Development and Evaluation of Fast‑Dissolving Oral Film of Poorly...

[Full title: Development and Evaluation of Fast‑Dissolving Oral Film of Poorly Water-Soluble Drug Felodipine]

Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 256 Development and Evaluation of Fast‑dissolving Oral Film of Poorly Water‑soluble Drug Felodipine Pravin Kumar Sharma 1 , Pankaj Kumar Sharma 1 , Gajanan N. Darwhekar 2 , Birendra Shrivastava 1 1 School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan, India, 2 Acropolis Institute of Pharmaceutical Education and Research, Indore, Madhya Pradesh, India Abstract Aim: The objective of the presented research work was to develop and evaluate fast-dissolving oral film containing solid dispersion of Felodipine (FnFDFs) for improvement of its water solubility, dissolution, and oral bioavailability by avoiding the first pass metabolism, providing faster onset of action and avoidance of problem of dysphasia. Materials and Methods: First, solid dispersion (SDP) of Felodipine was prepared using solvent evaporation method using PVP-K 30 as hydrophilic polymeric carrier in different proportions. FnFDFs were prepared using solvent casting method and optimized using Box–Behnken design by applying design-expert. The concentration of HPMC-E 5 (40–45% w/w) as a film forming polymer, propylene glycol (10–15% w/w of polymer) as a plasticizer, and croscarmellose (1–5% w/w) as disintegrating agent was selected as independent variables and tensile strength, disintegration time and percentage drug dissolution was selected as a response variables. Further, FnFDFs were evaluated based on uniformity of mass, thickness, percentage drug content, folding endurance, surface pH, moisture uptake, percentage swelling, percentage elongation, tensile strength, in vitro disintegration time, in vitro percentage drug dissolution or release study, stability study, surface morphology using scanning electron microscope, ex vivo permeation study, and in vivo pharmacokinetic study. Results and Discussions: Fourier transform infrared and differential scanning calorimetry analysis revealed the compatibility between drug and excipients. Results of evaluation of FnFDFs suggested satisfactory performance for all the parameters. FnFDFs indicated in vitro disintegration time of 22.84 ± 0.31 s and in vitro percentage drug dissolution of 97.09 ± 1.54% up to 10 min, thus suggested faster drug dissolution. Moreover, in vivo pharmacokinetic study in rats revealed faster absorption and around 90% of oral bioavailability up to 1–2 h for FnFDFs through the buccal administration in comparison with that slow absorption and around 20% of oral bioavailability up to 3 h for oral suspension of Felodipine through gastrointestinal tract. Conclusion: Based on the results, it was concluded that fast-dissolving oral film contained SDP of drug may provide the merits of faster onset of action, avoidance of extensive first-pass metabolism, enhanced bioavailability, and improved patient compliance for the delivery of poorly water-soluble drug such as Felodipine Keywords: Bioavailability, Felodipine, Solid dispersion and fast-dissolving oral film of solid dispersion of Felodipine Address for correspondence: Pravin Kumar Sharma, School of Pharmaceutical Sciences, Jaipur National University, Jaipur, Rajasthan, India. E-mail: praveensharma 910@gmail.com Received: 09-03-2018 Revised: 18-03-2018 Accepted: 23-03-2018 INTRODUCTION F elodipine is a calcium-channel blocker which is used as antihypertensive and antianginal drug. It is poorly watersoluble drug which undergoes extensive firstpass metabolism and thus has oral bioavailability of around 15% [1,2] Delivery of poorly watersoluble drugs by the oral route has been difficult due to insufficient amount of drug dissolved for absorption from the gastrointestinal tract [3] Although, there are number of formulation strategies have been employed to enhance the dissolution rate of poorly soluble drugs such as particle size reduction, modification of crystal habits, salts formation, complex RESEARCH AR TICLE

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 257 formation with cyclodextrin, use of surfactants, solid dispersions (SDP), fast-dissolving oral films (FDFs), lipidbased formulations, and prodrug approaches [4,5] Preparation of SDP is a better approach for improving drug solubility because it is easier to produce and more applicable [6] Fastdissolving dosage forms were initially prepared for providing substitute to conventional solid dosage forms to achieve better patient compliance [7] In addition, fast-dissolving oral films (FDFs) showed a great potential over other dosage forms for the delivery of poorly soluble drugs since they provide distinct advantages including rapid disintegration and dissolution in the oral cavity thus increase bioavailability with faster onset of action and avoidance of first-pass effect [8] Due to high permeability of oral mucosa, it allows direct access of drug to the systemic circulation and avoids the first pass metabolism [9] FDFs represent an advantageous dosage form, especially for geriatric and pediatric patients [10,11] Quality by design as a more holistic approach can be applied to assure the quality of the