Vol 3 | Issue 2 | 2013 | 94-101. e-ISSN 2249 – 7706 print-ISSN 2249– 7714

International Journal of

Advanced Pharmaceutics www.ijapjournal.com

DESIGN DEVELOPMENT AND EVALUATION OF NOVEL NANOEMULSION OF SIMVASTATIN Ankith Kumar Reddy B*, Subhashis Debnath, M. Niranjan Babu Department of Pharmaceutics, Seven Hills College of Pharmacy, Tirupati, Andhra Pradesh- 517 561, India. ABSTRACT Simvastatin is a synthetic anti hyperlipidemic drug which will solubilize the low density lipids accumulated in the arteries. Simvastatin is a lipophilic drug thus results in the poor bioavailability after oral administration. Therefore the clinical efficacy of these drugs is sometimes not realized. Therefore nanoemulsion containing Simvastatin was prepared with a view to increase its bioavailability. The pseudo-ternary phase diagrams were developed using the aqueous titration method. Surfactant (Tween 80) and co-surfactant (Ethanol) were mixed(Smix) in different volume ratios (1:1, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1). Isopropyl myristate optimized as an oil phase based on the solubility study. For each phase diagram, oil (olive oil) and specific smix ratios were mixed thoroughly in different volume ratios from 1:7 to 7:1. Different combinations of oil and smix (1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1) were made for the study to delineate the boundaries of the phases precisely formed in the phase diagrams.The formulated nanoemulsions has to be evaluated for drug release, viscosity, surfactant concentration, electro conductivity and TEM analysis. Keywords: Simvastatin, Nanoemulsion, Surfactant, oil, Co-surfactant. INTRODUCTION In drug discovery, about 40% of exciting new molecular entities (NMEs) displays low solubility in water leading to poor bioavailability, high intrasubject/ inter subject variability and lack of dose proportionality. Furthermore, oral delivery of numerous drugs is hindered owing to their high hydrophobicity. Therefore, producing suitable formulations is very important to improve the solubility and bioavailability of such drugs. Formulation and development of poorly water soluble drugs (PWSD) candidates continue to be a challenge to formulation scientists mainly because of the emerging new drug discovery programs. The various options available to overcome the hurdle include micronisation, salt formation, use of microspheres, solid dispersions, co-grinding, complexation, lipid-surfactant based formulations and many others. The lipid based formulation approach has attracted wide attention in order to enhance drug solubilization in the gastrointestinal tract (GIT) and to improve the oral bioavailability [1-5]. Thus in this research work a novel o/w Corresponding Author:- Ankith Kumar Reddy B

nanoemulsion formulation is tried which enhances the oral bioavailability of the poorly water soluble drug terbinafine HCl. The objectives of the present work include development and characterization of o/w nanoemulsion containing terbinafine HCl, improve oral bioavailability by lymphatic transport and reduce hepatic first-pass metabolism and to reduce the dose required to produce same pharmacological effect where by dose related side effects can be reduced [6-9]. MATERIALS AND METHODS Materials Chemicals used in this study were, Simvastatin (Aurobindo Pharma Ltd, Hyderabad), Isopropyl myristate (Hi media laboratories Pvt ltd), Tween 80 (S.D fine chemicals Ltd. Mumbai), Ethanol (Changshu Yangyuan chemicals), All other chemicals and reagents used were of analytical grade. Methods Solubility studies Email:- [email protected]

