Table 1. Composition of buccal films formulations

Formulation code	F1	F2	F3	F4	F5	F6	F7	F8
Losartan pot. mg	25	25	25	25	25	25	25	25
HPMC K(15M) mg	600	450	300	450	300	450	300	450
Eudragit RS100 mg	-	150	300	-	-	-	-	-
Eudragit RL100 mg	-	-	-	150	300	-	-	-
SCMC mg	-	-	-	-	-	150	300	-
EC mg	-	-	-	-	-	-	-	150
Glycerin ml	0.0588	0.0588	0.0588	0.0588	0.0588	0.0588	0.0588	0.0588

Drug incorporated in each film: 25 mg and Polymer incorporated in each film: 54.34 mg

Table 2. Physical characterization of prepared buccal film

Formulation Code	Weight Uniformity (mg)± SD	Thickness (mm) ± SD	Folding Endurance	Surface pH ± SD	Content Uniformity mgs ± SD
F1	78.75 ±1.32	0.20 ±0.04	216±1.8	5.6± 0.06	99.46±0.25
F2	79.48 ±1.14	0.22±0.01	207±1.3	5.6±0.05	98.67±0.21
F3	78.72 ±0.63	0.21±0.02	212±1.9	6.0±0.09	97.67±0.45
F4	78.01 ±0.42	0.23±0.01	254±1.7	5.3±0.07	98.75±0.28
F5	79.28 ±0.33	0.20±0.04	244±1.5	5.6±0.04	97.38±0.40
F6	79.24 ±0.69	0.23±0.08	238±1.4	6.0±0.08	98.89±0.35
F7	79.75 ±0.36	0.24±0.02	246±1.9	6.3±0.04	98.81±0.46
F8	77.85 ±0.92	0.21±0.03	260±2.1	6.0±0.11	88.46±0.50

Table 3: Physicochemical Characteristics of Buccal film

Formulation Code	Swelling Index	Tensile strength (kg/mm²)	Bioadhesive Strength ( g)	Force of Adhesion (n)
F1	1.20±0.01	1.850±0.012	8.87	0.087
F2	0.61±0.04	1.261±0.091	9.46	0.092
F3	0.57±0.03	0.985±0.014	8.72	0.085
F4	1.25±0.08	0.875±0.026	7.57	0.074
F5	0.99±0.06	1.050±0.038	8.86	0.086
F6	2.26±0.08	1.325±0.041	10.10	0.102
F7	2.35±0.02	0.985±0.046	12.10	0.118
F8	0.84±0.03	1.675±0.029	7.48	0.073

The in vitro drug release study for Losartan potassium films were carried out in phosphate buffer 6.6 pH for six hours.In order to find out the drug release and mechanism, this was predominantly influence, the drug release from the artificial membrane. The in vitro drug release data subjected to different mode of graphical treatment. The in vitro drug release data for Losartan potassium films were calculated in percentage. The formulations F1, F2, F3, F4, F5, F6, F7 and F8, Shown 80.25%, 81.24%, 75.68%, 79.73%, 88.46%, 86.33%, 94.49%, and 73.97% drug release respectively (fig.1). The maximum drug release was shown by formulation F7 due to higher concentration of SCMC compared to other formulation it having higher drug release values due to swelling property. The lower drug release was shown by formulation F8 (73.97%) due to ethyl cellulose which is water insoluble.

Preparation of buccal film:

Table No 1 contains the composition of various formulations used for the buccal film. For the formulation F1, F6 & F7 accurately weight quantity of hydrophilic polymer Hydroxypropyl methyl cellulose and Sodium carboxy methyl cellulose, dissolved in distilled water allow to swell for 5 min. The accurately weight quantity of losartan potassium dissolved in ethanol, the solution was mixed according to required proportion. Two drop of glycerin was added as plasticizer (0.058 ml). The polymeric solution was poured over the mercury surface in Petri dish. The formulation F2, F3, F4. F5 & F8 containing hydrophobic polymer Eudragit RS 100, Edragit RL100, and Ethyl cellulose was dissolved in acetone.

The solution of HPMC and hydrophobic polymer mixed together with drug was. Two drop of (0.058 ml) glycerin incorporated as plasticizer. The solution was poured over the mercury surface in Petridish. Then using inverted funnel over petri dish, allow controlled evaporation of solvent. These were left undisturbed at room temperature for three to four

EVALUATION OF BUCCAL PATCHES:

Thickness and Weight uniformity:

The thickness of each film was measured by using screw gauge at five different positions of the films and the average was calculated. Each film of 2 cm diameter was weighed individually on electronic balance and average weight of three films was found ⁷.