product. It provides a basis for optimizing and improving the manufacturing operation [12] MATERIALS AND METHODS Materials Felodipine was obtained as a gift sample from Cipla Ltd. HPMC-E 5, croscarmellose and methyl cellulose were obtained as a gift sample from Signet Chemicals. All other chemicals were purchased from Loba chemicals Methods Solubility determination The solubility of Felodipine was determined using saturation solubility method. An excess amount of Felodipine was added to 10 ml of distilled water, phosphate buffer pH 6.8, ethanol, and acetonitrile separately in a glass vials. The content of vials was mixed vigorously for 30 min, and further solutions were shaken mechanically to equilibrate. After 72 h, the content of each vial was centrifuged for 10 min at 2500 rpm. The supernatant of each vial was filtered through 0.45 µ membrane filter and then, filtrate was diluted suitably with solvent. The concentration of Felodipine was analyzed using double beam UV visible spectrophotometer (UV 1800, Shimadzu, Japan) at 237.1 nm against blank [13] FTIR analysis FTIR spectrum of the HPMC-E 5, PVP-K 30, Felodipine, physical mixture of Felodipine with HPMC-E 5, and physical mixture of Felodipine with PVP-K 30 was recorded using FTIR spectrophotometer (FTIR-84008, Shimadzu, Japan) to study the incompatibility over the wavelength range of 4000–400/cm by preparing a dispersion of samples in KBr [14,15] DSC analysis DSC-Thermogram of HPMC-E 5, PVP-K 30, Felodipine, physical mixture of Felodipine with HPMC-E 5, and physical mixture of Felodipine with PVP-K 30 were recorded using DSC (DSC-60, Shimadzu, Japan) to study the incompatibility. The heating rate of 10°C/min in the range of 3−400°C under inert nitrogen environment at a flow rate of 40 ml/min was used. The samples (2−3 mg) were put in aluminum sampling pan against empty aluminum pan as reference standard [14,16] Preparation of SDP of Felodipine SDP of Felodipine was prepared using solvent evaporation method using PVP-K 30 as hydrophilic polymeric carrier in drug:polymer ratio of 1:1, 1:2, 1:3, and 1:4 to find out the best ratio based on the improvement of water solubility. Weighed amount of Felodipine and PVP-K 30 was dissolved in ethanol to get a clear solution and was further stirred continuously at 40°C until complete solvent gets evaporated to obtain solid mass. Then, solid mass was passed through the sieve no. 44 and stored in a desiccator until used for further studies [15-17] Formulation of FnFDFs FnFDFs were prepared using solvent casting method by dissolving weighed amount of SDP of Felodipine (equivalent weight of 10 mg of Felodipine), HPMC-E 5 (40–45% w/w), and propylene glycol (10–15% w/w of polymer) in 10 ml of distilled water with continuous stirring on a digital magnetic stirrer at 800 rpm for 1 h. Subsequently, required amount of croscarmellose (1–5% w/w), methyl cellulose (1.2% w/w), tween-80 (2% w/w), citric acid (1% w/w), disodium EDTA (0.5% w/w), sorbitol (2% w/w), peppermint oil (Q.S.), and indigo carmine (Q.S.) was gradually added to the casting solution under constant stirring at 1200 rpm at room temperature until a clear solution was obtained. As solution became clear, casting solution was then stirred for 4 h at 100 rpm to disappear entrapped air blisters. The resulting solution was then casted on a fabricated glass mold lubricated with glycerin and allowed to dry completely at room temperature to form film. The dried films were carefully separated from the glass mold and cut to produce six squareshaped FnFDFs of 2 cm 2 with an approximate weight of 150 mg and were stored in double wrapped aluminum foils [18,19] Optimization of FnFDFs Formulation of FnFDFs was optimized using Box–Behnken design by applying design-expert to obtain desired and optimum characteristics. HPMC-E 5 (A), propylene glycol (B), and croscarmellose (C) were selected as an independent variable and tensile strength (R 1), disintegration time (R 2), and percentage drug dissolution (R 3) were selected as dependent or response variables. Subsequently, the experimental design was analyzed for various models such as quadratic, linear, 2 FI, and mean to find out the best fit model on the basis of

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 258 responses of the dependent variables. Further, statistical validity using ANOVA and cube plots were established to find the compositions of the optimized formulation [9,20,21] Evaluation of FnFDFs Uniformity of mass Twenty randomly selected FnFDFs were weighed on digital balance. Films of 2 cm 2 were cut from the different places of the casted film and then, average mass were calculated [18] Thickness It was determined using digital vernier caliper at five different points of films including four corners and middle point [18] Percentage drug content FnFDFs (2 cm 2 film containing equivalent of 10 mg of Felodipine) were dissolved in phosphate buffer pH 6.8. The samples were filtered using 0.45 μ membrane filter, diluted, and analyzed for percentage drug content by double beam UV visible spectrophotometer (UV 1800, Shimadzu, Japan) at 237.1 nm [22] Folding endurance It was determined by repeatedly folding the film at the same place until it broken. The number of folding