MATERIALS AND METHODS:

Materials:

Timolol maleate was received as gift sample from alcon labs, Banglore. Na CMC was procured from Rankem,Chennai. HPC was procured from Himedia laboratories, Mumbai and sodium alginate was procured from Loba Chemie Pvt. Ltd., Mumbai, India. All other chemicals were of analytical grade.

Methods:

Formulation of timolol maleate buccal films ⁵:

The films containing Timolol maleate were prepared using different ratios of (table no. 1) sodium carboxy methyl cellulose and hydroxy propyl cellulose, sodium alginateand sodium carboxy methyl cellulose. The polymers in different ratios were dissolved in the respective solvents. Then the drug was added slowly in the polymeric solution and stirred on the magnetic stirrer to obtain a uniform solution. Glycerol was used as plasticizer. Then the solution was poured on the glass moulds having surface area of 16 cm² and dried at the room temperature. Then the films were cut into 1x2 cm² films. Drug incorporated for each 1x2 cm² film was 10 mg.

Table No.1: Composition of buccal films of Timolol maleate

(D1 to D6)

Ingredients	Formulation code
Ingredients	D1	D2	D3	D4	D5	D6
Timolol maleate	80	80	80	80	80	80
SCMC	100	150	250	200	200	100
HPC	250	200	100	-	-	-
Sodium alginate	-	-	-	200	100	200
PVA	-	-	-	-	-	- -
PVP K-30	-	-	-	-	-	-
Xanthan gum	-	-	-	-	-	-
Carageenan gum	-	-	-	-	-	-
Glycerol (w/w)	1%	1%	1%	1%	1%	1%
Water(ml)	10	10	10	10	10	10

FTIR studies⁶:

FTIR spectra help to identify drug and to detect the interaction of the drug with the polymer and other excipents. IR spectroscopy of pure drug and physical mixture of drug with polymers was carried out using shimadzu FTIR to check the compatibility between drug and polymers. The FTIR spectra of drug with polymers were compared with the standard IR spectrum of the pure drug.

Evaluations of films:

Thickness ⁶

The thickness of films was measured by screw gauge micrometer with least count 0.01mm. The thickness uniformity was measured at three different sites and average of three readings was taken with standard deviation.

Uniformity of weight ⁷

The film of area 1x1 cm² was to be cut in different parts of the film and weigh in digital balance. The average weight and standard deviation values were calculated from the individual weights.

Content uniformity test ⁷

The film of area 1x2 cm² was dissolved in 100 ml phosphate buffer of pH 6.8. Then the solution was to be filtered through a filter medium. Then 1 ml was withdrawn from the above solution and diluted to 10 ml with phosphate buffer of pH 6.8. The absorbance of the solution was taken at 294nm and concentration was calculated. By correcting dilution factor, the drug content was calculated.

Moisture loss ⁸

The prepared films were weighed individually and kept in a desiccators containing anhydrous calcium chloride at room temperature. After 3 days the films were reweighed and the percentage moisture content was determined by using formula

Moisture uptake ⁹

The percent moisture absorption test was conducted to check the physical stability and integrity of the films at high humid conditions. The films were placed in the desiccators containing saturated solution of aluminium chloride, keeping the humidity inside the desiccators at 79.5 % R.H. After 3 days the films were taken and weighed the percentage moisture absorption of the films were calculated by using the formula.

Folding endurance ¹⁰

A specific area of strip was cut and repeatedly folded at the same place till it broke. The number of times the film could be folded without breaking gave the value of folding endurance.

Tensile Strength¹¹

The tensile strength was determined by the apparatus designed locally. The instrument was designed such that it had vertical wooden platform with fixed scale and attachments for two clips that holds buccal film under test. Out of the two clips one was fixed and other was movable. Weights were hanged to one end of pulley and the other end of pulley was attached with movable clip. The wooden platform was such fitted that it would not dislocate while the test is running. Three strips of film were cut having 2cm length and 2cm breadth. The thickness and breadth of strips were noted at three sites and average value was taken for calculation. The rate of change of stress was kept constant with the increment of 0.5g per 2 minutes. The elongation was observed and the total weights taken were used for calculation. The tensile strength was calculated by using following formula.