Folding endurance:

Folding endurance of the films was determined by repeatedly folding one film at the same place till it broke or fold manually which was considered satisfactory to reveal good films properties. The number of times of films could be folded at the same place without breaking gave the value of the folding endurance. This test was done on five films of size 2 cm X 4 cm ⁸.

Swelling studies:

Three films were tested for each formulation, buccal films of 2 cm diameter cut and placed in 2%agar plate and incubated at 37⁰C, at regular one hour interval (to 3 hr) the films was removed from Petri dish and excess water removed carefully using the filter paper. The swollen films were reweighed and the swelling index was calculated. The experiments were carried out in triplicate and average values were reported ⁹.

Swelling index = W₁ -- W₀/ W₀

Where, W₁= Final weight and W₀= Initial weight

Tensile strength:

Tensile strength of the films was determined with fabricated tensile apparatus containing one weighing pan which was attached to films fixed with clamps to both ends. A small strip measuring 4cm X 1cm was cut with sharp blade. One end of film was fixed by placing in the film holder clamp. Another end of the film was fixed with the help of another clamp. The strip straight while stretching and. A thread was tied to the clamp and passed over pulley and a small pan attached to another end to hold weight. The film was pulled by pulley system. Weight gradually added to the pan to increases the pulling force till the film was broken. The tensile strength values were calculated using the formula ³.

Tensile strength (kg/mm2) =

Force at break (kg) / Initial cross sectional area of the film

Surface pH:

Buccal films were left to swell for 2h on the surface of the agar plate, prepared by dissolving 2% (w/v) agar in warmed isotonic phosphate buffer of pH 6.6 under stirring and then poured the solution into the Petri dish allowed to stand till gelling at room temperature. The surface pH was measured by means of pH paper placed on the surface of the swollen films ⁷.

Content uniformity:

A film of size 2cm diameter was cut and dissolved in 100 ml of phosphate buffer solution in standard flask and it was shaking for 6 hrs at consecutive interval the solution was filtered and appropriately diluted and absorbance measured at 250 nm ⁷.

Measurement of Bioadhesion force:

The Sheep cheek pouch was carefully excised without removing connective and adipose tissue and washed with saline. The tissue was stored in saline; later the membrane was placed over the surface of glass slide mounted on lower Teflon block (B) and secured. The block (A) was then lowered in to the glass container, which was then filled with phosphate buffer of pH 6.6 and kept at 37 ± 1⁰ C, such that the buffer just reaches the surface of mucosal membrane and kept it moist. One formulation at a time was taken and struck to the lower surface of upper Teflon block (A) with a standard cyanoacrylate adhesive. The beaker containing mucosal tissue containing the lower block (B) was adjusted over the base of the balance so that the mucosal tissue exactly below the upper block. A preload weight of 5 gm was placed above the expanded cap, left for 5 and 10 min, after which the films binds with mucin. The weights were then removed slowly and gradually after binding the films with mucin the water were added slowly on the right side pan till the films separates from the mucosal surface/membrane. The weights required for complete detachment was noted the force required for detachment expressed in grams. Procedure was repeated for two more films of each formulation of different batches. Average was computed and recorded ¹⁰.

Bioadhesive force (F) = [W x g] / 1000

In Vitro Release Studies Of Buccal Films:

The diffusion study was carried out by using modified diffusion cell. The method used by commercial semipermeable membrane, a films of size 2 cm diameterwas cut and placed on semipermiable membrane, the membrane was placed between the donor compartment containing phosphate buffer solution 6.6 pH (salivary pH) and reservoir compartment phosphate buffer 7.4 (blood pH) the entire surface of membrane was in contact with receptor compartment was agitated magnetic stirrer at 37⁰C. Sample of 1ml were withdrawn from receptor compartment and replaced by equal volume of fresh media. The withdrawn sample was analyzed using ultra violet spectrophotometer (Systronic 118) at 250 nm using phosphate buffer as a blank¹¹.

