Development and In Vitro Evaluation of Mucoadhesive Buccal Gels of Betamethasone Sodium Phosphate

INTRODUCTION

Conventionally oral submucous fibrosis (OSMF) is treated using corticosteroids in the form of injections and gels.¹ Prevailing gel formulations for OSMF treatment are associated with poor retention at the absorption surface resulting in minimum effective treatment and patient noncompliance.² OSMF treatment requires specific and prolonged drug action on to the affected area of submucosa. In this view, corticosteroids at low dose seem to be better drug of choice.³ Hence, the present study includes formulation and evaluation of mucoadhesive gel of betamethasone sodium phosphate (BSP) using synthetic hydrophilic mucoadhesive polymers Na CMC and HPMC, which will bind with the underlying oral mucosa facilitating targeted drug delivery for better drug therapy and immediate onset of action.⁴

The sweet taste, easy dispersible and spreadable properties of glycerin used in the formulation leads to better patient compliance and at the same time it also has anti-inflammatory action⁵ , which effectively reduces burning sensation observed in OSMF.

OSMF is a generalized pathological condition of oral mucosa where in connective tissue of lamina propria and juxta- epithelial undergo drastic fibroelastic change causing mucosal rigidity of varying intensity leading to increased risk of oral cancer.⁶ The main symptoms and signs of OSMF include inflam¬mation, hypovascularity, depapillation, blanching, dryness, burning sensation, mouth ulceration, hypomobility of tongue and soft palate associated with difficulty in maintaining oral hygiene, improper speech, abnormal mastication and difficulty in swallowing.⁷ Increase in fibrinogen and its degradation products in plasma observed in OSMF cause excessive deposition of fibrin in connective tissue and this brings about progressive restriction of mouth opening. It also affects pharynx and oesophagus.⁸

OSMF is commonly observed with the use of areca nut in various forms with significant duration and frequency of chewing habits.⁹ The contents of the areca nut like alkaloid (arecoline) and flavonoids (catechin, tannin) enhance the collagen production, strengthen the cross-linking and reduce degradation of collagen.¹⁰ The friction and microtrauma produced by rough fibres of arecanut to oral mucosa facilitates alkaloid diffusion into sub epithelial connective tissue and cause juxtaepithelial inflammation.¹¹

OSMF can be effectively treated with submucosal injection of betamethasone.¹² Betamethasone has antagonistic activity on the soluble factors generated by sensitized lymphocytes after activation by nonspecific antigens. Fibrosis is prevented by a decrease in fibroblastic proliferation and deposition of collagen.¹³

Intralesional injection of betamethasone declines inflammatory reaction, trismus and burning sensation and at the same time, these injections cause pain and injury to already inflamed tissue.¹⁴ In this study a non - invasive method of drug delivery was planned.¹⁵

MATERIALS AND METHODS

Betamethasone sodium phosphate was obtained as a gift sample from Glenmark Pharmaceuticals Ltd. (Malegaon, India), hydroxylpropyl methyl cellulose K100 (HPMC K100) was purchased from N R chemicals (Mumbai, India), carboxymethyl cellulose sodium (Na CMC), sodium meta bisulphite and glycerin were purchased from S.D. Fine Chemicals (Mumbai, India).

Preparation of mucoadhesive buccal betamethasone sodium phosphate gels:

HPMC and Na CMC were used in alone and also in combination of both in equal proportions for preparing gels.¹⁶ The composition of gel formulations with different polymers are shown in Table 1. Previously dissolved BSP in glycerin, mixed with polymer and sodium meta bisulphite is further subjected to hydration for 24 h. The prepared gels were filled in empty aluminum tubes and labeled accordingly.¹⁷

Physicochemical evaluation mucoadhesive gels

The prepared formulations were subjected for the evaluation of the following physicochemical parameters:

Homogeneity: The prepared gels were placed to set in a clean glass beaker and inspected for proper appearance and presence of any aggregates.¹⁸

Grittiness:

The gels were evaluated microscopically for the presence of any particulate substance.¹⁸

Spreadability:

One gram of gel was placed between two horizontal plates of size 20 cm x 20 cm and on upper plate 125 g weight was applied. The diameter of the spread area of the gel was measured after 1 minute.¹⁸