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Vol 3 | Issue 2 | 2013 | 94-101. The solubility studies have to be done to detect the oil that solubilises maximum amount of drug. The solubility of Simvastatin in different oils was determined by adding an excess amount of drug to 1 ml of selected oils (oleic acid, isopropyl myristate, olive oil, triacetin, castor oil and ground nut oil) in stoppered vials. The vials were kept at 25± 0.50C in isothermal shaker for 72hours to reach equilibrium. The equilibrated samples were removed from the shaker and centrifuged at 3000rpm for 15min. The supernatant was taken and filtered through Whatman filter paper and concentration of Simvastatinwas determined in the oils after dilution using UV-Visible spectrophotometer at 238 nm [11]. Compatibility Study Compatibility studies have to be done all the excipients along with the drug that is used to formulate nano emulsion. The compatibility studies was done by using Bruker FTIR spectrophotometer. Compatibility studies were used for detection of any possible chemical interaction between the drug, oil, surfactant and the cosurfactant. A physical mixture of drug, oil, surfactant and cosurfactant was prepared and mixed with suitable quantity of potassium bromide. About 100 mg of this mixture was compressed to form a transparent pellet using a hydraulic press at 15 tons pressure. It was scanned from 4000 to 400 cm-1. The IR spectrum of the physical mixture was compared with those of pure drug, oil, surfactant and cosurfactant and matching was done to detect any appearance or disappearance of peaks [12-15]. PSEUDO-TERNARY PHASE DIAGRAM STUDY Constructing pseudo-ternary phase diagrams is time consuming, particularly when the aim is to accurately delineate the phase boundary [16-20]. Care was taken to ensure that observations are not made on metastable systems, although the free energy required to form an emulsion is very low, the formation is thermodynamically spontaneous [21]. The relationship between the phase behaviour of a mixture and its composition can be captured with the aid of a phase diagram. Isopropyl myristate (oil), Tween 80 (surfactants), and ethanol (co-surfactant) were selected to study the phase diagrams in detail. Pseudoternary phase diagrams were constructed separately for each smix ratio to identify the o/w nanoemulsion regions [22-24]. The pseudo-ternary phase diagrams were developed using the aqueous titration method. Surfactant (Tween 80) and co-surfactant (Ethanol) were mixed(Smix) in different volume ratios (1:1, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1). Isopropyl myristate optimized as an oil phase based on the solubility study. For each phase diagram, oil (olive oil) and specific smix ratios were mixed thoroughly in different volume ratios from 1:7 to 7:1. Different combinations of oil and smix (1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1) were made for the study to delineate the

boundaries of the phases precisely formed in the phase diagrams [25-27]. Aqueous phase was slowly titrated for each combination of oil and smix separately. 5ml of aqueous phase was added at each interval up to 50ml under magnetic stirring and visually observed for phase clarity and flowabilty. Calculations for other ratios of oil and smix were also done. The physical state was plotted on a pseudo-three-component phase diagram with one axis representing the aqueous phase, the second representing the oil phase, and third representing a mixture of surfactant and cosurfactant (SMIX) at a fixed volume ratio [28-31]. Selection of formulations Based on the NE region of the each phase diagram different formulations are selected in which drug is incorporated into the oil in following basis: 1. 10 mg of Simvastatin was selected as the dose for incorporation into the oil phase. 2. 2 mL was selected as the NE formulation for convenience. 3. The oil should be in such a concentration that it solubilizes the drug (single dose) completely. 10 mg of Simvastatin will dissolve easily in 0.2 mL of oil (10% of 2 mL). 4. Different concentration of oils were selected from each phase diagram, at a difference of (5% 10%, 15%,20%, 25%, etc) from the NE region. 5. For each 5 % of oil selected, the formula that used the minimum concentration of smix for its NE formulation was selected from the phase diagram. Evaluation of Nanoemulsion Thermodynamic stability tests Selected formulations were subjected thermodynamic stability tests.

to

different

Heating cooling cycle Between refrigerator temperature 4oC and 45oC of 6 cycles with storage at each temperature of not less than 48 h was studied. Formulations, which were stable at these temperatures, were subjected to centrifugation. Centrifugation Those formulations that passed were centrifused at 3500 rpm for about 30min by using centrifuge. The formulations that did not phase separated were taken to the further tests. Freeze thaw cycle Between – 210C and +250C three freeze thaw cycles with storage at each temperature for not less than 48 h was done for the formulations, which passed these thermodynamic stress tests, were further taken for the dispersibility tests [32]. Dispersibility tests

