with other antihypertensive drugs), or used in greater doses in acute and chronic renal

3.1. Introduction Furosemide is a very efficient loop diuretic used in draining all kinds of edemas (of cardiac, hepatic or renal origin), in mild or moderate hypertension (itself or combined with other antihypertensive drugs), or used in greater doses in acute and chronic renal failure, in oliguria (Berkó et al. 2002). Erratic oral absorption (11 90%) is the main problem associated with the formulation and effectiveness of the Furosemide (FSM) (Jackson 2006; Akbuĝa et al. 1988; Chungi et al. 1979). According to Biopharmaceutical Classification System (BCS), FSM is classified as a class IV drug having low solubility and low permeability (Ozdmir and Ordu 1998; Boles Ponto and Schoenwald 1990). SNEDDS can be utilized to enhance drug solubilization in GIT and it has also an impact on permeability (Date et al. 2010; Porter et al. 2008; Tang et al. 2007). SNEDDS strategy has been experimented for furosemice (FSM), and various carriers have been tested (Zvonar et al., 2010). However, at present, no FSM marketed products arising from this approach are available, probably because of the unsatisfactory performance of the studied systems. The core objectives of the present study were to develop and evaluate an optimized self emulsifying drug delivery system for FSM and to assess its pharmacodynamic effect in terms of diuretic efficacy. 3.2. Material and methods 3.2.1. Materials FSM was received as a gift sample from Torrent Pharmaceuticals Ltd., Ahmedabad, India. Maisine 35-1, Transcutol P and Lauroglycol 90 were generously provided by Gattefosse France. Cremophore RH 40 and Cremophore EL were received as gift sample from BASF, USA. Captex 355 EP/NF, Captex 300 EP/NF and Capmul MCM NF were a generous gift from Abitec Corporation, USA. Polyethylene glycol 400 (PEG Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 67

400) was purchased from Merck Limited, Mumbai, India. Oleic acid was purchased from S. D. Fine Chemicals Limited, Mumbai. Propylene glycol and Tween 80 were purchased from Thomas Baker Chemicals Limited, Mumbai. Castor oil USP was purchased from Arora Pharmaceuticals Private Limited, New Delhi. Empty hard gelatin capsules were obtained from Associated Capsules Pvt. Ltd, Mumbai. Dialysis Tubing (seamless cellulose tubing, MWCO 12000) was purchased from Sigma Chemical Co. USA. All other chemicals used were of analytical grade. 3.2.2. Preparations of solutions a. 0.1M sodium hydroxide solution: Solution of molarity 0.1M prepared by diluting 4 g of sodium hydroxide to 1000 ml with water. b. 0.1N hydrochloric acid solution: Solution of normality 0.1N prepared by diluting 8.5 ml of hydrochloric acid to 1000 ml with water. c. Phosphate buffer (ph 6.8): Dissolve 28.80 g of disodium hydrogen orthophosphate and 11.45 g of potassium dihydrogen orthophosphate in sufficient water to produce 1000 ml. 3.2.3. Identification of the drug The drug was identified by physical observations, melting point determination and Ultraviolet Spectroscopy (UV). a. Physical observations: The color and powder forms of drug were observed and identified as per IP 2007 specifications and compared significantly. b. Melting point: the melting point of furosemide sample was determined by melting point apparatus and compared with the melting point of reference sample as specified in the BP 2007. c. Ultraviolet Spectroscopy: A 0.0005 per cent w/v solution in 0.1 M sodium hydroxide was examined in the range of 220 nm to 360 nm. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 68

3.2.4. Calibration curve Standard calibration curve for furosemide was prepared by making solutions of different concentration in methanol as follows: 1, 2, 3, 4, 5 µg/ml and the absorbance was measured at 276 nm (UV-2202, Systronics, India). Standard calibration curve for furosemide was prepared by making solutions of different concentration in 1:1 ratio of methanol: phosphate buffer (ph 6.8) and ƛ max was determined (UV-2202, Systronics, India). 3.2.5. Solubility studies These studies were performed to determine the solubility in individual vehicle. Highest solubility showing vehicles were then used for formulation of SNEDDS. Initially the solubility of FSM was determined in oils (i. e., Maisine 35-1, Capmul MCM, Captex 355 EP/NF, Captex 300 EP/NF, Oleic acid, Castor oil), surfactant (i. e. Tween 80, Lauroglycol 90, Cremophore RH 40 and Cremophore EL) and co-surfactants (Transcutol P, PEG 400, Propylene glycol). 2 ml of each vehicle was added in capped vial containing excess of FSM. These vials were stirred on a water bath maintained at 30 C for 48 hours. After attainment of equilibrium each vial was centrifuged at 5000 RPM for 10 min to separate the insoluble FSM and excess of insoluble FSM was removed by membrane filter of 0.22 µ pore size (Pall Life Sciences, India). Dissolved FSM was quantified by UV-spectrophotometer (UV-2202, Systronics, India) at 276 nm. 3.2.6. Pseudoternary phase diagram studies Water titration method was used for construction of phase diagram using oil and surfactant/co-surfactant mix (S mix ). Based on solubility studies, two sets of S mix (i.e., Tween 80: Transcutol P and PEG 400: Transcutol P) were investigated with Capmul MCM as the oil phase. Surfactant and co-surfactant were added in the ratio of 1:1, 2:1, 3:1 and 4:1 for both of the sets. Distilled water was added in a drop wise manner to the Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 69

