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The main purpose of the present investigation is to enhance the solubility and bioavailability of poorly water-soluble atorvastatin calcium through the self nanoemulsifying drug delivery system (SNEDDS).The components for self-nanoemulsion were determined by solubility studies in different excipients. Sefsol 218, Cremophor RH 40 and Propylene glycol were chosen as oil, surfactant and cosurfactant respectively. The efficient nanoemulsion regions were identified by construction of pseudoternary phase diagrams . Prepared SNEDDS formulations were tested for nanoemulsifying properties including robustness to dilution, visual assessment of self-emulsification, viscosity, drug content, globule size and zeta potential. The SNEDDS formulation F3consist of 20% (w/w) Sefsol 218, 40% (w/w) Cremophor RH40 and 40% (w/w) Propylene glycol of each excipient showed the highest drug content (96.58%) and a small mean droplet size (12.7±0.3 nm) ,was selected as optimized SNEDDS formulation.For in vitro dissolution studies,the ATR SNEDDS was then formulated into soft gelatin capsules.The dissolution rate was significantly increased compared to the marketed ATR (Lipitor) tablet under the same conditions.The optimized formulation ATR soft gelatin capsules were then subjected to stability studies at 30 ± 2°C/65 ± 5% RH for a period of three months.The ATR-loaded SNEDDS was compared with the suspension of commercial tablet by oral administration in wistar rats. The bioavailability of ATR SNEDDS was significantly enhanced compared with that of the tablet (p<0.05).It was concluded that the SNEDDS can be use to improve the oral bioavailability of lip o p h ilic drug ATR. Keywords: Self-nanoemulsifying drug delivery system ( SNEDDS), atorvastatin calcium, ternary phase diagrams, droplet size, dissolution , oral bioavailability.
Improving oral bioavailability of drugs such as solid dosage forms remains a challenge for the formulation scientists due to a number of problems. Poor bioavailability can be due to poor solubility, degradation in GI lumen, poor membrane penetration and presystemic elimination [1,2]. Different approaches have been used for avoiding these problems. One of the most popular methods is lipid based formulation such as oils, surfactant dispersions, self-emulsifying formulations, emulsions, and liposomes . Lipid based formulations can highly improve the delivery of poorly soluble compounds. A typical lipid dosage form mostly contains one or more drugs that are dissolved in a mixture of lipophilic excipients such as triglycerides, partial glycerides, surfactants or co-surfactants . There are a number of potential advantages of self-emulsifying lipid formulations including physicochemical stability, greater oral bioavailability, dose reduction , consistent drug absorption profiles, selective targeting of drug in GIT (Gastrointestinal Tract), control of drug delivery profiles, ability to enhance Cmax and AUC, reduce Tmax, linear AUC-dose relationship, reduced variations due to effect of food, protecting sensitive drug substances, high drug payloads and flexibility of designing liquid or solid dosage forms.Lipid based drug delivery systems, chiefly self-nanoemulsifying drug delivery system (SNEDDS) [5 ,6] due to its ability to improve oral bioavailability of poorly water soluble drugs as it has high solvent capacity, ease of dispersion and forms very fine droplet size, it has gained great attention. Basically ,SNEDDS are isotropic mixtures of oil, surfactant, co-surfactant/co-solvent and drug that form fine oil-in-water nanoemulsion when added to aqueous phases under gentle agitation. There is one limitation for most self-emulsifying systems as they have to be administered in lipid-filled soft or hard-shelled gelatin capsules because of the liquid nature of the product. It is important to avoid interaction between the capsule shell and the emulsion so as to inhibit the hydroscopic contents from dehydrating or entering into the capsule shell. Atorvastatin calcium (ATR) is an Anti-hyper lipidemic agent and it is used in the treatment of obesity. As per its mechanism of action, it is an inhibitor of 3-hydroxy-3- methylglutaryl-coenzyme A reductase (HMG-CoA reductase), an enzyme which catalyzes the conversion of HMG-CoA to mevalonate. Mevalonate is a building block for biosynthesis of cholestrol . It reduces the level of LDLâ€cholesterol, apolipoprotein B, and triglycerides, and increases the HDLâ€cholesterol. So used for the treatment of hyperlipidaemias , such as hypercholesterolaemias, combined (mixed) hyperlipidaemia (type IIa or IIb hyperlipoproteinaemias), hypertriglyceridaemia (type IV), and dysbetalipoproteinaemia (type III). It is very slightly soluble in distilled water, pH 7.4 phosphate buffer, and acetonitrile, while slightly soluble in ethanol, and freely soluble in methanol  . The absolute bioavailability of ATR is approximately 14%, main reasons are thought to be the low solubility of it, presystemic clearance in gastrointestinal mucosa and/or first-pass metabolism in liver [7,10 ] . As determined by Cmax and area under the curve (AUC), rate and extent of its absorption reduce by approximately 25% and 9% respectively due to the presence of the food in GIT.The main objective of this research is to formulate, optimize and evaluate the performance of SNEDDS containing ATR with suitable excipients, in order to enhance its oral bioavailability.