times that film had taken to broke was noted and considered as folding endurance [23] Surface pH FnFDFs were moistened with distilled water and pH at the metaphase of water and film was recorded using pH meter (MKVI, Systronics, Ahmedabad) [24] Moisture uptake It was determined by placing FnFDFs (2 cm 2 ) in a desiccator for 24 h to ensure the complete drying of the film before the actual test. FnFDFs were then weighed in dry form and further placed at 75% of RH for 1 week. Then FnFDFs were reweighed and percentage increased weight as a moisture uptake was noted [22] Percentage swelling It was determined by placing previously weighed FnFDFs (2 cm 2 ) in a beaker and then poured 50 ml of phosphate buffer pH 6.8 on it. The increased weight of the FnFDFs was calculated after 60 min by removing it from beaker using the following formula: % S=(Ws-Wi)/Wi×100 Where, % S is percentage swelling, Ws is the weight of swollen film , and Wi is initial weight of film at zero time [25] Percentage elongation It was determined by stretching FnFDFs with sufficient force required to exceed the elastic limit of the film up to the breaking of the film. It was calculated by using following equation: [23] % Elongation=Increase in length at breaking point (cm)/ Original length (cm) ×100 Tensile strength FnFDFs (2 cm 2 ) were placed and fixed between two clamps of tensile tester positioned at the distance of 2 cm and load or force required to break the film was measured by pulling the bottom clamp with 30 inch/min. Then, tensile strength was calculated using the following formula: [26,27] Tensile strength (N/m 2 )=Load at failure × 100/cross-sectional area of the film In vitro disintegration time FnFDFs (2 cm 2 ) were allowed to disintegrate by putting them in a 20 ml of phosphate buffer pH 6.8 in glass petri plate with mild agitation. The time at which FnFDFs broke or disintegrated was noted as disintegration time with stopwatch [22] In vitro drug dissolution or release study The in vitro drug dissolution test of FnFDFs (2 cm 2 film containing equivalent of 10 mg of Felodipine) was carried out using USP paddle type dissolution testing apparatus (TDT-08 L, Electrolab, Mumbai) and phosphate buffer pH 6.8 (300 ml) as dissolution medium up to 10 min. A piece of metal wire was attached with FnFDFs to avoid their floating property in dissolution medium and then placed at the bottom of the vessel. This study was performed at 37 ± 0.5°C and stirring rate of 50 rpm. Further, aliquots of 1 ml was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min and 1 ml of the fresh dissolution medium was added to the vessel to maintain the volume of dissolution medium. Collected aliquots were filtered using 0.45 μ membrane filters and subsequently analyzed using double beam UV visible spectrophotometer (UV 1800, Shimadzu, Japan) at 237.1 nm against blank. The percentage drug dissolution was calculated and further mechanism of drug release was determined by fitting the dissolution data into various kinetic models using DDsolver 1.0 software [22,28] Stability study The stability study of optimized and validated FnFDFs was performed as per ICH Q 1 A guidelines to assess their physical and chemical stability. FnFDFs were stored in two different conditions at 25 ± 2°C/60 ± 5% RH and 40 ± 2°C/75 ± 5% RH up to 6 months. The samples were collected at 0, 30, 60, 90, 120, and 180 days and were analyzed for the estimation of physical parameters such as appearance and weight of film

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 259 and chemical parameter such as percentage drug content and surface pH [9,25] Surface morphology using scanning electron microscope (SEM) Surface morphology of optimized and validated FnFDFs was observed using SEM (JEOL, JSM 5600, USA) at accelerating voltage of 0.5 kv–30 kv, resolution of 3.5 nm, and magnification of X 18-1500 X. The sample of film was placed in a sample holder of SEM and images were developed to observe surface morphology [24,29] Ex vivo permeation study Ex vivo permeation study for optimized and validated FnFDFs (2 cm 2 film containing equivalent of 10 mg of Felodipine) was performed by using a Franz diffusion cell and membrane of porcine oral mucosa. Phosphate buffer pH 6.8 (13 ml) was used as a diffusion medium of receptor compartment and temperature was maintained at 37 ± 0.5°C. FnFDFs was fixed on the membrane at mid of receptor and donor compartment and diffusion medium was stirred at low speed. Further, aliquots of 1 ml was collected at 0, 1, 2, 3, 4, 6, 7, 8, 9, and 10 min and 1 ml of the fresh dissolution medium was added to the receptor compartment to maintain the volume of the diffusion medium. Collected aliquots were filtered using 0.45 μ membrane filter and subsequently analyzed using double beam UV visible spectrophotometer (UV 1800, Shimadzu, Japan) at 237.1 nm against blank [30] In vivo pharmacokinetic study In vivo pharmacokinetic study for FnFDFs was approved by the Institutional Animal Ethical Committee (IAEC) of Acropolis Institute of pharmaceutical education and research, Indore (Reg. No: 1627/PO/Re/S/2012/CPCSEA). Animals (7–8-week-old, male Sprague-Dawley rats were used and were accommodated with easy access of food and water at room