USE OF SIMPLEX LATTICE DESIGN IN DEVELOPMENT OF ORAL SELF-NANOEMULSIFYING DRUG DELIVERY SYSTEM CONTAINING ROSUVASTATIN CALCIUM

Objective : The aim of the present work was to enhance the solubility of rosuvastatin calcium by self-nano emulsifying drug delivery system (SNEDDS) using mixtures of oil, cosolvent, surfactant and cosurfactant. Methods: Based on solubility study and emulsification efficiency, Preliminary investigations of various oils, surfactants and cosurfactants were carried out for the selection of the proper SNEDDS ingredients. Pseudo-ternary phase diagrams were constructed to identify the efficient self-emulsification region. A series of SNEDDS formulations were prepared using labrasol: cremophor EL with a combination of peceol: ethyl oleate by using the simplex lattice design. Prepared formulation evaluated for refractive index, turbidimetric, droplet size, zeta potential and polydispersity index, self-emulsification, stability tests, viscosity and in vitro diffusion studies. Results: The best formula for SNEDDS in the current study were: 15% oil (peceol: ethyloleatein 1:1 ratio), 50% Labrasol and 35% Cremophor EL. All the SNEDDS batches globule size was found to be varied from 22.90±1.50 nm to 43.90±1.40 nm. and no significant variations in globule size were observed after 3 mo stability studies. All the batches % transparency was found to be varied from 95.40±1.40% to 99.50±1.10% and drug diffused in 10 min varied from 63.65±1.51% to 93.72±1.46 %. Conclusion: The data suggest the use of rosuvastatin calcium SNEDDS to offer the potential for delivery and it increases the aqueous solubility and bioavailability of the drug. SNEDDS, Rosuvastatin Calcium, Simplex lattice design, Peceol, Ethyl Oleate, Labrasol, Cremophore EL.


INTRODUCTION
Orally available drugs must be a sufficient soluble and permeable through the gastrointestinal tract. Almost two-thirds of the new drug candidates are poorly water-soluble, which is commonly associated with low bioavailability, high intra-and inter-subject variability, and lack of dose suitability. Lipid-based formulations offer the opportunity to enhance the absorption of lipophilic drugs. Being a nanosized, selfnano emulsifying drug delivery system (SNEDDS) offers a strong alternative to the more conventional oral formulations of lipophilic compounds. SNEDDS are isotropic mixtures of natural or synthetic oils, solid or liquid surfactants, one or more hydrophilic solvents and cosolvents: surfactants that have forming fine oil-in-water emulsions upon mild agitation followed by dilution in aqueous media [1,2].
The model drug for the current study had been selected from the biopharmaceutical classification system (BCS) class II. Rosuvastatin calcium is a lipid-lowering drug was an attractive candidate for the current study because it is a lipophilic compound with partition coefficient; log P = 4.81 and low aqueous solubility (0.00936 mg/ml). The current rosuvastatin calcium commercially marketed dosage forms is tablets and these show low (about 20%) and erratic oral bioavailability [3,4]. The aim of the present study is bioavailability enhancement of rosuvastatin calcium and find the optimum formula of rosuvastatin calcium SNEDDS followed by characterization.

Materials and reagents
Rosuvastatin calcium was gifted by Mepro Pharmaceuticals Pvt. Ltd., Surendranagar, Gujarat, India. Peceol, Labrasol, Transcutol P, Labrafil M, Labrafil M, Lauroglycol FCC and Capryol 90 were gifted by Gattefosse India Pvt. Ltd, Mumbai, India. Cremophore EL was gifted from BASF India Ltd., Mumbai, India. Sefsol was gifted from Nikko Chemicals, Japan. Polyethylene glycol 400, Propylene glycol, Tween 80, Tween 20, Span 20, Span 80, Oleic acid, Castor oil, Olive oil, Cotton-seed oil, Sesame oil and Almond oil were purchased from Seva fine chemical ltd, Ahmedabad, Gujarat, India. Methanol AR grade was purchased from SD fine chem Ltd, Mumbai, India. All other materials and chemicals used were of either pharmaceutical or analytical grade.

Solubility study and screening of surfactants, cosurfactant and oil
Screening of surfactants and oil was done by the equilibrium solubility method. An excess quantity of rosuvastatin calcium was added to 2 ml of excipients and mixed in a vial. The mixtures in vials were shaken at 25±1.0 °C for 48 h using a rotary shaker (Remi, Mumbai, India). Then, mixtures were centrifuged at 5000 rpm for 15 min. The supernatant was separated and the drug was extracted in methanol. The drug content was analyzed by using shimadzu 1700 UV-visible spectrophotometer at 244 nm. Several trials were taken with different ratios of surfactants, cosurfactants and oils to select the proper combination of surfactant: cosurfactant: oil. Preliminary selection of 0.5 ml surfactant: cosurfactant: oil (Smix: oil) ratios were prepared and diluted with water by water titration method. From the different trails, ratios which gave clear emulsion on dilution were selected for further study [5,6].

