATERIALS AND METHODS Materials Rosuvastatin Calcium was obtained as a gift sample by Zydus cadila pharmaceuticals Ltd, Ahmedabad. Oleic Acid, Cremephore RH40, Tween 80, Glycerin and Magnesium Aluminium silicate were obtained from S. D. Fine Chem., Mumbai. All the chemicals used were of analytical reagent grade and were used without further purification. Deionized water was prepared by a Milli-Q purification system from Millipore (Molsheim, France). Preformulation studies Preparation of calibration curve 100µg/ml Stock solution of Rosuvastatin Calcium was prepared in 6.8 ph Phosphate buffer and suitable dilutions were made i.e. 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml, 10µg/ml respectively. The sample was scanned using UV- spectrophotometer at the λ max 243nm. The absorbance of samples of different concentration at λ max 243 nm was measured. The graph was plotted between the absorbance and concentration as per the readings in Table 1. In the selected concentration range the graph obeyed the Beer-Lambert s law. A linear relationship was observed over the range of 0-10µg/ml as depicted in Fig1. P - 118

3 Figure 1 Calibration curve of Rosuvastatin in phosphate buffer ph 6.8 by UV-spectrophotometer. (λ max - 243nm) Thin layer chromatography Pre-coated silica gel-g plate was used for analysis of samples. The solution of Rosuvastatin Calcium was made in methanol. The sample was spotted by using capillary tube on TLC plates by keeping a distance of 2 cm above the base of the plate and was allowed to dry in air. The spot was observed under the UV chamber at the short wavelength of 243 nm, the principal spot in the chromatogram was obtained. The Rf value was calculated as shown in Table 2. Melting point Melting point was determined by digital auto melting point apparatus. Automatically the temperature at which drug starts to melt was shown by digital auto melting point apparatus, which was noted and compared with reported value. The melting point of the drug was found to be 116 C. Partition coefficient It is defined as the ratio of un- ionized drug distributed between the organic and aqueous phase at equilibrium. Po/w = (C oil /C water ) equilibrium Partition coefficient (oil/ water) is a measure of drugs lipophilicity and an indication of its ability to cross cell membranes. Drugs having values of log P much greater than 1 are classified as lipophilic, whereas those with partition coefficient much less than 1 are indicative of a hydrophilic drug Rosuvastatin Calcium used to show log P value of 2.4 against the log value of 2.1 as shown in Table 3. Log P = 2.1 Drug solubility study Excess amount of drug was added to few ml of each excipient placed in microtubes and the mixture was vortexed. The mixture was then heated to 40 C in a water bath to facilitate drug solubilization. The mixture was finally kept at ambient room temperature (25 C) under continuous shaking for 2 days to attain equilibrium. The mixtures were then centrifuged at rpm for min. Aliquots of supernatant were then diluted with alcohol, and the drug content was quantified using a UV spectroscopic method Pseudo-ternary phase diagram Phase diagrams were constructed in the presence of drug to obtain the optimum concentrations of oil, surfactant, and co-surfactant. Apex of the triangle represents each of them. SMEDDS form fine oil water emulsions with only gentle agitation, upon its introduction into aqueous media. The total of surfactant, co-surfactant and oil concentrations is always added to 100% for any mixture. The diagrams constructed were used to optimize the concentration of oil phase, surfactant and co-surfactant in different batches of varied concentration which were prepared and titrated with distilled water till transparency persisted. Ternary phase diagrams were thus constructed as the cosurfactant was increased by 5% for each composition, oil phase concentration was kept constant and the surfactant concentration was adjusted to make a total of 100% [Figure 2] P - 119