temperature under ambient condition) were divided into three groups as follows: Group-1 was the control group which was treated with saline, group-2 was the standard group which was treated with standard formulation, and group-3 was the test group which was treated with FnFDFs. Animals of standard group were treated with 1 ml of suspension containing 10 mg of Felodipine by oral administration. Then, FnFDFs of 2 cm 2 (containing equivalent of 10 mg of Felodipine) were cut into four small pieces and were placed between the cheeks of oral buccal cavity of rats of test group. Subsequently, aliquots (blood sample) of 0.25 ml were withdrawn from the tail vein and collected in a heparin-containing capillary tube at 0, 0.25, 0.5, 1, 2, 3, 6, 12, 24, and up to 48 h. Further, plasma was separated from blood using protein precipitation method by mixing it with 1 ml of acetonitrile and 5 ml of isopropyl alcohol (as internal standard) with vortexing and centrifuged for 10000 rpm up to 10 min. Then, supernatant was collected, mixed with mixture of acetonitrile:water (7:3) and analyzed by HPLC. Further, the concentration of Felodipine in plasma was determined and plotted with respect to time and meaningful in vivo pharmacokinetic parameters were calculated [31-33] RESULTS AND DISCUSSIONS Solubility determination Results of this study suggested the poor aqueous solubility of Felodipine in distilled water (0.0436 ± 0.001 mg/ml), phosphate buffer pH 6.8 (0.0424 ± 0.001 mg/ml), and suggested high solubility in organic solvents such as ethanol (0.916 ± 0.013 mg/ml) and acetonitrile (0.731 ± 0.011 mg/ml) FTIR analysis Recorded FTIR spectrums of different components are shown in Figure 1. FTIR spectrums of a physical mixture of Felodipine with HPMC-E 5 and physical mixture of Felodipine with PVP-K 30 showed the major peaks of both the components when compared to that of single-component spectrum. There were no incompatibilities found between drug and excipient in their physical mixture DSC analysis DSC-Thermograms of different components are shown in Figure 2. Thermogram of HPMC-E 5 showed a very small peak at 281.11°C which indicated its melting point. Thermogram of PVP-K 30 showed a broad endothermic peak at 92.34°C indicated the melting point of PVP-K 30. Thermogram of Felodipine showed the sharp endothermic peak at 147.32°C indicated the melting point of Felodipine and the high intensity of peak also revealed the highly crystalline nature of Felodipine. Thermogram of a physical mixture of HPMC-E 5 and Felodipine showed one small sharp endothermic peak at 146.83°C indicated the presence of Felodipine and suggested no incompatibility. Low intensity of the peak of Felodipine also revealed the suppression of its crystalline behavior in a mixed form with HPMC-E 5. Thermogram of a physical mixture of PVP-K 30 and Felodipine showed one endothermic peak at 146.24°C indicated the presence of Felodipine and suggested no incompatibility. The reduced intensity of the peak of Felodipine revealed its presence in molecular dispersion form with PVP-K 30 Preparation of SDP of Felodipine Results revealed a maximum increase in the water solubility for the drug–polymer ratio of 1:3 in comparison with 1:1 and 1:2. However, SDPs had not shown any significant increase in the solubility of Felodipine on further increasing ratio up to 1:4.

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 260 Formulation of FnFDFs FnFDFs were prepared by using solvent casting method. Each film contained SDP of the equivalent of 10 mg of Felodipine. The casting solution was prepared in distilled water without the use of organic solvent and was casted on fabricated glass mold to yield films of uniform size of 2 cm 2 with an approximate weight of 150 mg Optimization of FnFDFs There were total 13 runs for Box–Behnken experimental design as shown in Table 1, which were trialed in a randomized fashion to avoid chances of bias. The effects of different levels (low, medium and high) of independent variables on response variables were investigated. The linear model was found be the best fit for all response variables Effect of independent variables on tensile strength The effects A, B, and C on the tensile strength were determined and recorded in the form of cube plot as shown in Figure 3. It suggested the increased value of tensile strength with the increased concentration of both HPMC-E 5 and propylene glycol. However, the optimum combination of HPMC-E 5 and propylene glycol also affected the tensile strength significantly. The change in concentration of croscarmellose had not shown any significant variation on the tensile strength of FnFDFs. It revealed the improved mechanical strength of the FnFDFs at a higher concentration of HPMC-E 5 and propylene glycol Effect of independent variables on disintegration time The effects of A, B, and C on the disintegration time were determined and recorded in the form of cube plot as shown in Figure 4. It suggested decreased disintegration time with the increased concentration of croscarmellose and propylene glycol and increased disintegration time with the increased concentration of HPMC-E 5. However, HPMC-E 5 being a hydrophilic polymer in combination with propylene glycol had