mixture of certain weight ratios (i. e., 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9) of oil and surfactant/co-surfactant (S mix ). Then each mixture was observed for phase clarity and flowabilty. Phase diagrams were constructed by using trial version of CHEMIX School 3.50 software (Minnesota, USA). 3.2.7. Preparation of SNEDDS formulations From the solubility study and ternary phase diagram studies, SNEDDS components were selected for FSM incorporation and a series of SNEDDS were prepared (Table 3.1) with varying ratio of oil to S mix. The series contained Capmul MCM (oil) and Tween 80/Transcutol P (S mix ). FSM (6.67 % w/w) was loaded into each mixture. Table 3.1: Composition of developed formulations for FSM. Formulation codes Capmul MCM Tween 80 Transcutol P Furosemide %w/w mg %w/w mg %w/w mg %w/w mg F1 28.00 168 52.27 313.60 13.07 78.40 6.67 40 F2 18.67 112 49.78 298.67 24.89 149.33 6.67 40 F3 18.67 112 59.73 358.40 14.93 89.60 6.67 40 F4 18.67 112 56.00 336 18.67 112 6.67 40 F5 28.00 168 49.00 294 16.33 98 6.67 40 F6 28.00 168 43.56 261.33 21.78 130.67 6.67 40 The FSM-SNEDDS was prepared by dissolving FSM into S mix in glass vials and accurately weighed oil was added. Components were mixed and heated (45 50 C) to form a homogenous mixture and stored at room temperature till further use. 3.2.8. Characterization and evaluation of formulations 3.2.8.1. Dilution test SNEDDS formulation containing 40 mg of FSM (1 part) was diluted 10 times with distilled water, 0.1N HCl and phosphate buffer of ph 6.8 and observed. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 70

3.2.8.2. Drug content determination Pre weighted quantity of FSM containing SNEDDS were dissolved in 25 ml of methanol. FSM content was determined spectrophotometrically (UV-2202, Systronics, India) at 276 nm. 3.2.8.3. Emulsification time and precipitation assessment The emulsification time of SNEDDS formulation was assessed on USP II dissolution apparatus (Dolphin India). Each formulation (600 mg) was added drop wise to 500 ml of distilled water maintained at 37±0.5 C. Gentle agitation was provided by a standard stainless steel dissolution paddle rotating at 50 RPM. Precipitation was evaluated by visual assessment of the resultant emulsion after 24 h. The formulations were then categorized as clear (transparent or transparent with bluish tinge), non-clear (turbid), stable (no precipitation at the end of 24 h), or unstable (showing precipitation within 24 h) (Khoo et al. 1998). 3.2.8.4. Percentage transmittance (ƛ max 560 nm) 1 ml of SNEDDS formulation was diluted 100 times with distilled water. Percentage transmittance were measured spectrophotometrically (UV-2202, Systronics, India) at 560 nm using distilled water as a blank. 3.2.8.6. Viscosity determination SNEDDS (1 ml) was diluted 10 times and 100 times with distilled water in a beaker with constant stirring on a magnetic stirrer. Viscosity of resulting nanoemulsion and initial SNEDDS were determined by using Brookfield R/S plus rheometer (Brookfield Engineering, Middleboro, MA). Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 71

