Formulation and In-Vitro Evaluation of Mucoadhesive Buccal Patch of Ondansetron Hydrochloride

Oral drug delivery is the most common and preferred route of drug administration. It offers advantages like painless administration, cost-effectiveness and more patient compliance as compared to other routes such as intramuscular, intravenous and pulmonary^[1]. However, poor bioavailability is common with oral route.

Some of the reason for poor bioavailability are as follow:-

1. Poor solubility.
2. First pass metabolism.
3. Degradation in the gastrointestinal tract.

In recent approach to improve bioavailability and achieving modified release from drug. various novel drug delivery dosage form are being studies and research are being carried out. One of such novel drug delivery is the mucoadhesive drug delivery system. Mucoadhesive drug delivery system(MDDS) can be administered through various route such as^[3]:

• Oromucosal
• Nasal
• Ocular
• Intravesical
• Vaginal

Mucoadhesive buccal drug delivery system is a drug delivery system which involve the administration of drug through buccal mucosa of the oral cavity ^[5].Buccal mucosa lines the inner region of cheeks. Within oral mucosal cavity, the buccal region offers an attractive administration for the systemic drug delivery ^[10].The buccal mucosa is rich is in blood supply and absorption of drug occurs effectively. Buccal drug delivery system interacts with the mucus layer covering the mucosal epithelial surface, and mucin molecules and increase the residence time of the dosages from at the site of absorption.

Buccal patches are dosage forms that are applied to the inside of the cheeks (buccal mucosa). The patches are either for local or systemic treatment and adhere to the mucosa for an extended period. Because of the small size and thin layer of the patches, patient compliance was higher than with other kinds of medication. In general, patches are made up of three layers, each of which contributes to and controls the direction of drug release. The outer impermeable backing layer directs drug release and prevents drug loss away from the contact site. It also serves as a mechanical support and protects the layers. The medication is kept in the middle reservoir laver. The mucoadhesive is the last laver, which allows the mucoadhesion to adhere to the mucosa.

5. Reasons for mucoadhesive buccal patch:

1. MBDDS prolongs the residence time of dosage form at site of application or absorption.
2. High drug loading capacity.
3. Improve therapeutic efficacy of the drugs.
4. Controlled drug release.

5. Drug candidates for mucoadhesive buccal patch:

1. Molecular mass is less than 600 Daltons.
2. Diffusion coefficient is high.
3. Suitable partition coefficient, so that concentration gradient in the skin be high.
4. Drugs possess low melting point which relate good ideal solubility.
5. Half-life is 2-8 hrs or less.
6. Molecule is non-irritating and non-sensitizing.
7. Oral bioavailability is low.

5. Advantages of mucoadhesive buccal patch:

1. Rapid onset of action.
2. It has sustained action so reduce frequency of administration.
3. First pass metabolism is bypassed.
4. Directly enter systemic circulation.
5. Buccal mucosa provides efficient blood supply and has relatively low enzymatic activity.
6. It allows self- medication and hence is well accepted by patient.
7. Drug can be removed from the site of application in case of toxicity.

MUCOADHESION :

Mucoadhesion is the mechanism in which two biological materials are held together by interfacial force ^[3]. It describes the attractive forces between a biological material and mucus or mucous membrane. They are generally hydrophilic as they contain many hydrogen macromolecules due to the large amount of water (approximately 95%) within its composition. However, mucin also contains glycoproteins that enable the formation of a gel-like substances ^[3].

Theory of mucoadhesion :The theories includes wetting theory, electronic theory, adsorption theory diffusion. theory and fracture theory^[3].