not found to cause any significant increase in disintegration time. It revealed fast disintegration of the FnFDFs at a higher concentration of croscarmellose and propylene glycol Effect of independent variables on percentage drug dissolution The effects of A, B, and C on the percentage drug dissolution were determined and recorded in the form of cube plots as shown in Figure 5. It suggested increased percentage drug dissolution with the increased concentration of propylene glycol and croscarmellose. However, the increased concentration of HPMC-E 5 caused very slight decrease in percentage drug dissolution from FnFDFs but being hydrophilic in nature, it had not affected significantly and facilitated a better dissolution of Felodipine in proper combination with propylene glycol. It revealed over 97% of drug dissolution at a higher concentration of propylene glycol and croscarmellose The statistical data of ANOVA test and linear equations for tensile strength, disintegration time, and percentage drug dissolution are shown in Table 2. R 2 values were found to be more than 0.9 for all response variables and the difference between adjusted and predicted R 2 value was also found to be below 0.2. It indicated that linear model could be used to navigate the design space. Further, formulation of FnFDFs was validated using an optimized concentration of independent variable and validated results are shown in Table 3. Validated values of response variables were found to be close to that of optimized values Evaluation of FnFDFs Uniformity of mass Results of determination of uniformity of mass, thickness, % drug content and folding endurance of FnFDFs are shown in Table 4. The average mass of optimized and validated FnFDFs was found to be 148.13 ± 0.69 mg. The mass of the film cut from the different places was found to be uniform Figure 1: Recorded FTIR spectrums of different components Figure 2: Recorded DSC-thermograms of different components

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 261 Table 1: Box–behnken experimental design for FnFDFs Batch No. Independent variables HPMC‑E 5 (A) % Propylene glycol (B) % Croscarmellose (C) % FnFDF-1 42.5 15 1 FnFDF-2 40 12.5 1 FnFDF-3 42.5 10 5 FnFDF-4 40 15 3 FnFDF-5 45 10 3 FnFDF-6 42.5 12.5 3 FnFDF-7 42.5 15 5 FnFDF-8 40 10 3 FnFDF-9 45 15 3 FnFDF-10 40 12.5 1 FnFDF-11 40 12.5 5 FnFDF-12 45 12.5 5 FnFDF-13 42.5 10 1 Table 2: Summary of statistical analysis of linear model for FnFDFs Parameters Observed values for Tensile strength Disintegration time Percentage drug dissolution R 2 0.9232 0.9445 0.9783 Adjusted R 2 0.8976 0.9260 0.9711 Predicted R 2 0.8323 0.8755 0.9577 SD 0.0007 0.6515 0.2252 % CV 0.3357 3.00 0.2356 Linear equation R 1=+0.1589+0.0009 A+0.0004 B+ 0.00002 C R 2=−28.5663+1.1965 A+ 0.0515 B−0.2458 C R 3=+122.9653−0.6632 A+0.03 05 B+0.0547 C Thickness The thickness of optimized and validated FnFDFs was found to be 0.246 ± 0.013 mm. Results revealed increased thickness of FnFDFs with increased concentration of HPMC-E 5 and propylene glycol. The thickness of the film cut from the different places was found to be uniform Percent drug content Percent drug content of optimized and validated FnFDFs was found to be 98.62 ± 1.11%. Results suggested a good uniformity of content in the FnFDFs without any significant variation Figure 3: Cube plot of effects of A, B, and C on the tensile strength of FnFDFs

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 262 Table 3: Optimized and validated values of independent and response variables for FnFDFs Type of variable Variables Optimized value Validated value ( n =3) Independent HPMC-E 5 (% w/w) 44.6146 44.61 Propylene glycol (% w/w) 14.4107 14.41 Croscarmellose (% w/w) 4.6307 4.63 Response or dependent Tensile strength (N/m 2 ) 0.2059 0.201±0.002 Disintegration time (seconds) 24.4219 22.84±0.31 Percent drug dissolution (%) 94.0669 97.09±1.54 Table 4: Results of uniformity of mass, thickness, % drug content and folding endurance (n=3) S. No. Batch No. Uniformity of mass of 2 cm 2 of film (average weight of 20 films in mg±SD) Thickness (average in mm±SD) Percent drug content (average in %±SD) Folding endurance (average of times folded±SD) 1 Batch No. 1 144.16±1.06 0.232±0.014 98.45±1.11 216±1 2 Batch No. 2 148.72±1.13 0.247±0.011 98.73±1.01 219±2 3 Batch No. 3 143.38±0.98 0.231±0.008 98.32±1.03 213±1 4 Batch No. 4 141.14±1.13 0.216±0.012 97.95±1.12 203±2 5 Batch No. 5 149.25±0.92 0.245±0.014 98.64±1.05 219±3 6 Batch No. 6 145.83±1.11 0.234±0.013 98.42±1.11 216±1 7 Batch No. 7 146.17±1.15 0.238±0.011 98.71±1.12 217±1 8 Batch No. 8 142.26±1.17 0.219±0.012 98.15±1.13 197±2 9 Batch No. 9 149.18±1.02 0.248±0.015 99.02±0.82 221±1 10 Batch No. 10 143.23±1.21 0.212±0.009 98.13±1.08 206±1 11 Batch No. 11 142.91±1.18 0.216±0.011 98.11±1.04 199±2 12 Batch No. 12 149.84±1.11 0.247±0.014 98.13±0.73 220±1 13 Batch No. 13 146.57±1.23 0.231±0.016 98.51±1.07 215±2 14 Optimized 148.13±0.69 0.246±0.013 98.62±1.11 221±1 Folding endurance Folding endurance of optimized and validated FnFDFs was found to be 221 ± 1. Results suggested the increased value of folding endurance with increasing the concentration of HPMC-E 5 and propylene glycol Surface pH Results of determination of surface pH, moisture uptake, percentage swelling, and percent elongation are shown in Table 5. Surface pH of optimized and validated FnFDFs was found to be 6.75 ± 0.01. Results of this study indicated almost Figure 4: Cube plot of effects of A, B, and C on disintegration time of FnFDFs