3.2.8.7. Droplet size analysis and polydispersity index (PDI) determination SNEDDS formulation (600 mg) containing 40 mg of FSM was diluted to 100 ml and mixed gently by inverting the flask. The size of droplets hence formed and PDI was measured by using Zetasizer (Malvern Instruments). 3.2.8.8. Zeta potential determination SNEDDS was diluted 10 times and 100 times with distilled water by constant stirring on a magnetic stirrer. Zeta potential of the resulting emulsion was determined by using Zetasizer (Malvern Instruments). 3.2.9. In vitro dissolution studies In vitro dissolution studies were performed to evaluate the dissolution rate of SNEDDS. These studies were carried out in USP type II dissolution test apparatus (Dolphin, India) at 100 RPM in 900 ml of phosphate buffer (ph 6.8). The temperature was maintained at 37±0.5 C. SNEDDS formulations were filled in hard gelatin capsule (size 0) and used for dissolution studies; results were compared with plain FSM and marketed tablet of FSM (LASIX). Aliquots were withdrawn at 5, 10, 20, 30, 45 and 60 min intervals and filtered using 0.22 µ nylon membranes. The withdrawn samples were diluted suitably and analyzed for the FSM content UV spectrophotometrically at 276 nm against phosphate buffer (ph 6.8). An equal volume of the dissolution medium was replaced in the vessel after each withdrawal to maintain the sink condition. Each test was performed in triplicate (n=3), and calculated mean values of cumulative drug release were used while plotting the release curves. 3.2.10. In vitro diffusion studies Permeation of FSM through biological membrane was evaluated by in vitro diffusion studies carried out by using dialysis technique (Kadu et al. 2010; Paradkar et al. 2007). Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 72

One end of pretreated cellulose dialysis tubing (Tube was soaked for 3-4 hours in a solution of 2 % sodium bicarbonate and 1 mm EDTA. Then the tube was rinsed carefully with distilled water from inside as well outside) (7 cm in length) was tied with thread and 0.22 ml of SNEDDS formulation (equivalent to 15 mg FSM) was placed in it along with 0.78 ml of dialyzing medium (phosphate buffer ph 6.8). The other end of tubing was also tied with thread and was allowed to rotate freely in the dissolution vessel of a USP type II dissolution test apparatus that contained 900 ml dialyzing medium (phosphate buffer ph 6.8) maintained at 37 ± 0.5 C and stirred at 100 RPM. Aliquots were collected periodically and replaced with fresh dissolution medium and analyzed spectrophotometrically at 276 nm for FSM content. 3.2.11. In vivo pharmacodynamic studies In vivo study was approved and performed in accordance with the guideline of the animal ethics committee. All institutional and national guidelines for the care and use of laboratory animals were followed. The rats were housed individually in metabolic cages, controlled conditions of temperature (25 C) and a 12:12 h light/dark cycle. The study was conducted in four groups consisting of three male wistar rats weighing 250 280 g. Animals were grouped as: Group I: Three rats for plain FSM drug suspension in 0.25% Carboxy Methyl Cellulose (FSM) Group II: Three rats for optimized SNEDDS formulation (F3) of FSM (SNEDDS) Group III: Three rats for 0.25% Carboxy Methyl Cellulose (Control) Group IV: Three rats for blank SNEDDS formulation (Placebo) The rats were fasted 15 hrs prior to the experiment. Suspension of FSM (15 mg/kg) and optimized SNEDDS formulation F3 (equivalent to 15 mg of FSM) was administered to animals by gavage performing doses. The four groups of rats were allocated to one of Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 73

four different treatments as summarized in Table 3.2. The groups were inverted after providing washout period of 72 hours to each group (Pires et al. 2011; Vogel 2002). Table 3.2: In vivo study design of pure FSM and FSM-SNEDDS. Treatment FSM SNEDDS Control Placebo Period 1 Group 1 Group 2 Group 3 Group 4 Washout period of 72 hours Period 2 Group 2 Group 3 Group 4 Group 1 Washout period of 72 hours Period 3 Group 3 Group 4 Group 1 Group 2 Washout period of 72 hours Period 4 Group 4 Group 1 Group 2 Group 3 Cumulative urine output was recorded at 60, 120, 180 and 240 minutes after oral administration of compounds. The urine volume was measured and a urine sample was taken for further analysis. Urinary sodium was determined in a flame photometer (F129, Systronics, India). Results were presented as mean ± S.E.M. (standard error of mean) and were analyzed by two-way analysis of variance followed by a Boferroni post hoc test. A p<0.05 was considered significant. 3.2.12. Stability studies Chemical and physical stability of the optimized FSM SNEDDS formulation was assessed at 40 ± 2 C/75 ± 5% RH as per ICH Guidelines. SNEDDS equivalent to 40 mg FSM was filled in size 0 hard gelatin capsules, packed in aluminum strips and stored for three months in stability chamber (CHM 10S, REMI Instruments Ltd, India). Samples were analyzed at 0, 30, 60 and 90 days for clarity, drug content and time required for 90% drug release (t 90% ). Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 74