Ex Vivo Diffusion Study:

Diffusion study was carried out by using modified diffusion cell, by using fresh sheep cheek pouch membrane ¹¹. A film of size 2 cmdiameter was cut and placed on model mucosal embrane. The membrane was placed between the donor compartment containing phosphate buffer pH 6.6 (salivary pH) and reservoir compartment phosphate buffer 7.4 (blood pH) the entire surface of membrane was in contact with receptor compartment was agitated magnetic stirrer at 37⁰C. Sample of 1ml were withdrawn from receptor compartment and replaced by equal volume of fresh media. The withdrawn samples were analyzed using ultra violet spectrophotometer at 250 nm using phosphate buffer as a blank.

Scanning Electron Microscopy Study:

The morphological study of prepared buccal film was done by SEM study, using double-backed adhesive tape and then sputter coated for 8 min at 1.1 LV under argon atmosphere with gold palladium before examination under the scanning electron microscope (JEOL JSM-6100 Scanning Electron Microscope, Japan). The images were captured on an Ilford PANF 50 black and white 35mm film.

RESULTS AND DISCUSSION:

Physical characteristics of the prepared buccal film containing individual polymers are shown in Table 2. The patches were ranges 0.20±0.040 to 0.24±0.02 mm in thickness. The mass ranged from 77.85 to 79.75 mg. The surface pH of all formulations was within 5.3 to 6.3 units of the neutral pH and hence no mucosal irritation was expected. The recorded folding endurance of the patches was > 200 times.

The swelling behavior of formulations was observed on 2%agar plate. The swelling index was higher in formulation F6 and F7, i.e. 2.26 and 2.35 respectively the good swelling property shown by the formulation containing HPMC and SCMC due to high concentration of SCMC among all the formulation (Table 3).

The bioadhesive strength of formulation F7 was higher (i.e.12.10 gm), due to higher concentration of SCMC among all the formulation SCMC having higher mucoadhesive property and less in F8 (i.e.7.48gm). The formulation F7 containing 2:2 ratio of HPMC=SCMC the SCMC having anionic charge it bind to mucosal glycoprotein and form a strong bioadhesion to buccal mucosa Table 3.

The tensile strength were found for the all the formulation .The force required to break the film strip is more in formulation F1 containing HPMC 4:0 ratio, because of good cross-linking with drug molecules.

Table 4. Release Kinetic Study Of Prepared Buccal Film

Formulation code	In vitro drug release	Zero order drug release	Higuchis regression
F1	80.25%	0.950	0.986
F2	81.24%	0.908	0.994
F3	75.68%	0.958	0.954
F4	79.73%	0.987	0.973
F5	88.46%	0.944	0.968
F6	86.33%	0.963	0.973
F7	94.49%	0.945	0.993
F8	73.97%	0.894	0.982

Fig.1 Comparative dissolution profile

Table 4 indicates that, square root of time verses cumulative % drug release was maximum from formulation F2 (HPMC: EUDRAGIT RS 100=3:1 ratio) i.e. 0.994 followed matrix diffusion.

Ex vivo drug diffusion:

The ex vivo drug diffusion study was carried for formulation F7 the diffusion study was carried out by using freshly excised goat buccal pouch membrane. The ex vivo drug diffusion was 65.32%. for six hours. The ex vivo drug diffusion data further proceed for the regression coefficient using MS-excel statistical program. The R²value for formulation F7 was 0.995 it shown the zero order drug release. The formulation F7 was shown good Ex vivo-In vitro correlation i.e. 0.952. According to the Higuchi´s plot the all formulation had shown the matrix diffusion drug release pattern.

The surface morphology of optimized batch F7 found at different set. The SEM photograph (fig. 2) indicates the uniform dispersion of polymeric solution with drug molecule, which may be suitable for the matrix system (fig. 2).

Fig. 2: Scanning Electron Microscopy of prepared buccal film batch F7.

CONCLUSION:

The formulation F7 containing (HPMC:SCMC = 2:2) ratio was found to be best among the all formulation batches because of its consistent release rate for six hours and extent of drug release was 94.49%. Graphical treatment of formulation F7 according to Higuchi´s equations has shown the drug release was diffusion mediated. The good correlation observed between the In vitro and ex vivo profile revealed the ability of the formulation F7 to reproduce the in vitro release pattern through the biological membranes. Hence the polymer HPMC and SCMC the objectives of present study as improving bioavailability by avoiding first pass metabolism, reduced the side effect due to higher dosing and improve patient compliance.

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