Shake flask method is used to determine Equilibrium solubility of Atorvastatin calcium in various excipients. Briefly, an excess amount of ATR was added to each vial containing 2ml of each excipient, and mixed by vortexing in order to facilitate proper mixing of ATR with the vehicles. Vials were then shaken for 48 h in a Thermostatically controlled shaking water bath at 37 ± 1°C followed by equilibrium for 24 h. In order to separate the undissolved drug , the supersaturated sample was centrifuged at 3000 rpm for 10 min.
The supernatant was then filtered using a membrane filter (0.45 μm, Whatman) and suitably diluted with methanol .The drug concentration was obtained via UV validated method at 246 nm (model 752; Exact Science Apparatus Ltd., Shanghai, China). Pseudoternary phase diagram study Regarding the results of solubility studies of drug and the self emulsification tendency of excipients, we used Sefsol 218 as the oil phase. Cremophor RH 40 was selected as surfactant and Propylene glycol as cosurfactant . The water titration method was used to get the pseudo-ternary phase diagrams consisting of oil, surfactant, cosurfactant and water. The surfactant/cosurfactant ratio used was 1:1,2:1,3:1,1:2 .For each phase diagram, in different glass vials oil (Sefsol 218) and specific Smix ratio were mixed thoroughly in various volume ratios from 1:9 to 9:1.
Sixteen different combinations of oil and Smix (1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3.5, 1:3, 1:2.33, 1:2, 1:1.5, 1:1, 1:0.66, 1:0.43, 1:0.25, and 1:0.11) were blended and titrated with water by dropwise addition under gentle agitation. The suitable ratio of one excipient to another in the SNEDDS formulations was examined and according to the data, pseudoternary diagrams were mapped with Origin 8.
The nanoemulsion area in each phase diagram was plotted and the wider region showed the better self nanoemulsifying efficiency. Preparation of SNEDDS formulations Once the nanoemulsion region was identified, SNEDDS formulations with desired component ratios were prepared (Table 1). Firstly Atorvastatin calcium (10mg) was dissolved into cosurfactant or Smix in a beaker, heated at 37°C in a water-bath or if necessary a vortex mixer is used to facilitate the solubilization. The required weight of oil was added into the beaker and mixed until the drug was perfectly dissolved.
This mixture was then stored at room temperature for further study. Robustness to Dilution The effect of dilution on SNEDDS preconcentrate is determined by the dilution study .Robustness of ATR SNEDDS to dilution was done by diluting it 100 ,250 and 1000 times of distilled water, 0.1 N HCl and Phosphate buffer of pH 6.8 . These diluted nanoemulsions were kept for 24h and observed for any signs of phase separation or drug precipitation . Drug content For determining the ATR content, SNEDDS containing Atorvastatin Calcium was added in volumetric flask (VF) containing Methanol and mixed it well by shaking or inverting the VF two to three times [9,12].
By using a validated UV method at 246 nm after suitable dilution, this solution was analyzed spectrophotometrically for the ATR content (model 752; Exact Science Apparatus Ltd., Shanghai, China). Visual assessment Various compositions were categorized according to the speed of emulsification, clarity, and apparent stability of the obtained emulsion [13,11]. Visual assessment was done by drop wise addition of the preconcentrate (SNEDDS) into 100 ml of distilled water.
After equilibrium, following aspects were observed;self-emulsification time, dispersibility, appearance,then scored according to the five grading systems shown in Table 2[17,16,15]. Determination of droplet size,polydispersity index and zeta-potential For determining the droplet size and zeta-potential,approximately 0.2mL concentration of Atorvastatin SNEDDS was diluted with purified water (20mL) or 0.1M HCl (20mL) and moderately shaken in a volumetric flask at 37°C .
The average droplet size and zeta-potential were determined by dynamic light scattering (DLS) using a photon correlation spectrometer (Zetasizer 3000 HSA, Malvern Ltd, UK).The droplet size ,PDI and zeta-potential of various formulations are shown in Table 4. Viscosity determination The viscosity of the prepared SNEDDS formulations as such without being diluted was measured by Brookfield viscometer (Brookfield DV-III Ultra Rheometer ) using spindle C 16-1 at 25±0.5 -C [21,20] .The results are shown in Table 4. Transmission electron microscopy (TEM) of SNEDDS The morphology of the optimized ATR loaded SNEDDS was observed by TEM (H-7650, Hitachi, Japan).Briefly,nanoemulsion was formed by dilution of SNEDDS with distilled water then ,one drop of diluted sample was deposited on a film-coated copper grid and negatively stained with 2% (w/v) phosphotungstic solution.