3.3. Results & Discussion 3.3.1. Identification of drug (a) Physical observation of drug reveals that furosemide is white, crystalline powder. (b) Melting point of furosemide was found to be 208-210 C. (c) When examined in the range 220 nm to 360 nm, a 0.0005 per cent w/v solution in 0.1 M sodium hydroxide showed three absorption maxima at about 228 nm, 271 nm and 333 nm. The ratio of the absorbance at the maximum at about 271 nm to that at the maximum at about 228 nm is 0.52 to 0.57. The above mentioned tests confirm the identity of the received drug furosemide. 3.3.2. Calibration curve Calibration curve of furosemide was prepared in methanol. ƛ max was found to be 276 nm (Figure 3.1). Figure 3.1: UV spectrophotometrical scanning of furosemide in methanol showing ƛ max at 276 nm. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 75

The concentration was increased in a predetermined way. Regression equation was calculated and utilized for quantitative estimation of drug in samples. The correlation coefficient found is given in Figure 3.3. Results are expressed as mean±sd (Table 3.3). Table 3.3: Absorbance vs concentration data for calibration curve. Concentration Absorbance (µg/ml) 1 0.098±0.004 2 0.193±0.01 3 0.288±0.12 4 0.378±0.16 5 0.487±0.21 0.6 Absorbance 0.5 0.4 0.3 0.2 y = 0.096x - 0.000 R² = 0.998 0.1 0 0 1 2 3 4 5 Concentration (µg/ml) Figure 3.2: Calibration curve of furosemide in methanol. 3.3.3. Solubility Studies Solubility of drug substance is a key criterion for selection of components for developing a SNEDDS formulation. The self-emulsifying formulations consisting of oil, Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 76

surfactant, co-surfactant and drug should be a clear and monophasic liquid at ambient temperature. Solubility studies were performed to identify suitable oils, surfactants and co-surfactants that possess good solubilizing capacity for FSM (Table 3.4) (Results are expressed as mean±sd). As FSM was found to have maximum solubility in Capmul MCM, Tween 80, Transcutol P and PEG 400, further studies were conducted using various combinations of these oils and surfactants to identify the self emulsifying area. Two sets of S mix and oil in different ratios were used to construct ternary phase diagrams. They were (1) Tween 80 and PEG 400 as S mix and Capmul MCM as oil phase and (2) Tween 80 and Transcutol P as S mix and Capmul MCM as oil phase. For both the sets the selected ratios of S mix were 1:1, 2:1, 3:1 and 4:1. Table 3.4: Solubility studies of FSM in various vehicles. Vehicle Function in SNEDDS Solubility (mg/ml) Maisine 35-1 Oil 3.16±0.84 Oleic Acid Oil 1.81±0.49 Capmul MCM Oil 6.14±0.46 Castor oil Oil 2.19±0.68 Captex 355 EP/NF Oil 0.24±0.07 Tween 80 Surfactant 91.12±2.86 Cremophore EL Surfactant 49.10±1.73 Cremophore RH 40 Surfactant 27.11±1.06 Span 20 Surfactant 22.16±0.63 Lauroglycol 90 Surfactant 1.70±0.82 PEG 400 Co-surfactant 228.63±2.52 Propylene Glycol Co-surfactant 20.78±0.94 Transcutol P Co-surfactant 152.90±2.45 Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 77

3.3.4. Pseudoternary phase diagram studies Self-nanoemulsifying systems form fine oil water emulsions with gentle agitation, upon their introduction into aqueous media. Surfactant gets preferentially adsorbed at the interface, reducing the interfacial energy as well as providing a mechanical barrier to coalescence. The Figure 3.4A, B, C and D and Figure 3.5A, B, C and D show ternary phase diagrams of Tween 80-PEG 400 (S mix ) and Capmul MCM as oil phase and Tween 80-Transcutol P (S mix ) and Capmul MCM as oil phase, respectively. The area in the shade indicates micro/nano emulsion region. Wider region indicates better selfemulsifying ability. The co-surfactant helps to achieve prerequisites of emulsion formation it helps in keeping the film flexible, fluid and tightly packed (Constantinides 1995). From the phase diagram studies it can be observed that as the S mix ratio increases the emulsion area decreases. Therefore the co-surfactant plays vital role in the emulsion formation for both of the S mix combinations. The phase study revealed that the emulsion region was more with Tween 80 Transcutol P (S mix ) in comparison to Tween 80-PEG 400 combination. Hence the Tween 80 Transcutol P (S mix ) was selected for FSM loading and further studies. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 78

Figure 3.3: Pseudo ternary phase diagrams with following components: Oil=Capmul MCM, Surfactant=Tween 80, Co-surfactant=PEG 400. S/Cos ratio of A is 1:1, B is 2:1, C is 3:1 and D is 4:1. S/Cos indicates Surfactant/Co-surfactant. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 79