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Vol 3 | Issue 2 | 2013 | 94-101. Dispersibilty tests were done using a dissolution apparatus 2. 1mL of each formulation was added to 500 mL of water at 37 ± 0.5oC. A stainless steel dissolution paddle rotating at 50 rpm provided gentle agitation. In vitro performance of the formulation was assessed visually using the following grading system: Grade A: Clear and bluish appearance rapidly forms with in I min. Grade B: Slightly less clear emulsion having bluish white colour forms rapidly. Grade C: Fine milky emulsion that formed within 2 min. Grade D: Dull, greyish white emulsion having slightly oily appearance that is slow to emulsify (longer than 2 min). Grade E: Poor or minimal emulsification with large oil globules present on the surface. The formulations that passed the thermodynamic stability and also dispersibility tests in Grade A and B were selected for further studies. The selected formulations were prepared by dissolving 10 mg (single dose) of Simvastatin in oil (10%, 15%, 20%, 25% etc.). Respective smix ratio was added to the oil, mixed using magnetic stirrer and aqueous phase was added. The resulting mixture gave nanoemulsion [33]. Viscosity determination Viscosity of the formulations (0.5 g) was determined as such without dilution using Brookfield DVII ultra+ viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) using spindle # CPE 40 at 25 ± 0.5ºC. The software used for the calculations was Rheocalc V2.6. Electroconductivity study (Ghosh PK, et al., 2006): For the conductivity measurements, the tested nanoemulsions were prepared with a 0.01 N aqueous solution of NaCl instead of distilled water. This test system was measured by an electroconductometer (Conductivity meter 305, Systronic). Refractive index and percent transmittance The refractive index of the system was measured by an Abbe refractometer (Bausch and Lomb optical company, NY) by placing 1 drop of nanoemulsion on the slide. The percent transmittance of the system was measured at 650 nm using a UV spectrophotometer (Shimadzu, Japan) keeping distilled water as blank [34]. Drug content The drug content was calculated by UV visible spectrophotometer. The formulation was diluted to required concentration using methanol as solvent and the absorbance was measured at 238nm against a solvent blank. The drug content was calculated as: Drug content = Analyzed content x 100 Theoretical content Transmission electron microscopy (TEM)

Tem analysis should be done for the formulated nanoemulsion to determine the globule size of oil present in the nanoemulsion. This can be done by TOPCON 002B operating at 200 kV capable of point to point resolution. To perform the TEM observations, the nanoemulsion formulation was diluted with water (1/100). A drop of the diluted nanoemulsion was then directly deposited on the holey film grid and observed after drying. In vitro drug release In vitro drug release for the nanoemulsion formulation should be done in order to measure and detect the formulation that release the maximum amount drug (simvastatin) release from the nanoemulsion formulation. This test was performed in 500 mL of Phosphate buffer pH 7.4 using USP Dissolution apparatus Type II at 75 rpm and 37±0.5oC. 2 mL of nanoemulsion formulation containing single dose 10mg of Simvastatin was placed in a dialysis bag (Himedia dialysis membrane 150). Samples (5mL) were withdrawn at regular time intervals (0, 0.5, 1, 1.5, 2, 4, 6 ,8 h) and an aliquot amount of phosphate buffer was replaced. The release of drug from nanoemulsion formulation was compared with the conventional tablet formulation (Ozovas TM 10) and the suspension of pure drug. The samples were analyzed for the drug content using UV-Visible spectrophotometer at 238nm [35]. RESULT AND DISCUSSION Solubility studies The solubility of Simvastatin was found to be highest in isopropyimyristate (22.2±0.95mg/ml) as compared to other oils. This may be attributed to the polarity of the hydrophobic drug that favour their solubilization in the oil. Thus, Isopropyl myristate was selected as the oil phase for the development of the formulation(Table no 1) Compatibility studies The spectra obtained from IR studies at wavelength from 4000 cm-1 to 400 cm-1. After interpretation of the above spectra it was confirmed that there was no major shifting, loss or appearance of functional peaks between the spectra of drug, oil, physical mixture of drug and oil and surfactants, cosurfactants. From the spectra it was concluded that the drug was encapsulated into the oil without any chemical interaction. Pseudo-Ternary Phase diagram study When cosurfactant was added with surfactant in equal amount When cosurfactant was added with surfactant in equal amount [Smix ratio 1:1, Fig. 1(a)], the nanoemulsion region in the phase diagrams increased and maximum oil that could be solubilized was 10% w/w using Smix concentration of 13% w/w. This may be attributed to the fact that the addition of co-surfactant may lead to greater penetration of the oil phase in the hydrophobic region of the surfactant monomer thereby further