1. Wetting theory: This theory is applied to liquid system which present affinity to the surface to spread over it. The affinity can be found by measuring contact angle. Lower the contact angle, greater the affinity. Spreading coefficient must be positive in order to adhere to biological membrane and contact angle between cells and polymer should be equal or zero for proper spreadibility.
2. Electronic theory: According to this theory both mucoadhesive material and biological material consists of opposite electrical charges. When each material comes in contact then they transfer electrons. This result in formation of electrical double layer of charges that are attracted to each other and thus lead to mucoadhesion.
3. Adsorption theory: It is most accepted theory. According to this theory mucoadhesion occurs between the mucoadhesive material and mucus by bonding such van der Waals and hydrogen bonds. electrostatic attraction or hydrophobic interaction.
4. Diffusion theory: Both polymer and mucin chain lead to interpenetrate to a sufficient depth and leads to mucoadhesion. The adhesion force increases with the degree of penetration of the polymer chain size. The penetration rate depends on the diffusion coefficient, mobility and contact time, flexibility and nature of mucoadhesive chain.
5. Fracture theory: This theory analyzed the force required for the separation of the two surfaces after adhesion. Fracture theory is the major mechanism by which to determine the mechanical strength of a particular mucoadhesive, and describe the force necessary to separate the two materials after muco-adhesion has occurred.

STAGES OF MUCOADHESION :

The mechanism of mucoadhesion is mainly divided into two steps :

Contact stage: The mucoadhesive material is attached with the mucus membrane. Then the formulation is swell and initsate it deep contact with the mucus membrane,

Consolidation stage: The mucoadhesive formulation is activated by the moisture. Van der Waals force and hydrogen bond are linked with the mucoadhesive material and then adhesive interaction is established^[4].

Fig.no 1: Stages of Mucoadhesion

FORMULATING COMPONENTS:

POLYMERS:

Polymer is a macromolecule composed of many repeated subunits. Polymers are backbone of MDDS. Buccal patches are designed as multilayered polymeric laminates in which the drugs are sandwiched between two polymeric layers i.e. the backing layer (outer layer) which prevents the loss of drug to outer membrane and inner polymeric. layer that function as an adhesive and/or rate controlling. Mucoadhesive polymer are water soluble and water insoluble polymers which are swellable networks jointed by cross linking agents^[7].

Table no.1: Types of polymers

AIM:

To formulate and evaluate mucoadhesive buccal patch of Onadansetron Hydrochloride.

OBJECTIVES:

• To determine physical properties such as thickness, weight variation, folding endurance, surface pH, thickness of buccal mucoadhesive patch.
• To determine drug content and in vitro permeation/drug release of the formulation.
• To optimize patch from different formulation.

MATERIALS AND METHODS

MATERIALS:

API and all other excipients for the preparation of mucoadhesive buccal patches were provided by Department of Pharmacy, JF Institute of Health Serences/LACHS, Hatt?ban, Lahtpur.

Following mentioned chemicals will be used during the project.

Table 2: List of chemicals

INSTRUMENTS AND DEVICES:

• Electronic balance
• Magnetic Stirrer
• Micrometer Screw Guage
• UV/Visible Spectrophotometer
• Petri dish
• Franz Diffusion Cell

METHODOLOGY:

PREPARATION OF BUCCAL PATCH :

Buccal patch of ondansetron was prepared by solvent casting method using different proportions and combinations of HPMC ES (1000-1250 mg), HPMC K100 M (500-1000 mg), and Eudragit® NE 30 D (150-1000 mg), propylene glycol (0.2ml), sucrose and ondansetron hydrochloride (81 mg).

HPMC was weighed and suspended in 10ml of ethanol with constant stirring and about 2ml of water was added to it. This solution was mixed with Eudragit NE 30 D and homogenized. Plasticizer was added to the mixture and mixed. Required amount of ondansetron was dissolved in propylene glycol before addition into the polymer blend. The above mixture was stirred by magnetic stirrer gently until a clear solution was obtained. Then sucrose was added. The solution was smeared to remove any entrapped air. Then the solution was poured into petri dish of diameter 9 cm; dried in an oven at 37°C for 16 h. The prepared films were then removed from the petri dish and cut 1x1cm². Then stored in vacuum desiccators^[8].