Figure 3.4: Pseudo ternary phase diagrams with following components: Oil=Capmul MCM, Surfactant=Tween 80, Co-surfactant=Transcutol P. S/Cos ratio of A is 1:1, B is 2:1, C is 3:1 and D is 4:1. S/Cos indicates Surfactant/Cosurfactant. 3.3.5. Characterization and evaluation of formulations 3.3.5.1. Dilution test The objective of dilution study was to study the degree of emulsification and recrystallization of the FSM, if any. Dilution may better mimic conditions in the stomach following oral administration of SNEDDS pre-concentrate. An accurate mixture of emulsifier is necessary to form a stable nano emulsion, for the development of SNEDDS formulation when one part of each SNEDDS formulation was diluted with Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 80

10 parts of distilled water, 0.1 HCl and phosphate buffer (ph 6.8) (Table 3.5). It was observed that the formulations F1, F3, F5 and F6 were found to be most stable because they do not show any precipitation or phase separation on storage in various dilution media. Self emulsified formulation F3 after dilution with distilled water is shown in Figure 3.6. Figure 3.5: Furosemide Self Emulsified formulation F3 in distilled water producing bluish tinge. Table 3.5: Observation of dilution test of FSM-SNEDDS formulations. Formulation Distilled Water 0.1N HCl Phosphate buffer ph 6.8 F1 Stable up to 6 hr Stable up to 6 hr Stable up to 6 hr F2 Unclear within 30 min Unclear within 30 min Stable up to 6 hr F3 Stable up to 6 hr Stable up to 6 hr Stable up to 6 hr F4 Stable up to 6 hr Unclear within 30 min Stable up to 6 hr F5 Stable up to 6 hr Stable up to 6 hr Stable up to 6 hr F6 Stable up to 6 hr Stable up to 6 hr Stable up to 6 hr 3.3.5.2. Drug content determination FSM content of the SNEDDS formulations is shown in Table 3.6 (Results are expressed as mean±sd), which was in the limit (96 102%). Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 81

Table 3.6: Characterization of FSM-SNEDDS formulations Parameters F1 F2 F3 F4 F5 F6 Drug Content (%) 97.41±2.06 96.45±1.64 97.49±1.58 101.37±1.86 99.43±1.67 98.03±1.26 Self Emulsification 12±2 12±1 16±1 13±1 10±1 9±1 Time (s) Precipitation Stable Stable Stable Stable Unstable Unstable Clarity Bluish Bluish Bluish Bluish Turbid Turbid Viscosity (cps) 0 times dilution 342 334 367 353 326 317 10 times dilution 1.08 1.02 1.19 1.14 0.992 0.986 100 times dilution 0.876 0.863 0.893 0.883 0.858 0.853 % Transmittance 68.48±0.22 78.34±0.34 86.12±0.67 85.42±0.53 68.21±0.81 71.92±0.39 Droplet Size (nm) 154.4±2.10 47.56±1.22 26.8±1.02 28.9±2.21 147.6±3.21 57.44±2.78 Polydispersity 0.566±0.02 1±0.06 0.215±0.03 0.56±0.02 0.678±0.03 0.565±0.02 index Zeta (mv) Potential -20.9±0.54-15.1±0.27-10.4±0.12-17.8±0.31-19.3±0.51-16.3±0.22 3.3.5.3. Emulsification time and precipitation assessment The rate of emulsification is an important parameter for the assessment of the efficiency or spontaneous emulsification of formulation without aid of any external thermal or mechanical energy source. Formulation should disperse completely and quickly when subjected to aqueous dilution under mild agitation of GIT due to peristaltic activity. It has been reported that self emulsification mechanism involves the erosion of a fine cloud of small droplets from the monolayer around emulsion droplets, rather than progressive reduction in droplet size (Pouton 1997). The ease of emulsification was suggested to be related to the ease of water penetration into the colloidal or gel phases formed on the surface of the droplet (Rang and Miller 1999, Groves et al., 1974). It was Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 82

observed that an increase in the proportion of Tween 80 from 43.56 to 59.73 % w/w in the composition resulted in increased self-emulsification time from 9 to 16 seconds (Table 3.6) (Results are expressed as mean±sd). This might be because of high viscosity imparted by Tween 80 which increases the free surface energy of system thereby increasing the emulsification time with increase in content of surfactant. Below 49.78% concentration of surfactant, there was turbid and unstable dispersion (Table 3.6). This may be due to excess penetration of water into the bulk oil causing massive interfacial disruption and ejection of droplets into the bulk aqueous phase (Kadu et al. 2010). 3.3.5.4. Percentage transmittance (ƛ max 560 nm) The percentage transmittance of the six selected optimized formulation was determined. As the value closer to 100% is formulation which is isotropic in nature therefore, optimized formulation of F3 from S mix ratio of 4:1 gave maximum percentage transmittance (Table 3.6) (Results are expressed as mean±sd). As SNEDDS form nano emulsion in GIT, it meets with patient acceptability but isotropic nature of formulations or percentage transmittance closer to 100% gives an indication of globule size in nanometer range. The droplet size of the emulsion is a crucial factor in selfemulsification performance, because it determines the rate and extent of drug release as well as absorption (Parmar et al. 2011). Thus, the formulation has the capacity to undergo enhanced absorption and thus ability to have increased oral bioavailability. 3.3.5.5. Viscosity determination Viscosity of SNEDDS without dilution was found to be in between 317 and 367 cp, which was suitable for filling in hard gelatin capsule without risk of leaking problem. As SNEDDS was diluted 10 and 100 times with water, viscosity of the system was decreased, which indicates that oral administration of SNEDDS formulation will be Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 83