Percent Swelling Index ¹²

The polymeric films cut into 1 x 1 cm were weighed accurately and kept immersed in 50 ml of water. The films were taken out carefully at 5, 10, 30 and 60 minutes intervals blotted with filter paper to remove the water present on their surface and weighed accurately, the percent swelling is calculated using formula:

Surface pH ¹³

It is imperative that the buccal formulation should have good patient acceptance and compliance. For this, film should not cause any irritation on the mucosal membrane and hence, these parameters assume significance in development of any buccoadhesive drug delivery system. The surface pH was determined by the method similar to that used by Botten Berg et al. The combined glass electrode was used for this purpose. The films were kept in contact with distilled water for 1 hour in glass tubes. The surface pH was then noted by bringing a single glass electrode near the surface of film and allowing it to equilibrate for 1 minute.

Mucoadhesion strengths ¹¹

Mucoadhesion strength of the film was measured by using sheep buccal mucosa as model mucosal membrane. Fresh sheep buccal mucosa was obtained from a local slaughter house and was used within 2 hr of slaughtering. The mucosal membrane was washed with distilled water and then with phosphate buffer of pH 6.8.

The mucoadhesive strength measurement apparatus was fabricated locally. The mucoadhesive strength of the films was determined using this locally fabricated apparatus. The weight at which the film was detached was recorded. The mean value of three trials was taken for each set of formulations. After each measurement, the tissue was gently and thoroughly washed with phosphate buffer and left for 5 minutes before placing a new film to get appropriate results for the formulation.

Diffusion studies ¹⁴

Diffusion cell:

The diffusion studies were done to get an idea of permeation of drug through barrier from the buccal film. In the present study, fabricated diffusion cell was used. Diffusion cells generally comprise two compartments, one containing the active compartment (donor compartment) and the other containing receptor solution (receptor compartment), separated by barrier i.e. himedia dialysis membarane. The cell consisted of sampling port and temperature maintaining jacket. The outlet and inlet was connected with latex tube so the jacket had stagnant water inside and heat was provided by hot plate. The magnetic bead was used to stir the receptor solution using magnetic stirrer. The dialysis membarane was placed on receptor compartment and both compartments held tight by clamps.

Method:

Phosphate buffer of pH 6.8 was used as receptor solution. The volume of diffusion cell was 20 ml and stirred with magnetic bead. The temperature was maintained at 37 ± 1°C with the help of magnetic stirrer. The diffusion was carried out for 9 hours and 3 ml sample was withdrawn at an interval of 1 hour. The same volume of phosphate buffer pH of 6.8 was added to receptor compartment to maintain sink conditions and the samples were analyzed at 294nm.

Buccal acceptance study on human volunteers ¹⁵:

Buccal acceptance study on human volunteers of selected formulations without drug (placebo films) was carried out to,

a) Investigate bioadhesive performance of different polymers loaded in the films.

b) Investigate the acceptability of different polymers for use in mucoadhesive films and

c) Determine any irritation or any side effects produce by films.

Permission for buccal acceptance study on human volunteers was obtained from the institutional ethical committee. And the tests were conducted under the guidance of physician. The study was conducted on 10 human volunteers. Written consent was obtained from the volunteers before study. The volunteers were not allowed to take water or food starting from half an hour before the study. The volunteers were given different coded films along with written instruction sheets. They were instructed to press films against cheek for about 1 min without moistening the tablet after application. The volunteers were asked to record time of film insertion time of adhesion and event that occurs after the adhesion. At the end the volunteers were also asked to report for the irritation, taste, discomfortness, dryness of mouth, salivation and heaviness of cheek.