After drying the sample was photographed by transmission electron microscopy. In vitro dissolution studies The optimized ATR SNEDDS F3 was filled into soft gelatin capsules and was stored at room temperaturefor 24 h to allow complete solidification of the systems before use  . The in vitro drug release of ATR from the capsules was developed using USP dissolution apparatus II (ZRS-8G , Tianjin,China ). The dissolution medium consisted of 900 mL of freshly prepared pH 6.8 phosphate buffer maintained at 37 ± 0.5° C and the speed of the paddle was set at 100 rpm. Capsules were held to the bottom of the vessel using copper sinkers.
At regular time intervals,5 ml samples were withdrawn and replaced with equal volumes of fresh medium to maintain the volume and sink conditions.Samples were then filtered using a membrane filter (0.45 μm, Whatman) and drug concentration was obtained via UV validated method at 246 nm. The release of drug from SNEDDS formulation was compared with the conventional tablet (Lipitor ® ).All measurements were done in triplicate. In vitro drug diffusion study The diffusion of atorvastatin calcium from the SNEDDS was investigated by a dialysis membrane method .
One end of pretreated cellulose dialysis bag (MWCO 12.000 Da) was sealed firmly with clamp and 0.5 mL of self-nanoemulsifying formulation F3(equivalent to 10 mg ATR) was introduced in it along with 0.5 mL of dialyzing medium (phosphate buffer pH 6.8). The other end of bag was also secured with clamp and was allowed to rotate freely . The bags were incubated in beakers containing 500 ml phosphate buffer (pH 6.8)at 37± 0.5 ° C and shaken at a speed of 100 rpm  .Samples were withdrawn individually at 0.5, 1, 2, 4, 6, 8,10,12and 24 h respectively and replaced with equal volumes of fresh medium at same time . The diffusion of drug from optimized formulation was compared with the pure drug.
The drug content was determined spectrophotometrically at 246 nm. Pharmacokinetic The in vivo study was conducted in two groups consisting of six male albino rats weighing 150-200 g. Animals were purchased from the Animal Center of China Pharmaceutical University (Nanjing, China) and approved by the Ethics Committee of the university. They were housed and handled according to National Institutes of Health guidelines. The rats were starved for 24 h before to the experiment with free access to water ad libitum.
The formulations (optimized SNEDDS, conventional Tablet suspension ) were given orally using oral feeding needle (25 mg/kg body weight). Blood samples(0.5ml) were collected from the retro-orbital vein into heparin-rinsed vials according to a programmed schedule just before dosing (0 h) and after 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24h. The blood samples were immediately centrifuged at 10000 rpm for 10 min and plasma samples were collected and stored at -20°C until drug analysis . Frozen plasma samples were thawed at room temperature just prior to extraction. Briefly, 150μL of internal standard (Indomethacin) was added into 150μL of plasma containing ATR and mixed for1 min.
Then 150 μL of ammonium acetate buffer pH4 was added and vortexed for1 min. The drug was extracted with 2 ml of Di-ethyl ether by mixing vigorously for 3 min. After centrifuging at 4000 rpm for 15min, the organic layer was transferred to a clean tube and evaporated under a stream of nitrogen at 40 °C. The residue was dissolved in 150 μL of methanol and injected into the HPLC system. The chromatographic column used was Kromasil C18 (150mm- 4.6 mm, 5 μm) and the mobile phase was acetonitrile-0.1M ammonium acetate buffer, pH 4.0 (50:50).It was run at a flow rate of 1.0 ml/min.
Eluents were monitored using UV detection at a wavelength of 246 nm. Pharmacokinetic analysis The plasma concentrations versus time profiles were analyzed using Kinetica™ software (version 4.4.1,Thermoelectron corporation, Philadelphia, USA). Data from the plasma concentration-time curve within 24 h after drug intake were employed to estimate the following pharmacokinetic parameters for individual rat in each group, peak plasma concentration (Cmax), the time to reach Cmax (Tmax), area under the plasma concentration versus time curve from zero to last sampling time 24 h (AUC0-24h).
Student's t-test was performed to evaluate the significant differences between the two formulations.Values are reported as mean±S.D. and the data were considered statistically significant at P < 0.05. Stability studies The optimized SNEDDS formulations were filled into soft gelatin capsules kept in glass bottles and subjected to stability studies at 30 ±2-C/65±5% RH for a period of three months . Samples were charged in stability chambers with humidity and temperature control .The SNEDDS was evaluated at 0, 30, 60 and 90 days for drug content,disintegration time and in vitro dissolution profile