diluted with the stomach fluid and viscosity will be decreased and therefore absorption from the stomach will be fast (Table 3.6). Results are expressed as mean±sd. 3.3.5.6. Droplet size analysis and PDI determination The droplet size of the emulsion is an essential factor in self emulsification performance because it determines the rate and extent of drug release as well as drug absorption. Also, it has been reported that the smaller particle size of the emulsion droplets may lead to more rapid absorption and improve the bioavailability (Liu et al. 2007). It is well known that in nano emulsion systems the addition of surfactants stabilize and condense the interfacial film, while the addition of co-surfactant causes the film to expand; thus, the relative proportion of surfactant to co-surfactant has varied effects on the droplet size. From Table 3.6 (Results are expressed as mean±sd), it can be seen that formulation F3 has the smallest droplet size of 26.8 nm. Size distribution reports of formulations F1-F6 are shown in Figure 3.7-3.12. Polydispersity is the ratio of standard deviation to the mean droplet size. This signifies the uniformity of droplet size within the formulation. The higher the value of polydispersity, the lower is the uniformity of the droplet size in the formulation (Dixit et al. 2010). The lowest value of polydispersity was found to be 0.215 for SNEDDS formulation F3, which indicates uniformity of droplet size within the formulation. Formulation F2 has highest polydispersity value of 1, signifies non uniformity of droplets (Table 3.6) (Results are expressed as mean±sd). PDI values of more than 0.7 indicate that the sample has a very broad size distribution. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 84

Figure 3.6: Size distribution report of FSM-SNEDDS formulation F1. Figure 3.7: Size distribution report of FSM-SNEDDS formulation F2. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 85

Figure 3.8: Size distribution report of FSM-SNEDDS formulation F3. Figure 3.9: Size distribution report of FSM-SNEDDS formulation F4. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 86

Figure 3.10: Size distribution report of FSM-SNEDDS formulation F5. Figure 3.11: Size distribution report of FSM-SNEDDS formulation F6. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 87

3.3.5.7. Zeta potential determination Emulsion droplet polarity is also a very essential factor in characterizing emulsification efficiency (Shah et al. 1994). Zeta potential is the potential difference between the surface of tightly bound layer (shear plane and electroneutral region of the solution). The significance of zeta potential is that its value can be related to the stability of colloidal dispersions. Zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in dispersion. For molecules and particles that are small enough, a high zeta potential will present stability. When the potential is low, attraction exceeds repulsion and the dispersion will break and flocculate. So, colloids with high zeta potential (negative or positive) are electrically stabilized. Negative values of zeta potential of the optimized formulations indicated that the formulations were negatively charged. Formulation F1 was found to be most stable formulation (Table 3.6) (Results are expressed as mean±sd). Zeta potential reports of formulation F1-F6 are shown in Figure 3.13-3.18. Figure 3.12: Zeta potential report of FSM-SNEDDS formulation F1. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 88

Figure 3.13: Zeta potential report of FSM-SNEDDS formulation F2. Figure 3.14: Zeta potential report of FSM-SNEDDS formulation F3. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 89

Figure 3.15: Zeta potential report of FSM-SNEDDS formulation F4. Figure 3.16: Zeta potential report of FSM-SNEDDS formulation F5. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 90

Figure 3.17: Zeta potential report of FSM-SNEDDS formulation F6. 3.3.6. In vitro dissolution studies SNEDDS formulation F3 showed significantly higher drug release as compared to plain FSM and marketed FSM tablet (LASIX) (Table 3.7, Figure 3.19) (Results are expressed as mean±sd). F3 showed more than 90% of drug release in 10 minutes while plain FSM and marketed tablet showed 57 and 64 %, respectively. Spontaneous formation of nanoemulsion of SNEDDS formulation F3 could be the reason for the faster rate of drug release into the aqueous medium. Dramatic increase in the rate of release of FSM from SNEDDS compared to plain FSM and marketed formulation can be attributed to its quick dispersibility and ability to keep drug in solubilized state. Thus, this greater availability of dissolved FSM from the SNEDDS formulation could lead to higher absorption and higher oral bioavailability. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 91