Curve fitting analysis¹⁶:

The mechanism of Timolol maleate release from the films was studied by fitting the dissolution data of optimized formulation in following models

1. Time versus cumulative percent drug released (zero-order kinetic model).

2. Time versus log cumulative percent drug remaining (First-order kinetic model).

3. Square root of time versus cumulative percent drug released (Higuchi’s model).

4. Log time versus log cumulative percent drug released (Korsmeyer-Peppas equation).

Based on the slope and the R² values obtained from the above models the mechanism of drug release was decided.

Stability studies ¹⁷

Stability studies were performed for 3 months for optimized formulation. All the stability samples (packed in the backing membrane (Aluminum foil) were prepared in triplicates and were kept for stability testing conditions, 25 ◦C/60%RH in Stability Chamber, serving as test condition as per ICH Guideline Q1A. Stability samples were evaluated for physicochemical parameters, drug content and diffusion study at each sampling point (1, 2 and 3 months).

RESULT AND DISCUSSION:

As a preformulation study for drug polymer compatibility by FTIR gave conformation about their purity and showed no interaction between drug and selected polymers. The physicomechanical evaluation (Table 2 and 3) indicates that the thickness of these films varied between 0.116±0.011 and 0.196±0.005 mm, the thinnest being formulation D1 and the thickest being formulation D 6. The weight variation of these formulated buccal films varied between 116.07 ± 0.833(D6) and 170.27 ± 0.813 g (D 2). Folding endurance was measured manually. The drug content (%) in all formulations varied between the range 89.74± 0.10% and 93.28±2.88%. This indicates that the drug dispersed uniformly throughout the polymeric film. Checking the physical stability of film at high humid conditions and integrity of the film at dry conditions, the films were evaluated for % moisture loss and % moisture uptake. Amongst all the formulation the high value of % moisture loss was observed in D5 and lowest value was observed in D2 .The high and low value of % moisture uptake was observed in D4 and D1 respectively. The highest folding endurance was observed in the case of D 2 (306.66±1.52) and the lowest in the case of D 4 (291.33±1.52). The range of folding endurance study ensured flexibility of these formulated buccal films. Considering the fact that acidic and alkaline pH may affect or cause irritation to the buccal mucosa and influence the rate of hydration of the polymers, the surface pH of films was determined by digital pH meter. The all prepared formulation of timolol maleate buccal film showing the pH range within the range of 5.97±0.05 to 6.58±0.12. All of formulation provides an acceptable pH in the range of salivary pH (5.0 to 7.0) hence cannot produce any irritation to buccal mucosa. Table 2 and 3 shows the results of physicochemical characters of timolol maleate buccal films.

Mucoadhesive strength:

The strength of film was dependent on the property of mucoadhesive polymers, which adheres to the mucosal surface and also on the concentration of polymer used. Among all the formulations D3 show maximum mucoadhesion strength of 20.67±0.49 and D6 show lowest mucoadhesion strength of 15.43±0.97. Mucoadhesive strength and mucoadhesive force of all formulations were calculated and mentioned in the Table. 3. It is observed that overall mucoadhesion strength of all the formulations was increased with increase in polymer concentration. The tensile strength was found to be in the range of 2.43±0.03 to 1.57±0.03 the formulation D2 (2.43±0.03) showed the best tensile strength. The tensile strength values for all the films are tabulated in the table 3.