METHOD  OF STANDARD CALIBRATION CURVE PREPARATION

A stock solution of Ondansetron hydrochloride (100mcg/ml) was prepared in phosphate buffer pH 6.8. Five samples comprising 5, 10, 15, 20, 25 mcg/ml were prepared from stock solution in 50 ml volumetric flask. The sample were then analyzed in UV visible spectrophotometer at maximum wavelength of 310nm. The calibration curve was prepared by plotting absorbance versus concentration of Ondansetron hydrochloride,

Analytical method development: Analytical method validation is done to demonstrate that the method is suitable for intended purpose. It was based on the validation parameters like linearity, accuracy, precision, and specificity as per ICH Guidelines.

Linearity: The linearity of an analytical procedures is its ability (within the given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. In order to demonstrate linearity, various concentrations of Ondansetron were prepared in phosphate buffer (pH 6.8) i.e. 5µg/ml, 10µg/ml, 15µg/ml, 20µg/ml. 25µg/ml.

Phosphate buffer was prepared as per IP. All concentration were first scanned in UV range (200-400) nanometer for the determination of maximum wavelength (max). Then the absorbance of all these solutions were measured in maximum wavelength.

Accuracy: The closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value. For accuracy test, the different concentration of sample was prepared (5, 10, 15 mcg/ml).

Precision: It expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the sample homogeneous sample under the prescribed condition.

For demonstration of precision, six different solutions of reference standard of Ondansetron hydrochloride were prepared in phosphate buffer solution with the concentration of 5µg/ml. All the solution were scanned at max of 310 nanometers and the concentration of sample was calculated with reference standard of Ondansetron hydrochloride.

Specificity: Specificity is defined as ability of the analytical method to evaluate unequivocally the analyte in presence of those components expected to be in sample, It helps to ensure there is no interfere in analysis by other components of the mixture. If the analytical method that is being performed is specific, then the concentration of the component can be measured regardless the presence of the other compound. The specificity test was carried out by measuring the spectrum of Ondansetron hydrochloride and excipients used in the formulation at 310 mm in UV visible spectrophotometer using phosphate buffer of 6.8.

Table no.3: Formulation Table

1. Physical appearance: The patches were observed visually for their physical appearance such as transparency and color.

2. Surface texture: The surface texture of the patch was evaluated by simply touching the surface of the patch.

3. Mass uniformity: Mass uniformity was tested in 10 different randomly selected patches from each batch using electronic balance and average weight was calculated.

4. Thickness: Micrometer screw gauge was used to measure thickness of patch. For each formulation, three randomly selected films were used. Film of size (3x3cm²) were cut, and all the measurement were done in triplicate. The average patch thickness and standard deviation was calculated^[8].

5. Folding endurance: The folding endurance of patch was determined by folding the patch repeatedly at the same place till it broke. The number of times the patch could be folded without breaking gives the value of the folding endurance^[8].

6. Surface pH: A combined glass electrode was used for this purpose. The patches were allowed to swell by keeping them in contact with 1 ml of distilled water for 2 hrs at room temperature, and pH was noted down by bringing the electrode in contact with the surface of the patch, allowing it to equilibrium for minute.

7. Content uniformity: To ensure the uniformity of distribution of ondansetron in the film, a content uniformity test was done. Films (11 cm³ equivalent to 4 mg of ondansetron) were cut at three different locations and dissolved in 10 ml of phosphate buffer saline (pH 6.8) by continuous shaking on a water bath at room temperature for 8 3.5.8 The solution was filtered through Whatman filter paper, and the samples were diluted suitably and analyzed using UV spectrophotometer at a max 310 nm against a blank. A calibration curve was constructed, and the drug content was estimated from the curve. The method validation was done for linearity, precision, and accuracy^[8].

8. Determination of moisture content: The prepared film was marked, then weighed individually and kept in desiccator containing activated silica at room temperature for 24 hours. The film was weighed again after 24 hrs. The percentage of moisture content was calculated as a difference between initial and final weight.