Drug excipient interaction study
Drug excipient interaction study was carried out by differential scanning calorimetric (DSC). DSC thermograms of the rosuvastatin calcium and formulation were derived from a DSC with a thermal analysis performed by an automatic thermal analyzer system (DSC 60, Shimadzu, Japan). The analysis was performed at a rate of 10 °C/min from 50 °C to 250 °C under a nitrogen flow of 20 ml/min [7,8].

Development of pseudo-ternary phase diagram
Pseudo ternary phase diagrams of oil, surfactant: cosurfactant (S: CoS) and water were developed using the water titration method. Aliquot of surfactant: cosurfactant mixture (Smix) mixed with oil at room temperature (25 °C). The ratio of Smix to oil was varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9 (%v/v). Deionized water was added in small increments (≤5% v/v) to the mixture of Smix/oil and stirred in a vortex shaker for 2 min (Remi, Mumbai, India). Concentration of water at which turbidity to transparency and transparency to turbidity transitions occurred was derived from weight measurements. These values were used to determine the boundaries of the nanoemulsion domain corresponding to choose the value of oils and surfactant: cosurfactant mixing ratio. To determine the effect of rosuvastatin calcium on nanoemulsion boundary, phase diagrams were constructed with the drug. Pseudo ternary phase diagrams were plotted using Tri plot version 4.1.2 [9][10][11].

Evaluation of rosuvastatin calcium SNEDDS
Drug Content: Drug was extracted from SNEDDS by dissolving in 25 ml methanol. Then the methanolic extract was separated out and drug content in methanolic extract was analyzed spectrophotometrically UV Visible spectrophotometer (Shimadzu 1700) at 244 nm, against the standard methanolic solution of Rosuvastatin calcium.
Self-Emulsification Time: The emulsification time of SNEDDS was determined by USP-II, dissolution apparatus. Each formulation was added dropwise into 500 ml with purified water at 37˚C and 50 rpm. Emulsification time was assessed visually.
Refractive Index: SNEDDS was added to 250 ml 0.1 N hydrochloric acid and 250 ml purified water at 50 rpm on a magnetic plate at ambient temperature. Then Refractive index of the system was measured by using an Abbe's Refractometer [12,13].
Turbidimetric: SNEDDS was added to 250 ml 0.1 N hydrochloric acid and 250 ml purified water at 50 rpm on a magnetic plate at ambient temperature. Turbidity of the system was measured by measuring % transmittance at 694 nm in the UV-Visible spectrophotometer.
Droplet Size, Zeta Potential and Polydispersity Index (PDI): Droplet size and zeta potential were determined using Particle size analyzer (Zetatrac, Microtrac). It is controlled by Microtrac FLEX Operating Software Particle size analyzer uses a high-frequency AC electric field to oscillate the charged particles. The Brownian motion power spectrum is analyzed with the Modulated Power Spectrum (MPS) technique, a component of the power spectrum resulting from oscillating particles. Samples were diluted to 250 ml with purified water and placed into cuvette to measure particle size, PDI and zeta potential [14,15].
Dilution and Aqueous Phase Composition: Robustness of SNEDDS to the dilution and effect of aqueous phase composition were studied. Optimized formulation was dispersed in 250 ml of distilled water and 0.1 N HCL with gentle stirring. Resulting emulsion was kept at 25±2 °C. Emulsion was evaluated for drug precipitation, phase separation and size over the period of 24 h. Viscosity: Viscosity was measured by using Brookfield viscometer (Middleboro, USA) at 25 °C. Spindle S61 was selected for the measurement of various formulations. Viscosity of SNEDDS was measured at 30 rpm before dilution and after dilution with aqueous phase (250 ml).

In vitro Diffusion Studies:
In vitro diffusion studies were carried out by dialysis technique. In this method, one end of dialysis membrane tubing (12 cm in length) was with thread and diluted SNEDDS was filled in it. Then, another end of the tubing was also secured with thread and it was allowed rotating freely in the dissolution vessel of USP-II, dissolution test apparatus (Electrolab TDT-08L, USP). Dissolution apparatus contained 250 ml pH 6.8 phosphate buffer maintained at 37±0.5 °C and stirred at 50 rpm. Aliquots were collected periodically and replaced with fresh dissolution medium. Aliquots, after filtration through Whatman filter paper (No. 41), were analyzed spectrophotometrically at 244 nm for drug content [16,17].
Stability Study: Chemical and physical stability of rosuvastatin calcium SNEDDS were assessed at 40±2 °C/75±5% RH and 25±3 °C as per ICH guidelines. It was stored in a glass vial and subject to a stability chamber over a period of 3 mo. Samples were withdrawn after 3 mo and assessed for physical appearance, dispersion time, % transmittance, viscosity and drug content.

Accelerated Stability Tests by Centrifugation and Freeze-Thaw Cycle:
Rosuvastatin calcium SNEDDS were diluted with 250 ml aqueous phases (distilled water and 0.1 N HCL) and centrifuged (Remi, Mumbai, India) at 5000 rpm for 30 min. In addition, it was subjected to a freeze-thaw cycle by storing it at-20 °C for 24 h and then for another 24 h at 40 °C. Nanoemulsions were observed visually for phase separation and drug precipitation, whereas their physical stability was assessed by measuring globule size before and after centrifugation and freeze-thaw cycle [18,19].