Table 5: In vitro drug release data for formulation D1 to D6

Time (hr)	Cumulative % drug released(n=3) Mean±SD
Time (hr)	D1	D2	D3	D4	D5	D6
1	16.521±0.819	19.252 ±0.914	24.372 ±0.834	15.565 ±0.653	18.023 ±0.626	13.176 ±1.246
2	29.308 ±0.827	32.176 ±.917	38.678 ±0.924	29.370 ±0.722	32.674 ±0.720	26.417 ±1.574
3	38.023±0.627	42.283 ±0.819	47.718 ±0.321	41.331 ±1.007	44.092 ±0.445	38.757 ±1.740
4	45.243±0.738	48.284 ±1.036	54.500 ±0.719	47.578 ±1.018	51.203 ±0.465	44.724 ±1.194
5	52.104±0.929	54.315 ±1.048	62.109 ±0.536	55.043 ±0.658	59.221 ±0.185	52.002 ±1.471
6	59.959 ±0.627	62.283 ± 1.018	68.653 ±0.629	62.201 ±0.907	65.212 ±0.744	59.617 ±1.213
7	64.758 ±0.814	67.113 ±1.012	73.920 ±0.951	69.199 ±0.559	72.503 ±1.285	66.209 ±1.083
8	72.285 ±0.728	75.459 ±0.849	81.276 ±1.076	74.753 ±0.486	78.265 ±0.469	70.599 ±0.544
9	80.498 ±0.932	83.181 ±0.728	89.175 ±1.021	85.007 ±0.802	88.199 ±1.025	80.211 ±0.957

Figure 3: Time Vs cumulative % drug release (zero order kinetics) of formulation D1 to D3

Figure 4: Time Vs cumulative % drug release (zero order kinetics) of formulation D4 to D6

Table.7. Response of healthy human volunteers to various subjective parameters for D3 formulations.

SL NO

Criteria

Volunteer’s response (%)

Irritation

1. Nil

2. Mild

3. Moderate

4. Maximum

100

---

Fragment loss

1. Nil

2. Mild

3. Moderate

4. Maximum

---

Taste

1. Tasteless

2. Bitter

3. Sour

4. Sweet

5. Astringent

6. Any other

100

---

Heaviness/ discomforts

1. Less

2. Moderate

3. High

---

Secretion of saliva

1. Normal

2. Enhance

3. Decrease

---

Dryness of mouth

1. Yes

2. No

---

100

Detachment at the end

1. Easy

2. Normal

3. Difficult

---

Buccal acceptance study on human volunteers:

Buccal acceptance study on human volunteers of selected formulations without drug (placebo films) was carried out to,

a) for buccal acceptance study on human volunteers was obtained from the institutional ethical committee. The tests were conducted under the guidance of physician. The study was conducted on 10 human volunteers. Written consent was obtained from the volunteers before study.

Response of Healthy Human Volunteers to Various Subjective Parameters for D3 and D5 formulations shows in table.7 and 8.

Table.8. Response of healthy human volunteers to various subjective parameters for D5 formulation.

SL NO

Criteria

Volunteer’s response (%)

Irritation

1. Nil

2. Mild

3. Moderate

4. Maximum

100

---

Fragment loss

1. Nil

2. Mild

3. Moderate

4. Maximum

---

Taste

1. Tasteless

2. Bitter

3. Sour

4. Sweet

5. Astringent

6. Any other

---

Heaviness/ discomforts

1. Less

2. Moderate

3. High

---

Secretion of saliva

1. Normal

2. Enhance

3. Decrease

---

Dryness of mouth

1. Yes

2. No

---

100

Detachment at the end

1. Easy

2. Normal

3. Difficult

---

Table .9. Stability study for D3

Time (month)	Physical appearance	Drug content	% CDR at 9^thhr
0	No changes	89.74 %	89.17%
One month	No changes	89.39%	89.65%
Two months	No changes	89.41%	90.02%
Three months	No changes	88.96%	89.06%

Table .10. Stability study for D5

Time (month)	Physical appearance	Drug content	% CDR at 9^th hr
0	No changes	91.42 %	88.19%
One month	No changes	91.15%	88.03%
Two months	No changes	91.96%	88.76%
Three months	No changes	90.92%	89.08%

Human acceptance study was carried out for four optimized formulation D3 and D5 for various parameter as tested in table.7 and 8. There were no complaints for irritation on applicant of films and 100% response was seen for taste. Almost 75% of candidates commented that there was no fragment loss and there was normal secretion of saliva. There were average results for heaviness/ discomfort. There were no complaints from the volunteers for the dryness of mouth and detachment of the films were acceptable where in 65% volunteers showed it was normal and 40% reveled it was easy.