Table 3.7: In vitro dissolution studies data of plain FSM, marketed formulation and developed FSM-SNEDDS formulations. Time % Cumulative Drug Release (min) F1 F2 F3 F4 F5 F6 Plain FSM LASIX 0 0 0 0 0 0 0 0 0 5 69.18±1.22 79.68±2.22 87.85±3.21 85.29±2.55 73.38±2.11 79.92±1.67 48.17±1.04 57.28±0.64 10 75.48±1.34 89.25±3.22 95.79±2.04 91.12±2.76 79.45±2.33 84.12±2.32 56.81±1.44 63.81±0.52 20 78.98±1.22 90.65±2.11 98.82±2.12 93.22±3.22 81.32±3.31 85.52±2.43 61.24±1.26 65.21±0.92 30 81.32±1.71 91.82±2.43 99.99±2.56 94.85±2.33 83.88±3.1 88.09±2.11 63.81±1.32 66.85±1.02 45 83.65±1.53 93.45±2.67 99.06±2.43 96.96±2.8 85.99±2.11 89.72±2.33 65.91±1.08 68.01±1.16 60 86.22±2.34 93.22±3.22 98.36±2.66 96.49±3.22 87.39±3.53 90.65±1.34 67.31±2.01 69.65±1.32 Figure 3.18: In vitro dissolution studies of plain FSM, marketed formulation and developed FSM-SNEDDS formulations. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 92

3.3.7. In vitro diffusion studies Conventional dissolution testing can only provide a measure of dispersibility of SNEDDS in the dissolution medium. Alternatively, in vitro performance of SNEDDS can be evaluated by drug diffusion studies using the dialysis technique. It is very popular and well documented in many literatures (Kadu et al. 2010; Paradkar et al. 2007). SNEDDS formulations F1, F3 and F4 were selected for diffusion studies, as these formulations show smaller droplet size among other formulations. Though formulations F2, F5 and F6 has less droplet size than F1 but formulations F5 and F6 found to be unstable and turbid on precipitation and clarity test while F2 has non uniformity of droplets due to very high PDI. The release of FSM from these dosage forms was evaluated in phosphate buffer ph 6.8; the release percentage of F3 was higher than that of F1 and F4 (Table 3.8, Figure 3.20) (Results are expressed as mean±sd). It suggests that FSM dissolved perfectly in SNEDDS form and could be released due to the small droplet size, which permits a faster rate of FSM release into aqueous phase. The release rate of FSM from SNEDDS F3 (mean droplet size: 26.8 nm) was faster than SNEDDS F1 and F4 (mean droplet size: 154.4 and 28.9 nm, respectively). In this study, diffusion profiles of all three formulations (F1, F3 and F4) did not show any differences during initial 1 h, however, at the end of 12 h, formulation F3 showed 98.5 % diffusion while F1 and F4 showed 86 % and 97 % diffusion, respectively (Figure 3.20). Results clearly indicate the effect of mean droplet size on FSM diffusion across dialyzing membrane. Hence decreasing the particle size of nano emulsion could increase the release rate of FSM. Therefore, F3 was selected as optimized formulation for in vivo studies having the smallest droplet size and thus higher diffusion than formulation F4. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 93

Table 3.8: In vitro diffusion studies data of FSM-SNEDDS F1, F3 and F4. Time (Hrs) % Diffusion F1 F3 F4 0 0 0 0 1 21.19±1.23 22.43±1.26 21.19±1.02 2 29.90±1.21 31.15±1.26 33.01±1.08 3 42.97±2.1 46.08±1.42 44.22±1.11 4 54.40±1.03 57.35±1.33 51.68±1.19 5 60±1.08 61.87±1.75 57.51±1.22 6 64.98±2.03 67.47±1.64 63.11±1.44 7 69.96±2.13 73.07±1.81 69.96±1.54 8 72.45±2.01 79.29±1.4 77.43±1.8 9 78.05±2.03 84.90±2.22 83.65±1.77 10 79.92±2.00 92.37±2.42 88.01±2.33 11 83.65±2.43 96.10±2.13 94.23±2.13 12 86.14±2.3 98.59±2.26 97.34±2.09 Figure 3.19: In vitro diffusion studies of FSM-SNEDDS F1, F3 and F4. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 94