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Vol 3 | Issue 2 | 2013 | 94-101. decreasing the interfacial tension, which will lead to increase in the fluidity of the interface thus increasing the entropy of the system. With further increase in cosurfactant i.e. smix ratio 1:2, 1:3 and 1:4 [Fig. 1(b),(c),(d)] , it was observed that nanoemulsion area was found to increase and percentage of oil solubilized was 15%,30% and 35% w/w respectively [36]. When surfactant concentration was increased with respect to co-surfactant [Smix ratio 2:1, Fig. 1 (e) ] it was seen that nanoemulsion area was increased compared to 1:1 and nearly 15% w/w/ oil could be solubilized with the smix concentration of 25% w/w. When the surfactant was further increased to 3 parts is to 1 part of cosurfactant [Fig. 1(f)], the nanoemulsion area increased and the maximum oil that could be solubilized was 20% w/w. When the smix ratio was 4:1 [Fig. 1 (g)], nanoemulsion area was found to increased further with 30% w/w of oil being solubilized at smix concentration of 12% w/w. Selection of formulations from phase diagrams Hundreds of formulations can be prepared from the nanoemulsion region of the phase diagram. While going through pseudoternary phase diagrams, oil could be solubilized up to the extent of 40% w/w. Therefore, from each phase diagram different concentrations of oil that formed a nanoemulsion was selected at 5% intervals (10%, 15%, 20%, 25%, 30%, 35%, 40%). So that, largest number of formulations could be selected covering the nanoemulsion are of the phase diagram (Table 2). For each percentage of oil selected, only those formulations were taken from the phase diagram which used minimum concentration of smix [37]. Evaluation of nanoemulsion Thermodynamic stability tests Nanoemulsions are thermodynamically stable systems and are formed at a particular concentration of oil, surfactant and water, with no phase separation, creaming or cracking. It is the thermostability which differentiates nanoemulsions from emulsions that have kinetic stability and will eventually phase separate. Thus, the selected formulations were subjected to different thermodynamic stability by using heating cooling cycle, centrifugation and freeze thaw cycle stress tests. Those formulations, which survived thermodynamic stability tests (Table 15), were taken for dispersibility test [38]. Dispersibility tests Table 1. Solubility of Simvastatin in different oils (n=3) S. No. Oil 1 Oleic acid 2 Isopropyl myristate 3 Castor oil 4 Olive oil 5 Groundnut oil 6 Triacetin

From(table 2)Formulations that passed dispersibility test in Grade A and B (Table 2) were taken for further study, as Grade A and B formulations will remain as nanoemulsions when dispersed in GIT. From (table 3) Optimized formulations were taken for viscosity, electro conductivity, refractive index, transmittance and in vitro release studies. Viscosity determination The viscosity of the optimized formulations was determined. The values are shown in (Table 4). It was observed that viscosity of all the formulations is less than 26 cP. Formulation NE2 has the minimum viscosity (16.80 cP), perhaps because of its higher aqueous content. Lower viscosity is an ideal characteristic of the o/w nanoemulsion [39]. Electro conductivity test Conductivity of the optimized formulations was found in range of 459.3-560.3 µS/cm (Table 4). From the viscosity and the Electro conductivity study it can be concluded that the system is of o/w type. Refractive index and transmittance The refractive index of the formulated nanoemulsion was similar to the refractive index of the water (1.333) and thpercent transmittance > 97%. Drug content Drug content of the optimized formulations was found in range of 96.48-99.62%. The drug content varied for upto 3.09% between formulations NE1 to NE5 [40]. Transmission Electron microscopy TEM analysis for the formulation has to be done by using zetasizer. A “positive” image is seen using TEM. Some droplet sizes were measured using TEM, as it is capable of point to point resolution. The droplets in the nanoemulsion appear dark and the surroundings are bright (Fig 2). In vitro Release studies The highest release i.e. 99.28 % was obtained in NE2 can be detected by performing Dissolution studies from 5 different nanoemulsion formulations (NE1 to NE 5), and simple drug suspension, having same quantity 10mg of Tebinafine HCl (Table 5, Fig. 3) and the amount of drug release is compared [41].