Optimization of rosuvastatin calcium SNEDDS by using simplex design
A simplex lattice design was used to optimize for SNEDDS. In this design, three factors were evaluated by changing their concentrations simultaneously and keeping their total concentration constant. The simplex lattice design is a three

Drug-excipient interaction study
The DSC results provided both qualitative and quantitative information about the physicochemical state of the drug present in the formulation. The thermogram of rosuvastatin calcium showed a melting endothermic peak at 85.34 °C and a formulation mixture containing rosuvastatin calcium showed a melting endothermic peak at 80.97 ⁰C as shown in fig. 4. The thermogram of the drug does not change after mixing with oil, surfactant and cosurfactant indicates the compatibility of oil, surfactant and co-surfactant with the drug. The peaks in both the thermogram show that there is no significant interaction between drug and excipients [23,24].

Pseudo ternary phase diagram
Pseudo ternary phase diagrams were constructed to identify the self-nano emulsifying regions and optimize the concentration of oil as shown in fig 5. The efficiency of emulsification was good when Labrasol: Cremophor EL concentration was more than 50% in a formulation. It was observed that increasing concentration of surfactants also increased the spontaneity of the self-emulsification region. Therefore, a higher concentration of surfactant higher selfemulsifying region in phase diagrams. The ratio of surfactant: cosurfactant was very effective in a stable and efficient SNEDDS formation. The phase diagrams were constructed at ratio of surfactant: cosurfactant 1:1, 2:1, 3:1 and 4:1. However, the stability of self-emulsifying droplets 1:1, 3:1 and 4:1 was decreased and precipitation after a few hour. So, ratio of 2:1 was chosen in the formulation. To determine the effect of drug addiction on the nanoemulsion boundary, phase diagram was constructed in the presence of the drug. No significant changes were observed in phase diagram regions after drug loading [25,26].     table 3 and table 4 respectively. It suggested that F, as well as P values, are significant. Counter plots as shown in fig 6, it reveals that an inverse relationship exists between mean globule size and % transparency. As the globule size of SNEDDS increases, % transparency decreases. Direct relationship exists between % transparency and % amount of drug diffusion. As the % transparency of formulation increases, the amount of rosuvastatin calcium diffused in 10 min were also increases. In order to obtain both high % transparency, high amount of rosuvastatin calcium diffused in 10 min and smallest possible mean globule size, the appropriate ratio of components was chosen for the optimized formulation, which consisting of oil (15%), surfactant (35%), cosurfactant (50%).

Validation of design
One extra checkpoint was taken and the checkpoint batch was prepared as shown in table 5. The checkpoint batch was evaluated for all three dependent variables. The practically obtained responses of the checkpoint batch were compared with the calculated responses from the simplex equations shown in table 6. Practically, obtained responses are closer to the predicted response. Closeness of the value justifies the validation of design [28,29].

Selection of optimized batch
Batch F2 was selected as an optimized batch in order to obtain high % transparency and higher % diffusion and the smallest mean globule size. The appropriate ratio of components for optimized formulation F2 was, oil (15%), surfactant (35%), cosurfactant (50%).  fig. 7. Generally, an increase of electrostatic repulsive forces between droplets prevents the coalescence of droplets. On the contrary, a decrease of electrostatic repulsive forces will cause phase separation. Rosuvastatin calcium SNEDDS (F2) was diluted with distilled water and resulted in zeta potential was found-8.40±0.02mV. According to the study, positively charged droplets could have better interaction with the mucus of the gastrointestinal tract, because intestinal cell interior carry negative charges with the presence of mucosal fluid. Here, F2 formulation has a positive potential, it was likely to facilitate intestinal absorption of rosuvastatin calcium. [30,31] Effect of Dilution and aqueous phase composition results indicated that SNEDDS can be diluted up to 1,000 fold without any phase separation or drug precipitation and it's remained stable over a 24 h. Aqueous phase composition also did not affect the physical stability of the resulting emulsion. Viscosity data were shown in table 8. It was observed that before dilution the formulation having higher viscosity and after dilutions with water up to 250 ml the emulation viscosity near to the water.

Stability studies of rosuvastatin calcium SNEDDS
No change in physical parameters such as homogeneity and clarity of SNEDDS was observed during stability studies. The stability data of rosuvastatin calcium SNEDDS at stated storage conditions is shown in table 9. Interestingly, it was shown that no decline in rosuvastatin calcium content which was observed at the end of three months indicating that rosuvastatin calcium remained chemically stable in SNEDDS. Furthermore, other parameters such as self nanoemulsion efficiency, % transmittance viscosity and dispersion time remained unchanged at all storage conditions during the entire period of study.

Accelerated stability study by centrifugation and freeze-thaw cycle
The effect of centrifugation and freeze-thaw cycling on emulsion is shown in table 10. Accelerated tests were carried under stress conditions. Optimized SNEDDS (F2) did not exhibit any drug precipitation and phase separation after centrifugation. Similarly, it survived freeze-thaw cycling and it was reconstituted without any phase separation or drug precipitation.