Moisture content : Initial weight - final weight/ Initial weight x 100 %

9. In-vitro permeation test: The in-vitro permeation test was carried out using Franz diffusion cell. The buccal mucosa of sheep cheek was used as the membrane. The membrane was fitted between donor and receptor compartment of Franz diffusion cell. The donor compartment was empty and receptor compartment was containing 5 ml of phosphate buffer of pH 6.8. the sample were collected at different time interval for analyzing the drug content in the receptor compartment and replaced with equal volume of freshly prepared phosphate buffer of pH 6.8. The drug content was analyzed at 310 nm using UV Spectrophotometer.

RESULT AND DISCUSSION

1. Determination of maximum wavelength: Ondansetron hydrochloride solution was scanned in the range of 200-400 nm. The maximum absorbance was observed at 310 nm.

2. Calibration curve : Calibration curve was prepared by plotting concentration versus absorbance for varying concentration of 5,10, 15, 20, 25 meg/ml of Ondansetron hydrochloride in phosphate buffer of pH 6.8 at 310 nm wavelength.

Fig no.2 :Calibration curve

3 .Analytical Method Validation

a) Linearity

The absorbance vs concentration value was plotted as in figure, based on the data on table below: Various concentration of Ondansetron hydrochloride and their absorbance.

Table no.4: Concentration vs absorbance

b) Accuracy

Accuracy of the analytical procedure was assessed using three different concentration level covering the specific range of 90% to 110% accuracy was then reported as percentage recovery by the assay of the known added amount of a result is shown in ANNEX I.

Table no.5: Concentration of Ondansetron HCl

To demonstrate the procedure is precise, 6 different solutions of same concentration of standard Ondansetron hydrochloride were prepared in phosphate buffer solution. All the solution was scanned for absorbance and the sample was calculated with reference to standard Ondansetron hydrochloride. All the data obtained is within the RSD of 2%. The result is shown in Annex II.

Table no. 6: Absorbance of Ondansetron HCl

d) Specificity

The spectrum showed maximum absorbance at 310 nm for the sample of the formulated product, but excipient did not show any absorbance at 310 nm. Thus, the result suggest that the excipient did not show interference. Therefore, method was specific to Ondansetron hydrochloride.

EVALUATION OF POST FORMULATION PARAMATER :

1. Physical Evaluation: The prepared patches were smooth, uniform, and flexible. The color of film was white. There was no sign of cracking of patch.

2. Thickness of patch: The thickness of patch ranges between 0.52.40.57mm to 0.56 0.001 mm. The small value of SD indicates that the patch within the formulations is uniform.

3. Weight uniformity: The weight of the patch's ranges between 108.162.13mg to 122.5±0.517mg, the weight variation in different formulation may be due to difference in total polymer content.

4. Folding endurance: Folding endurance of all patches were found to be very good for all formulation. The formulation containing polymer HPMC E15 and HPMC K100 M in the ratio 1:2 formulation showed better folding endurance among others.

5. Surface pH: The surface pH ranges from 6.23.10.15 to 6.8±0.05 in the patches formulated. The pH of all the formulation were in the range of buccal pH 1c 6.8 thus we can say that the patches formulated will not irritate the buccal mucosa as the pH were near to the pH of buccal mucosa.

6. Drug content: The drug content of formulations ranged from 92.27+0.252 to 96.23+0.251%. formulation F4 showed high drug content as compared to other formulations.

7. In-vitro Permeation test: The drug permeated through the mucosa of sheep check after 3 hours ranged from 62.2% to 82%. Tn-vitro permeation study was done for up to 3 hours. The formulation F4 showed more permeation value as compared to others formulation.

8. Moisture content: The moisture content in all the formulations were less than 5%. The formulation F8 Showed less moisture content as compared to other formulations

Fig no:3 Moisture content of F1 to F9