Extrudability:

The extrudability of the gels was determined by filling the prepared gel in one-ounce aluminum collapsible tube having a nasal opening of 5 mm. To this, 1 kg of load was constantly applied and the amount of gel extruded through the tip was weighed.¹⁸

pH:

In about 45 ml water 5 g of gel was dispersed and pH of this suspension was determined at 27 °C using the pH meter (pH ep@ - pocket sized pH meter, model no. S221504, Italy).¹⁸

Drug content uniformity:

In 100 ml of 6.4 pH phosphate buffer solution, about 1 g of gel (containing 1000 µg of BSP) was dissolved to give 10 mcg / ml. The absorbance was measured at 240 nm by U.V. Spectrophotometer (Shimadzu UV Spectrometer, model no. 1800 240V, Japan) against blank. The blank solution without drug was prepared in the same manner as above using gel containing respective polymers and other additives.¹⁸

Viscosity:

Brookfield Capcalc V3.0 Build 20.0 viscometer was used to determine viscosity of the gels using spindle-01.¹⁹ The readings were recorded over the speed ranging from 10, 15, 20, 25 and 30 rpm at 30 s between two successive speeds as equilibration time and then in a descending order, with shear rate of 133, 200, 267, 333, 400 s^-1.

The viscosity data were plotted for rheograms (Fig. 1-3)

1.Shear rate versus shear stress

2.Log of shear rate versus log of shear stress

3.Viscosity versus speed

Mucoadhesive studies:

An assembled device fabricated in laboratory was used to determine bioadhesive force of the gels. Fresh goat buccal tissue sections obtained from local slaughterhouse were fixed using cyanoacrylate adhesive allowing the mucosal surface outside upon two glass vials separately which were maintained at 36.5 °C for 10 m (Animal ethical committee clearance numberHKES/MTRIPS/IAEC/93/2017-18). One vial was connected to the balance, the other vial was placed on a height-adjustable pan. About 1 g gel was applied onto the buccal tissue of one vial. Subsequently the height of the other vial was adjusted so that the gel applied on the mucosal surface of one vial should coincide and adhere to the mucosal tissue surface of the other vial vertically. The weights were gradually added in ascending rate till the two vials separated. Bioadhesive force was determined based on the minimal weights required to separate the two vials. The buccal tissue was changed for each measurement²⁰.

In vitro drug diffusion studies:

A glass cylinder of 10 cm height, 3.7 cm outer diameter, 3.1 cm inner diameter having both ends open was taken. Cellophane membrane soaked in distilled water (24 h before use) was fixed to one end of this cylinder with an adhesive to result the permeation cell. One gram of the gel under study was kept in it. A beaker containing 100 ml 6.4 pH phosphate buffer solution acted as a receptor compartment. The sample was immersed to a depth of below the surface of medium in the receptor compartment. The medium in the receptor compartment was agitated using a magnetic stirrer at the temperature 37±1 °C. The 10 ml samples were withdrawn after every 10 minute interval and assayed at 240 nm. The volume withdrawn each time was replaced by equal amount of medium. All the studies were conducted in triplicate and standard deviation was calculated²1.

The results of in vitro release were fitted into four models of data treatment as follows (Fig. 4-7).

1. Percent cumulative drug release versus time.

2. Log percent cumulative drug remaining versus time.

3. Percent cumulative drug release versus square root of time.

4. Log percent cumulative drug release versus log of time.

Drug polymer interaction:

The interaction study was carried using using IR spectrophotometer (FTIR Burker, model Alpha E, Germany). IR spectrum of pure drug BSP (Fig. 8) and with excipients (Fig. 9) used in gel formulations were studied for their interactions. 

Stability Studies:

The gels were stored in a stability testing chamber at 30±2 °C temperature and 35±5% relative humidity for 6 months, to confirm any changes in physical appearance, pH, drug content uniformity, viscosity and mucoadhesive strength.

RESULTS AND DISCUSSION

The characterization of prepared mucoadhesive buccal BSP gels exhibited the following results.

All the gel formulations were free from lumps and gritty particulate substances (Table 2).