4 General representation for phase ternary diagram Int J Pharm Bio Sci 2016 Jan; 7(1): (P) Figure 2 The apex of the triangle depicts oil, surfactant and the co-surfactant, the parallelogram depicts the region of emulsification, and the inner circle depicts the region of Nano-emulsification. Formulation of SMEDDS The SMEDDS were formulated using oleic acid as the lipid component and three different surfactants (Cremophor RH40, Tween 80 and Span 80) and cosurfactants (PEG 400, Glycerin, Propylene Glycol) were used to formulate the SMEDDS. Nine different combinations were prepared and evaluated for their emulsifying property 23. These nine different ratios were obtained by phase ternary diagram and their respective ratios are shown in Table 4. Two batches were concluded for the final formulation after successful screening of excipients. Batch 1- Oleic acid: Cremophor RH40: PEG 400 Batch 2- Oleic acid: Tween 80: Glycerin Preparation of liquid SMEDDS Rosuvastatin calcium was accurately weighed and added to oil, surfactant and co-surfactant. Then the components were mixed by gentle stirring followed by vortex mixing and were heated at 40 o C on a magnetic stirrer, until Rosuvastatin Calcium was perfectly dissolved. The mixture was stored at room temperature until further use. Preparation of solid SMEDDS The liquid SMEDDS prepared previously were adsorbed onto Magnesium Aluminum Silicate by physical mixing in a small mortar and pestle. The resulting solid SMEDDS was a free flowing powder that was subsequently subjected to solid state characterization and dissolution studies. The formula optimized for solid SMEDDS is depicted in Table CHARACTERIZATION OF SMEDDS Self-emulsification and precipitation Study Emulsification time i.e. time required to reach the emulsified and homogenous mixture upon dilution was determined. All the different compositions were categorized on the speed of emulsification, clarity, and apparent stability of the resultant emulsion. It was assessed visually by drop wise addition of the pre concentrate (SMEDDS) into 250 ml of distilled water. This was done in a glass beaker at room temperature, and the contents were gently stirred magnetically at 100 rpm. Precipitation was observed by visual inspection of the resultant emulsion after 24 hours 25. Droplet Size and zeta potential of Nano/Micro Emulsion Measured quantity of liquid SMEDDS and solid SMEDDS were dispersed in 200 ml of distilled water to obtain an emulsion. The droplet size, zeta potential and size was measured using Dynamic Light Scattering Photon Correlation Spectroscopy (Malvern instrument, Made in Australia). The microemulsion samples were taken in disposable zeta cells with a scattering angle of 90 0 and a temperature of Powder X-Ray diffraction (P-XRD) of solid SMEDDS The physical state of Rosuvastatin Calcium in Solid SMEDDS was characterized by X-ray powder scattering (PXRD) measurements using X ray diffractometer (Model D2 Phaser, M/s Bruker, Germany). The measurements were performed at room temperature using monochromatic Cu Kα-radiation at 10mA and at 30kV over a 2Ө range of 5 to 80 with a continuous scanning speed of 5 /min. The analyzed samples were compactly packed in the cavity of an aluminum sample holder using P - 120

5 a glass slide. Powder XRD was performed on sample of pure Rosuvastatin calcium, solid SMEDDS and physical mixture of Rosuvastatin calcium and magnesium aluminum silicate 26. SEM analysis The surface morphology of solid SMEDDS and Rosuvastatin calcium was analyzed in an FEI Quanta 3D FEG Dual Beam Electron Microscope. The samples were fixed on an aluminum stub using a double sided carbon adhesive tape and were made electrically conductive by coating with palladium under vacuum. An accelerating voltage of 5 kv was used to visualize the samples. In vitro drug release study In vitro drug release studies from solid SMEDDS were performed using USP Type II dissolution apparatus with number of paddle rotations set to 50 rpm. The dissolution medium consisted of 900 ml of phosphate buffer ph6.8 maintained at 37±0.5 C. Solid SMEDDS containing 20 mg of Rosuvastatin Calcium was introduced into the dissolution medium. At predetermined time intervals 5ml of aliquot was withdrawn, filtered using 0.45µm syringe filter and an equivalent volume of fresh dissolution medium was immediately added. The amount of drug released was estimated by measuring absorbance at 243 nm using a single beam spectrophotometer. The calibration curve of Rosuvastatin calcium was made in phosphate buffer ph 6.8 and at 243 nm. In vitro drug release study of pure Rosuvastatin Calcium drug was performed under similar condition 27. RESULTS Solubility study The highest solubility of Rosuvastatin Calcium was observed in Tween 80 (59.70 mg/ml) followed by Span 80 and Cremophor RH The solubility of drug in various solvents is shown in Table 6. Determination of emulsification time All the formulations showed a rapid rate of emulsification ranging from secs. The results are presented in Table 7 & 8. The formulations X 9, X10, Y 8, Y9 were found to be stable in self emulsification test these were taken for further studies. Droplet size and zeta potential analysis The mean droplet size obtained from liquid SME mixture of Batch-1 (X9 and X10) was about µm, while the mean droplet size obtained from liquid SME mixture of Batch-2 (Y8 and Y9) was about µm. The zeta potential of the micro emulsion formulated from dispersion of liquid SMEDDS in water was found to be -3.5 and -6.7 mv for X9 and X10; and mv for Y8 and Y9 respectively. (Fig 6-17) The reports for the globule size measurement are presented below: Figure 4 Size distribution by intensity for formulation X 9 P - 121