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 263 normal pH which revealed no chances of irritation to the oral mucosa after its administration Moisture uptake Percentage moisture uptake of optimized and validated FnFDFs was found to be 2.15 ± 0.04%. Results of this study revealed increased moisture uptake with increased concentration of propylene glycol. It also suggested the mild hygroscopic behavior of the FnFDFs when kept openly at high humidity Percentage swelling Percentage swelling of optimized and validated FnFDFs was found to be 42.68 ± 0.52%. Higher percentage swelling of FnFDFs suggested its suitability for rapid release of Felodipine due to increased absorption of phosphate buffer pH 6.8 Percent elongation Percent elongation of optimized and validated FnFDFs was found to be 13.98 ± 0.92%. Results suggested the increased mechanical strength of FnFDFs with increased concentration of HPMC-E 5 and propylene glycol Tensile strength Overall results of determination of tensile strength, in-vitro disintegration time and in-vitro percent drug dissolution or release studies of FnFDFs are shown in Table 6. The tensile strength of optimized and validated FnFDFs was found to be 0.201 ± 0.002 N/m 2 . It revealed the good mechanical strength of FDFs against rupture and breaks In-vitro disintegration time In-vitro disintegration time of optimized and validated FnFDFs was found to be 22.84 ± 0.31 s. It revealed the Table 5: Results of surface pH, moisture uptake, percentage swelling and percent elongation ( n = 3) S. No. Batch No. Surface pH (average pH ±SD) Moisture uptake (average %±SD) Percentage Swelling (average %±SD) Percent elongation (average %±SD) 1 Batch No. 1 6.65 ± 0.01 2.15 ± 0.03 40.65 ± 0.32 11.97 ± 1.11 2 Batch No. 2 6.62 ± 0.06 2.23 ± 0.02 39.41 ± 0.51 13.43 ± 1.12 3 Batch No. 3 6.69 ± 0.04 1.96 ± 0.02 41.07 ± 0.46 12.15 ± 1.11 4 Batch No. 4 6.71 ± 0.02 1.87 ± 0.01 41.76 ± 0.48 10.46 ± 1.02 5 Batch No. 5 6.74 ± 0.01 1.94 ± 0.03 42.37 ± 0.52 13.58 ± 1.11 6 Batch No. 6 6.61 ± 0.03 2.08 ± 0.01 41.09 ± 0.37 12.24 ± 1.13 7 Batch No. 7 6.68 ± 0.04 1.83 ± 0.04 40.75 ± 0.41 12.41 ± 1.12 8 Batch No. 8 6.63 ± 0.02 1.67 ± 0.05 41.49 ± 0.34 10.73 ± 1.11 9 Batch No. 9 6.65 ± 0.01 1.83 ± 0.04 40.98 ± 0.36 13.48 ± 1.12 10 Batch No. 10 6.66 ± 0.05 2.12 ± 0.06 41.07 ± 0.28 10.82 ± 1.14 11 Batch No. 11 6.73 ± 0.01 2.19 ± 0.01 41.16 ± 0.31 11.99 ± 1.12 12 Batch No. 12 6.62 ± 0.06 2.06 ± 0.01 42.78 ± 0.58 13.21 ± 1.12 13 Batch No. 13 6.69 ± 0.03 1.92 ± 0.02 39.97 ± 0.47 12.67 ± 1.11 14 Optimized 6.75 ± 0.01 2.15 ± 0.04 42.68 ± 0.52 13.98 ± 0.92 Figure 5: Cube plot of effects of A, B, and C on percentage drug dissolution of FnFDFs

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 264 Table 6: Results of tensile strength, disintegration time and % drug dissolution of FnFDFs (n=3) S. No. Batch No. Tensile strength (average in N/m 2 ±SD) In-vitro disintegration time (average seconds±SD) In-vitro percent drug dissolution (average %±SD) 1 Batch No. 1 0.204±0.002 22.21±0.05 95.37±1.14 2 Batch No. 2 0.201±0.001 20.19±0.09 96.98±1.13 3 Batch No. 3 0.202±0.001 22.20±0.11 95.26±1.08 4 Batch No. 4 0.202±0.002 19.18±0.08 97.14±1.05 5 Batch No. 5 0.205±0.003 25.17±0.07 93.69±1.11 6 Batch No. 6 0.204±0.002 22.18±0.09 95.32±1.12 7 Batch No. 7 0.205±0.001 21.22±0.10 95.67±1.06 8 Batch No. 8 0.200±0.002 18.19±0.06 97.04±1.12 9 Batch No. 9 0.206±0.001 26.17±0.04 93.73±1.11 10 Batch No. 10 0.201±0.002 20.21±0.07 96.35±1.13 11 Batch No. 11 0.200±0.002 19.22±0.08 97.12±1.09 12 Batch No. 12 0.205±0.001 24.21±0.09 93.52±1.11 13 Batch No. 13 0.201±0.002 22.19±0.11 95.31±1.12 14 Optimized 0.201±0.002 22.84±0.31 97.09±1.54 Figure 7: SEM image of FnFDFs fast disintegration of the FnFDFs, and it facilitated faster dissolution of Felodipine In-vitro percent drug dissolution or release study In-vitro percent drug dissolution of optimized and validated FnFDFs was found to be 97.09 ± 1.54%. Graph of in-vitro Figure 6: In-vitro percent drug dissolution of optimized and validated FnFDFs percent drug dissolution study for optimized and validated FnFDFs is shown in Figure 6. It revealed more than 97% drug dissolution up to 10 min and thus indicated faster and almost complete drug dissolution. Further, to study the drug release mechanism from FnFDFs, the data of in-vitro percent drug dissolution were fitted into various release kinetic models and R 2 values are shown in Table 7. The maximum R 2 value for FnFDFs was found to be 0.9844 in first-order model and it revealed first-order drug release from FnFDFs Stability study Results of stability study of optimized and validated FnFDFs are shown in Table 8. Observations of study for both storage conditions indicated good physical and chemical stability based on the physical appearance, weight of film, percent drug content, and surface pH up to 6 months. However, FnFDFs stored at 25 ± 2°C/60 ± 5% RH had shown better stability in comparison with that stored at 40 ± 2°C/75 ± 5% RH.