The spreadability of the gels was in the range of 27±2 to 63±1 mm after 1 minute (Table 2). Viscosity of gel formulations F₅ and F₆ prepared by using equal proportions of sodium CMC and HPMC was low, hence showed high spreadability and good extrudability.

The mucoadhesive strength of the gels was between 12.300±0.004 to 13.850±0.003 g (Table 2). The gel formulations F₅ and F₆ containing equal proportions of sodium CMC and HPMC exhibited satisfactory mucoadhesive strength even though less viscous than other formulations. The mucoadhesive strength depends upon the type, concentration and combination of the polymers used.

The pH of the gels was between 6.4±0.3 to 6.9±0.1 (Table 2). Since the pH of the gels was same as the oral pH of saliva, they were free from irritation.

The percentage drug content of all the formulations was from 98.53±0.185 to 99.94±0.211 (Table 2). The gels showed uniformity of drug content.

The viscosity of the gels observed was 8420 to 12788 cps at low shear rate and 3670 to 6997 cps at high rate of shear (Table 2).

The viscosity measurements were made at varying speed and shear rates. All the prepared gel formulations showed shear thinning / pseudoplastic behavior at room temperature where in viscosity of the gels decreases as the shear rate is increased. Upon plotting log of shear stress versus log of shear rate, a straight line obtained with slope N (Fig. 1). The slope value N for F₁ , F₂ , F₃ , F₄ , F₅ and F₆ were 3.348, 2.216, 3.847, 3.957, 4.109 and 5.166 respectively. For the pseudoplastic property, the N value of all the prepared gels was greater than one signifying better pseudoplastic behavior of all the formulations.

For all shear thinning systems, rheogram obtained by plotting shear rates at various shear stresses, wherein the ‘up curve’ and ‘down curve’ lie separate (Fig. 2).

When viscosity was plotted against spindle speed, it was found that viscosity of the prepared gels is inversely proportional to speed. At highest speed, viscosity of the gels was lowest and the curve appeared straight (Fig. 3).

Increase in viscosity of the gels decreased the drug release, the order of decreasing percentage drug release in 2 h were F₅ > F₆ > F₃ > F₄ > F₁ > F₂ .

The formulations exhibited cumulative percent drug release from 73.070±0.586 to 86.869±0.380%. The regression coefficient values of the kinetic equations like zero order plots (Fig. 4), first order plots (Fig. 5), Higuchi diffusion plots (Fig. 6) and Korsmeyer-Peppas plots (Fig. 7) were nearer to one, suggesting that plots are fairly linear. The drug release is by non-Fickian release mechanism as the slope values of Korsmeyer-Peppas plots were in the range of 0.8836 to 0.9060. The regression coefficient values were calculated for both the zero order plots and first order plots, the results reveals that drug was released by zero order kinetics (Table 3).

FTIR of BSP was adopted to characterize the potential interactions with the excipients used (Fig. 8 and 9). 

In the spectrum of BSP, the broad band at 3417 cm^-1 was due to the free hydroxyl groups. The peak at 2941 cm^-1 was caused by –OH stretching. The peaks at 1093 and 1047 cm^-1 were due to secondary –OH group (characteristic peak of –CHOH in cyclic alcohols, C-O stretch) and primary –OH group (characteristic peak of –CH₂ -OH in primary alcohol, C-O stretch). In addition the bands at 1606 and 1454 cm^-1 were due to asymmetric and symmetric stretching of carboxylate salt groups.

After 6 months of stability studies, physical changes like color fading or separation of liquid exudates of the gels were not observed. There was no alteration in pH and drug content of all the gels.

CONCLUSION

The F₂ , F₄ and F₆ formulations containing 3% polymer had high mucoadhesive strength but exhibited slow release rate and poor spreadability. However F₁ , F₃ and F₅ containing 2.5% polymer showed significant release rate, mucoadhesive strength, spreadability and extrudability. The F₅ formulation exhibited prominent mucoadhesive strength of 12.300±0.004 g and drug release of 86.869±0.380% compared to F₆ . Hence, it was considered to be promising formulation and can be further subjected for in vivo studies.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.