6 Figure 5 Size distribution by intensity for formulation X 10 Figure 6 Size distribution by intensity for formulation Y 8 P - 122

7 Figure 7 Size distribution by intensity for formulation Y 9 Figure 8 Statistics graph for formulation X 9 P - 123

8 Figure 9 Statistics graph for formulation X 10 Figure 10 Statistics graph for formulation Y 8 P - 124

9 Figure 11 Statistics graph for formulation Y 9 Figure 12 Zeta potential distribution for formulation X 9 P - 125

10 Figure 13 Zeta potential distribution for formulation X 10 Figure 14 Zeta potential distribution for formulation Y8 P - 126

11 Figure 15 Zeta potential distribution for formulation Y9 Powder X ray diffraction studies (PXRD) PXRD of Rosuvastatin Calcium consists of sharp peaks. Magnesium Aluminum Silicate PXRD shows absence of any sharp diffraction patterns. The physical mixture (1:1) of Rosuvastatin Calcium and Magnesium Aluminum Silicate showed some crystalline peaks. The solid SMEDDS did not show significant crystalline peaks. (Fig 16-18) P - 127

12 Figure 16 Powder X-ray diffraction (PXRD) of Rosuvastatin calcium Figure 17 Powder X-ray diffraction (PXRD) of physical mixture of Rosuvastatin calcium and Magnesium aluminum silicate (Carried Out by NRLC-Lucknow Uttar Pradesh) P - 128

13 Figure 18 Powder X-ray diffraction (PXRD) of solid-smedds Scanning electron microscopy Scanning electron microscopy reveals the morphology of Magnesium Aluminum Silicate and solid SMEDDS. Micrographs of Solid SMEDDS shows liquid SMEDDS adsorbed onto the surface of Magnesium aluminum silicate particles. 31 (Figure 19-20) P - 129

14 Figure 19 SEM image of Magnesium aluminum silicate (Carried Out by Birbal Sahni Institute of Palaeobotany Lucknow, Uttar Pradesh) P - 130

15 Figure 20 SEM image of liquid SMEDDS adsorbed onto Magnesium aluminum silicate (Carried Out by Birbal Sahni Institute of Palaeobotany Lucknow, Uttar Pradesh) In vitro drug release study The results of percent drug release of all the batches of solid SMEDDS are given in Table 23. In vitro release profile of all the batches of solid SMEDDS and pure drug Rosuvastatin are given in Figure 21. P - 131