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 265 Figure 9: In-vivo study of an oral suspension of Felodipine and FnFDFs Figure 8: Ex-vivo drug permeation study of optimized and validated FnFDFs Surface morphology using SEM SEM image of FnFDFs is shown in Figure 7. It indicated smooth surface with pores of around 5-10 µ. The porous structure of the film suggested increased amorphous nature of the FnFDFs which could predict to be disintegrated and dissolved rapidly in the presence of dissolution medium Ex-vivo permeation study The result of ex-vivo permeation study of optimized and validated FnFDFs is shown in Figure 8. Ex-vivo drug permeation of optimized and validated FnFDFs was found to be 89.98 ± 1.11% up to 10 min. It indicated around 90% drug permeation up to 10 min and thus suggested good tissue permeability of Felodipine from FnFDFs In-vivo pharmacokinetic study Comparative results of in-vivo study for oral suspension of Felodipine and FnFDFs are shown in Table 9 and Figure 9. Various in-vivo pharmacokinetic parameters were calculated for oral suspension of Felodipine and FnFDFs as shown in Table 10. Maximum absorption of Felodipine from oral suspension was found to be 242.14 ± 0.027 µg/ml up to 3 h. However, maximum absorption of Felodipine from FnFDFs through oral buccal cavity was found to be 579.80 ± 0.046 µg/ml up to 1–2 h. The result of the present study revealed the fast and rapid absorption of Felodipine from FnFDFs through oral buccal cavity with the improved bioavailability of approximately 90% by avoidance of first-pass metabolism in comparison with its oral suspension Table 7: Linear correlation coefficient (R 2 ) values of drug release kinetic modeling (n=3) Drug release model R 2 values Zero-order model 0.8766 First order model 0.9844 Higuchi model 0.9609 Korsmeyer-Peppas modeland n values 0.9806 and 0.627

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 266 Table 8: Results of stability study of FnFDFs ( n =3) Days Storage at 25±2°C/60±5% RH for 6 months Appearance Weight (Avg. mg±SD) Drug content (Avg. %±SD) Surface pH (Avg. pH±SD) 0 Transparent 148.13±0.69 98.62±1.11 6.75±0.01 30 No Change 148.10±0.97 98.59±0.59 6.74±0.03 60 No Change 148.08±1.02 98.57±0.83 6.73±0.01 90 No Change 148.06±1.05 98.55±0.64 6.70±0.01 120 No Change 148.03±0.93 98.53±0.95 6.69±0.03 180 No Change 147.99±1.12 98.49±0.87 6.68±0.02 Storage at 40±2°C/75±5% RH for 6 months 0 Transparent 148.13±0.69 98.62±1.11 6.75±0.01 30 No Change 148.08±0.67 98.53±0.74 6.72±0.02 60 No Change 148.04±0.84 98.47±0.86 6.70±0.01 90 No Change 147.98±1.11 98.41±0.92 6.69±0.02 120 No Change 147.93±1.03 98.36±0.89 6.65±0.01 180 No Change 147.88±1.08 98.32±0.95 6.62±0.01 CONCLUSION Preparation of SDP of the Felodipine using solvent evaporation method with PVP-K 30 up to 1:3 (drug to polymer ratio) found to remarkably increase the aqueous solubility of Felodipine. Results of evaluation parameters of optimized and validated FnFDFs revealed good mechanical strength, uniformity of content, optimum surface pH, faster disintegration time, almost complete drug dissolution or release, good ex-vivo permeation, and good stability up to six months. In-vivo pharmacokinetic study indicated faster absorption and around 90% oral bioavailability up to 1–2 h for Felodipine in form of FnFDFs through the buccal administration due to avoidance of first pass metabolism. Thus it can be concluded that FnFDFs could be commercially exploited for the treatment of hypertension using Felodipine with merits of faster onset of action, avoidance of extensive first pass metabolism, low dosage regimen, enhanced bioavailability and improved patient compliance REFERENCES 1. Hariprasanna RC, Kulkarni U, Ahmad QJ, Gururaj SK, Srinath B. A study on formulation and processing factor influencing the release of Felodipine. Int J Curr Pharm Res 2010;2:86-9 2. Bukka R, Dwivedi M, Nargund