Stability studies:

The accelerated stability studies were carried out according to ICH guidelines. Optimized formulations D3 and D5 were packed in aluminum foil and this packed formulation was stored in ICH certified stability chambers (Thermo labs, Mumbai) maintained at 25⁰C ± 2⁰C and 60 % RH ± 5 % for 3 month. The films were evaluated before and after one month interval for period of three months to access any change in appearance, the drug content, and In vitro drug release.

The results of stability studies did not show any significant change in the physical appearance, drug content and in-vitro drug release studies of above two formulations as shown in the Table 9 and Table 10.

CONCLUSION:

Hence, we may conclude that, timolol maleate can be developed in the form of buccal films, with having good permeability. The drug release could be retarded for 9 hrs.

ACKNOWLEDGEMENT:

The authors are highly thankful to the chair person and management of Maratha Madal’s College of Pharmacy for providing all the facilities to carry out the research work.

REFERENCES:

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Received on 28.04.2012

Accepted on 08.05.2012

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(3): May-June 2012, 177-184

Formulation code	Thickness (mm) (n=3) Mean ±SD	Weight uniformity (n=3) Mean ±SD	% Drug content (n=3) Mean ±SD	% Moisture loss (n=3) Mean ±SD	% Moisture Uptake (n=3) Mean ±SD
D1	0.116±0.011	139.00±0.365	91.48±2.91	1.90±0.2	2.85±1.42
D2	0.116±0.005	170.27±0.813	91.56±2.93	1.85±0.80	2.77±1.38
D3	0.123±0.020	156.00±0.597	89.74±0.10	2.15±0.93	3.22±1.61
D4	0.183±0.057	150.25±0.577	91.39±2.92	4.16±1.80	5.72±2.38
D5	0.193±0.037	162.00±0.024	91.42±2.96	4.76±2.06	7.14±3.57
D6	0.196±0.005	116.07±0.833	93.28±2.88	3.17±1.37	4.76±2.38

Formulation code	Folding endurance (n=3) Mean±SD	Surface pH (n=3) Mean±SD	Tensile strength Kg/mm²(n=3) Mean± SD	Mucoadhesive strength (gm) (n=3) Mean±SD	Mucoadhesive force (n=3) Mean±SD
D1	306.33±1.52	6.35±0.08	2.28±0.04	16.77±0.48	1.64±0.04
D2	306.66±1.52	6.55±0.15	2.43±0.03	17.81±0.49	1.74±0.04
D3	300.66±0.57	6.58±0.12	2.54±0.03	20.67±0.49	2.02±0.06
D4	291.33±1.52	5.97±0.05	1.64±0.01	16.60±0.57	1.62±0.05
D5	297.33±1.15	6.13±0.15	1.61±0.01	18.82±0.63	1.84±0.06
D6	297.66±2.08	6.15±0.09	1.57±0.03	15.43±0.97	1.51±0.09

Sr. NO.	Formulation code	Percent swelling index in time (min) (n=3) Mean±SD
Sr. NO.	Formulation code	5	10	30	60
1	D1	3.80±1.64	7.22±0.93	29.62±1.82	37.34±0.93
2	D2	3.69±1.60	7.20±1.11	31.88±1.80	39.35±1.11
3	D3	4.29±1.85	8.33±1.60	34.10±1.76	41.27±1.60
4	D4	8.26±1.75	15.68±1.79	38.26±1.33	43.12±1.58
5	D5	9.44±1.09	17.77±1.69	31.65±1.08	46.67±1.69
6	D6	6.26±1.71	12.12±1.54	41.48±1.39	51.94±1.43

Formulation code	Zero Order	First Order	Higuchi	Korsemeyer peppas
Formulation code	R²	R²	R²	n	R²
D1	0.937	0.978	0.979	0.695	0.996
D2	0.908	0.985	0.972	0.640	0.995
D3	0.852	0.962	0.995	0.568	0.997
D4	0.941	0.965	0.976	0.738	0.976
D5	0.920	0.958	0.983	0.691	0.992
D6	0.950	0.981	0.972	0.787	0.988