16 Figure 21 Comparative results of percent drug release from pure Rosuvastatin calcium and all successful Batch 1 and Batch 2 solid SMEDDS DISCUSSION Solubility Study Proper selections of lipid excipients are the basic criteria on which lipid based drug delivery is dependant. Self micro emulsifying (SME) performance was evaluated for seven SME mixture and were selected for drug delivery based on its self-micro emulsifying efficiency, drug solubility and drug stability studies. All the materials selected for the study were FDA generally regarded as safe (GRAS) material. Solubility of Rosuvastatin Calcium was determined in different oils and surfactants to choose the excipient showing highest solubility for the drug. The highest solubility of Rosuvastatin Calcium was observed in Tween 80 (59.70 mg/ml) followed by Span 80 and Cremophor RH 40. The rate of emulsification of different formulation was taken as an important factor for the assessment of the efficiency of self emulsification that is the SMEDDS should disperse completely and quickly when subjected to dilution under mild agitation. The formulations were then categorized as clear (transparent or transparent with bluish tinge), nonclear (turbid), stable (no precipitation at the end of 24 hours), or unstable (showing precipitation within 24 hours). Based on the results of solubility study, ternary phase diagrams were constructed. Construction of Ternary phase diagrams helps to choose the proper concentration of excipients, i.e. oil concentration and optimum surfactant/ Co surfactant ratio in the formulation to produce emulsions with good stability. As the formulations X 9, X10, Y 8, Y9 were found to be stable in self emulsification test these were taken for further studies. Droplet Size and Zeta Potential Analysis The droplet size of the resulting microemulsion is important since it determines the rate and extent of drug release and absorption. The drug can diffuse faster from smaller droplets into the aqueous phase, thereby increasing the drug dissolution. Smaller droplet size presents large surface area for drug absorption. Increase in surfactant concentration decreases the droplet size up to a certain extent but thereafter further increase in surfactant concentration results in an increase in droplet size. Increase in surfactant concentration causes enhanced water penetration into oil droplets leading to breakdown of oil droplets and resultant bigger droplets. The droplet size of the microemulsion was measured using dynamic light scattering The surface charge (zeta potential) of the microemulsion formed from SMEDDS is believed to play a role in its bioavailability. Because of the presence of fatty acids in the structure of the excipients used, generally the surface charge of the droplet is negative. Powder X Ray Diffraction Studies (PXRD) PXRD further verified the physical state of the drug in the solid SMEDDS. In figure 16 the presence of sharp peaks is indicative of the presence of Rosuvastatin calcium in a highly crystalline form. Magnesium Aluminum Silicate is in amorphous state as demonstrated by the absence of sharp diffraction patterns. The physical mixture (1:1) of Rosuvastatin calcium and Magnesium Aluminum Silicate showed some crystalline peaks due to the presence of Rosuvastatin calcium in the mixture. In contrast to the physical mixture of Rosuvastatin calcium and Magnesium aluminum silicate, the solid SMEDDS did not show significant crystalline peaks, which further confirms the molecularly dispersed state of Rosuvastatin calcium in the formulation. (Fig 16-18) Scanning Electron Microscopy Scanning electron microscopy reveals the morphology of P - 132

17 solid SMEDDS. Micrographs of Solid SMEDDS (Figure 19-20) shows liquid SMEDDS adsorbed onto the surface of Magnesium Aluminum Silicate particles. Since the formulation process involved facilitating adsorption through physical mixing, partially covered magnesium Aluminum Silicate is also visible in the field of vision. No crystalline structures characteristic of solid Rosuvastatin calcium are found in solid SMEDDS micrographs suggesting that the drug is present in a completely dissolved state in the solid SMEDDS. In vitro Drug Release study For both the Batches, it was observed that about 60 % of the drug was released within 30 minutes. 35 % of the drug was released within first five minutes of the dissolution. However, the total amount of drug released from the Batch-1(X9) solid-smedds was 80 %, while that from Batch-2(X 10 ) solid SMEDDS was 70 % (Table 23, Figure 21). On comparison with in vitro drug release study of pure drug Rosuvastatin calcium, it showed only 21% drug release within five minutes and 60% in one hour. (Figure 22) The initial release of the drug from the formulations was faster that presents the faster drug release in intestinal basic ph Table 1 Preparation of standard curve of Rosuvastatin in phosphate buffer ph 6.8 S.No. Conc.(µg/ml) Absorbance(Avg±SD) ± ± ± ± ± ± ± ± ± ± The results are mean + SD (n=3), SD: Standard Deviation Table 2 Thin layer chromatogram of drug excipient interaction S. No Drug + Excipient Rf Value 1. Drug (Rosuvastatin) Drug + Oleic acid Drug + Cremophor RH Drug + Tween Drug + Glycerine 0.37 Table 3 Partition coefficient of Rosuvastatin Drug Log P Reported Log P Value Rosuvastatin Calcium P - 133