3.3.8. In vivo pharmacodynamic studies This study was performed to evaluate the pharmacodynamic potential of an optimized formulation (F3) against plain FSM. Cumulative volumes of excreted urine after oral administration compounds are shown in Figure 3.21A (Table 3.9). Statistically significant diuretic effect of SNEDDS was observed after 180 minutes in comparison to FSM, control and placebo. Sodium and chloride ions quantification is one of the best methods to determine diuretic effect of drugs (Opie and Kaplan 1991; Field et al. 1984). Values of concentration of sodium in excreted urine are shown in Figure 3.21B (Table 3.9). SNEDDS group showed significant increase in the amounts of electrolyte in comparison to control and placebo groups after 120 minutes of administration. However, effect on sodium concentration by SNEDDS was more but not statistically significant as compared to FSM group. Table 3.9: Time course of urine and sodium output in different groups. Values are reported as mean ± S.E.M. for twelve rats in each group. Time Control Placebo FSM SNEDDS (min) Cumulative Sodium Cumulative Sodium Cumulative Sodium Cumulative Sodium Urine Concentration Urine Concentration Output (µl)x10 4 (meq/l) X10 2 Output (µl) X10 4 (meq/l) X10 2 Urine Output Concentration Urine Concentration (µl)x10 4 (meq/l) X10 2 Output (meq/l) X10 2 (µl)x10 4 60 0.6±0.23 0.76±0.25 0.57±0.20 0.75±0.25 1±0.05 1.02±0.01 1.42±0.13 1.08±0.009 120 1.175±0.43 1.00±0.009 1.02±0.32 0.99±0.007 2±0.10 1.17±0.03 2.87±0.14** 1.18±0.01** 180 2±0.43 1.01±0.01 1.72±0.12 1.00±0.01 3.72±0.16** 1.22±0.02 6.25±0.35 *** 240 2.25±0.20 1.02±0.009 2.25±0.36 1.02±0.01 5.07±0.37** 1.39±0.02** 8.52±0.41 *** **Statistically significant from control and placebo group. p<0.05 1.35±0.010** 1.67±0.02** ***Statistically significant from control, placebo and FSM group. p<0.05 Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 95

Figure 3.20: (A) Time course of urine output in different groups. (B) Time course of sodium output in different groups. Values are reported as mean ± S.E.M. for twelve rats in each group. **Statistically significant from control and placebo group. p<0.05 ***Statistically significant from control, placebo and FSM group. p<0.05 Diuretic activity data suggest that SNEDDS formulation increased the pharmacological effect of FSM. The higher diuretic activity of the SNEDDS is due to complete dissolution of FSM in SNEDDS, which could have increased absorption. Solubility is a crucial characteristic for increasing the bioavailability of drugs according to the BCS (Amidon et al. 1995). Moreover, SNEDDS play an important role in the improvement of permeability too. Higher permeability may be attributed to Capmul MCM, Tween 80 and Transcutol P as these components have the ability to enhance the permeability (Date et al. 2010; Singh et al. 2009; Porter et al. 2008; Tang et al. 2007). However the present work did not deal with the permeation experiments using cell models and this aspect will be developed in future studies. 3.3.9. Stability studies Optimized SNEDDS formulation (F3) filled into hard gelatin capsules as the final dosage form. Liquid-filled hard gelatin capsules are prone to leakage and the entire system has a very limited shelf life owing to its liquid characteristics and the possibility Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 96

of precipitation of the FSM from the system. Thus, to evaluate its stability and the integrity of the dosage form, the optimized formulation (F3) was subjected to stability studies. No change in the physical parameters such as homogeneity and clarity was observed during the stability studies. There was no major change in the FSM content, drug release (t 90% ) and % transmittance. It was also observed that the formulation was compatible with the hard gelatin capsule shells. Also, there was no phase separation and drug precipitation was found at the end of three-month stability studies indicating that FSM remained chemically stable in SNEDDS (Table 3.10) (Results are expressed as mean±sd). Table 3.10: Evaluation data of FSM-SNEDDS formulation subjected to stability studies. Sampling Points (Days) % Drug Content t 90% (min) % Transmittance 0 97.49±1.58 <10 86.12 30 97.11±1.46 <10 86.17 60 96.77±1.77 <10 86.31 90 96.13±2.02 <10 86.04 3.4. Conclusions SNEDDS was successfully emerged as appealing approach to improve the bioavailability of furosemide. Increased solubility, Increased dissolution rate and ultimately increased bioavailability of a poorly water-soluble drug, furosemide, was observed with an optimized SNEDDS formulation consisting of Capmul MCM (18.67 % w/w), Tween 80 (59.73 % w/w), Transcutol P (14.93 % w/w) and FSM (6.67 % w/w). The developed formulation showed higher pharmacodynamic potential as compared with plain FSM. Results from stability studies established the stability of the developed formulation. Therefore, our study confirmed that the SNEDDS formulation Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 97

can be used as a possible alternative to traditional oral formulations of FSM to improve its bioavailability. Design Development & Evaluation of SEDDS for Poorly Water Soluble Drugs 98