LV, Prakasam K. Formulation and evaluation of Felodipine buccal films containing polyethylene oxide. Int J Res Pharm Biom Sci 2012;3:1153-8 3. Teofilo V, Bruno S, Paulo C. Solid dispersion as strategy to improve oral bioavailability poor water soluble drugs. Drug Discov Today 2007;12:1068-75 4. Shah, NH, Phuapradit W, Zhang YE, Sandhu H, Zhang L, Table 9: Results of in-vivo pharmacokinetic study ( n =3) S. No. Time (h) Rat plasma drug concentration (average µ g/ml±SD) Oral suspension of Felodipine FnFDFs 1 0 0±0.00 0±0.00 2 0.25 62.23±0.016 177.39±0.007 3 0.5 98.19±0.028 462.17±0.040 4 1 147.17±0.038 530.38±0.063 5 2 202.38±0.069 579.80±0.046 6 3 242.14±0.027 394.19±0.010 7 6 183.26±0.041 209.31±0.033 8 12 93.28±0.039 70.18±0.031 9 24 22.17±0.020 51.03±0.047 10 48 5.257±0.035 11.52±0.059 Table 10: In-vivo pharmacokinetic parameters Parameters Oral suspension of Felodipine FnFDFs C max ( µ g/ml) 242.14±0.03 579.80±0.04 t max (h) 3.14±0.05 1.34±0.03 Ka ( µ g/h) 0.691±0.18 3.694±0.02 Ke ( µ g/h) 0.112±0.03 0.026±0.002 AUC ( µ g/ml/h) 2975.75±3.75 4613.91±2.14 Vd (l) 0.037±0.002 0.014±0.003 At 1/2 (h) 1.00±0.04 0.187±0.019 Et 1/2 (h) 6.184±0.017 26.66±0.27 Cl T (l/h) 0.0033±0.0003 0.0021±0.0001

Sharma, et al .: Development and evaluation of fast dissolving oral film of felodipine Asian Journal of Pharmaceutic s • Jan-Mar 2018 (Suppl ) • 12 (1) | S 267 Malick W. Approaches for improving bioavailability of poorly soluble drugs. Inf Healthcare 2008;3:51-104 5. Siddiqui MD, Garg G, Sharma PK. A short review on novel approach in oral fast dissolving drug delivery system and their patents. Adv Biol Res 2011;5:291-303 6. Park K. Dissolution mechanisms of felodipine solid dispersions. J Control Release 2014;188:101-3 7. Naziya K, Rao NG, Mahipal RB. Overview on fast dissolving oral films. Int J Chem Pharm Sci 2013;1:63-75 8. Figueroa LS, Bhakay A, Jackeline I, Rozo J, Pandya N, Romanach RJ, et al . Preparation and characterization of hydroxyl propyl methyl cellulose films containing stable BCS class II drug nanoparticles for pharmaceutical applications. Int J Pharm 2012;423:496-508 9. Chaudhary H, Gauri S, Rathee P, Kumar V. 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Preparation and evaluation of oral fast dissolving films of citalopram hydrobromide. Int J Biopharm 2012;3:103-6 20. Rathore AS, Winkle H. Quality by design for biopharmaceuticals: Commentary. Nat Biotech 2009;27:26-34 21. Low AQ, Parmentier J, Khong YM, Chai CC, Tun TY, Beraniaa JE, et al . Effect of type and ratio of solubilizing polymer on characteristics of hot melt extruded orodispersible films. Int J Pharm 2013;455:138-47 22. Meshad AN, Hagrasy AS. Characterization and optimization of orodispersible mosapride film formulations. AAPS J 2011;12:1384-92 23. Ketul P, Patel K, Patel M, Patel NM. Design and development of telmisartan fast dissolving film. Int J Adv Pharm 2014;4:1-5 24. Kumar GP, Phani AR, Prasad RG, Sanganal JS, Manali N, Gupta R, et al. Polyvinylpyrrolidone oral films of enrofloxacin: Film characterization and drug release. Int J Pharm 2014;471:146-52 25. Nagar M, Nagar M, Chopra V. Formulation and evaluation of mouth dissolving film of aripiprazole. 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Preparation of a fast dissolving oral thin film containing dexamethasone: A possible application to antiemesis during cancer chemotherapy. Eur J Pharm Biopharm 2009;73:361-5 32. Kumar SK, Nagabhushanam MV, Rao KR, Bhikshapathi DV. Preparation and in vivo evaluation of oral dissolving films containing sumatriptan succinate. Pharm Lett 2013;5:27-38 33. Nishimura M, Matsuura K, Tsukioka T, Yamashita H, Inagaki N, Sugiyama T, et al . In-vitro and in-vivo characteristics of prochlorperazine oral disintegrating film. Int J Pharm 2009;368:98-102 Source of Support: Nil. Conflict of Interest: None declared.

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