18 Table 4 SMEDDS excipient combination for emulsification property Oil Surfactant Co-surfactant Surfactant: Co- Total (ml) (ml) (ml) surfactant Ratioformulation volume(ml) : : : : : : : : : :1 5 Table 5 Composition of optimized solid SMEDDS Formula Components Liquid SMEDDS Magnesium Aluminum Silicate Amount 1 ml 2 gm Table 6 Solubility of Rosuvastatin calcium in different excipients Excipient Solubility of Rosuvastatin (mg/ml) ±SD Oleic Acid ±0.909 Cremophor RH ±1.625 PEG ±1.357 Tween ±0.893 Span ±0.460 Glycerine ±0.545 Propylene Glycol ±0.852 The results are mean + SD (n=3), SD: Standard Deviation Table 7 Self emulsification and precipitation test of Batch 1 Formulation Self-emulsification Clarity Precipitation Code Time (Sec) X1 91±2 Clear Unstable X2 92±2 Clear Unstable X3 89±5 Clear Unstable X4 84±1 Clear Unstable X5 75±3 non clear X6 72±1 Clear Unstable X7 63±4 Clear Unstable X8 59±1 Clear Unstable X9 55±2 Clear Stable X10 57±1 Clear Stable The results are mean + SD (n=3), SD: Standard Deviation P - 134

19 Table 8 Self emulsification and precipitation test of Batch 2 Formulation Self-emulsification Code Time (Sec) Clarity Precipitation Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10 117±2 103±1 68±3 108±4 98±4 91±1 86±2 79±3 71±1 69±3 Clear unstable Nonclear Clear unstable Clear unstable Nonclear Clear unstable Clear unstable Clear stable Clear stable Clear unstable Table 9: Combined % drug release of successful Batch 1 and Batch 2 solid SMEDDS Time X9 X10 Y8 Y9 (min) CONCLUSION In the present research work, self-microemulsifying (SME) mixtures containing surfactant, cosurfactant and oil were prepared and their property to efficiently emulsify was evaluated. Excipients evaluated for selfmicroemulsification were Tween 80 and Cremophor RH 40 as surfactants, PEG 400 as cosurfactants and oleic acid as oil. All the excipients showed a tendency to form a microemulsion with varying degrees of efficiency. A particular SME mixture comprising of Tween 80, PEG 400, Oleic acid was selected and optimized and can be potentially used for delivering Rosuvastatin calcium, a poorly water soluble drug. When the drug loaded SMEDDS reaches in the GI tract the system take water from the surrounding and spontaneously form oil in water emulsion which disperses into fine droplets. The finer droplets provides a higher surface area for the drug to dissolve or permeate in surrounding medium. Powder X-Ray Diffractometry confirmed the molecularly dispersed state of Rosuvastatin Calcium in the formulation. In vitro drug release studies also showed faster drug release from SMEDDS formulation as compared with plain drug. Thus our research work confirmed that the solid SMEDDS formulation can be potentially used for delivering poorly water soluble drug and can be given in the form of suitable formulation. ACKNOWLEDGEMENT Authors thank Zydus cadila pharmaceuticals Ltd, Ahmedabad (India) for providing the gift sample of Rosuvastatin and Director, Amity Institute of Pharmacy, Amity University, Luck now Campus for providing the necessary facilities for the experimental work. CONFLICT OF INTEREST The authors report no conflict of interest regarding the publication of this paper. P - 135

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