Solubility (mg/ml) 5.01±1.67 22.2±o.95 11.35±0.84 7.25±0.45 4.25±1.52 0.281±1.69

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Vol 3 | Issue 2 | 2013 | 94-101. Table 2. Thermodynamic stability and dispersibility tests of different formulations selected from phase diagrams at a difference of5%w/w of oil. Smix Oil SMIX Aqueous Freeze H/C Cent. DT grade INF. Ratio (%) (%) (%) Thaw 1:1 5 13 82 X X √ A Fail 10 13 77 XXXXXXX X X C Fail 5 13 82 XX X C 10 13 77 X X 1:2 5 24 71 √ √ X B FAIL 10 34 56 √ √ √ A FAIL 15 25 60 √ √ √ B FAIL 1:3 15 8 77 √ √ √ A FAIL 20 8 72 √ √ √ A PASS 25 13 62 √ √ √ B FAIL 30 12 58 √ √ √ D FAIL 5 14 71 √ √ √ A FAIL 1:4 10 39 51 √ √ X D FAIL 15 8 77 √ √ √ A PASS 20 5 75 √ √ √ A PASS 25 13 62 √ √ √ C FAIL 30 7 63 √ √ √ A FAIL 35 12 53 √ √ √ A FAIL 5 16 79 √ X √ B FAIL 2:1 10 34 56 X X X E FAIL 15 25 60 √ √ √ B FAIL 3:1 5 10 85 √ √ √ A PASS 10 34 56 X X √ A FAIL 15 25 60 √ √ √ C FAIL 20 8 72 4:1 5 20 75 √ √ X E FAIL 10 20 70 √ X X C FAIL 15 8 77 √ √ √ B PASS 20 8 72 √ X √ A FAIL 25 13 62 √ √ √ A FAIL 30 12 58 √ √ √ A FAIL Heating cooling cycle (H/C), Centrifugation (Cent.), Dispersibility test (DT), Inference (Inf.) Table 3. Optimized formulations selected from phase diagram at a difference of 5% w/w of oil having least smix concentration Smix ratio Oil: Smix Dispersibili CODE Oil(%) S(%) CoS (%) Aqueous(%) (ml) ratio ty grade NE1 3:1 5 7.5 2.5 85 1:2 A NE2 1:4 15 1.6 6.4 77 1.875:1 A NE3 4:1 20 6.4 1.6 77 1.875:1 B NE4 1:3 25 2.0 6.0 72 2.5:1 A NE5 1:4 30 1.0 4.0 75 4:1 A Table 4. Characterization of optimized formulations (NE1 to NE5) Code Conductivity(µS/cm) Refractive index Transmittance (%) NE1 478.0 1.32 98.27 NE2 560.3 1.33 97.88 NE3 512.2 1.41 99.35 NE4 528.6 1.36 98.46 NE5 459.5 1.49 99.43

Drug content (%) 98.84 97.68 98.20 96.48 99.62

Viscosity (cP) 23.12. 16.80 18.25 24.26 25.08

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Vol 3 | Issue 2 | 2013 | 94-101. Table 5. Comparative In vitro release data for various formulations (n=3) Cumulative percentage release (%) Time (hours) NE1 NE2 NE3 NE4 NE5 0 0 0 0 0 0 0.5 28.9±0.8 35.2±1.1 24.8±1.6 26.8±0.9 19.2± 1.0 1 43.8±0.9 56.88±1.3 36.67±1.1 39.44±1.18 29.0±1.05 1.5 57.2±0.6 63.18±1.28 42.9±1.2 48.9±1.1 38.4± 1.6 2 63.8±1.8 69.80±1.11 49.9±0.9 57.53±1.7 44.63±1.7 4 73.6±1.2 81.06±1.9 57.53±1.3 67.9±0.9 51.68±1.9 6 81.0±1.3 87.14±2.3 68.02±1.6 72.5±1.8 54.22±2.4 8 88.2±1.4 96.18±1.2 76.5±1.4 84.65±1.1 69.28±2.1 12 93.1±1.4 99.08±0.8 81.28±1.1 86.27±1.1 75.8±2.3

Suspension 0 14.2±1.1 18.1±1.08 21.09±1.5 25.1±1.6 28.9±1.7 35.1±1.8 40.08±1.3 45.9 ±2.8

Tablet 0 13.5±1 16.2±1.09 21.1±1.9 24.5±2.1 26.9±2.8 29.1±1.4 38.2±1.8 43.4±0.8

Figure 1. Pseudoternary phase diagrams indicating o/w nanoemulsion region at different smix ratios A B C

D

E

F

G

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Vol 3 | Issue 2 | 2013 | 94-101. Figure 2. TEM analysis

SUMMARY AND CONCLUSION This thesis deals with the investigations carried out on the preparation and characterisation of oil-in-water nanoemulsion containing Simvastatin with minimum surfactant concentration that could improve its solubility and oral bioavailability. Higher drug release, optimum globule size, minimum polydispersity, lower viscosity, lower surfactant concentration, high electroconductivity and higher bioavailability has been optimized as NE formulation of Simvastatin containing Isopropyl myristate as oil, Tween80 surfactant and ethanol as cosurfactant respectively.

Figure 3. Comparative In vitro release profile in Phosphate buffer pH7.4

FUTURE PLANS Future studies are needed to establish its potential such as 1. Scale up studies of the optimized formulations. 2. Estimation of lipids in blood using animal models. 3. Characterisation by X-ray diffraction and Differential scanning calorimetry. 4. Stability studies as per ICH guidelines. 5. Shelf-life determination. 6. In vivo-in vitro correlation.

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