Transdermal Delivery of Ondansetron Hydrochloride in the management of Hyperemesis Gravidarum

Transdermal route has always been a route of choice for those drugs having limitations through oral route. Transdermal delivery of Ondansetron Hydrochloride (HCl) is one of the potential strategies, capable of maximizing the drug performance in patients with Hyperemesis Gravidarum facing issues with oral drug delivery. Current study focused on the development of matrix transdermal patches of Ondansetron HCl using solvent casting method with combination of different polymers and eugenol as penetration enhancer followed by physical characterization for various parameters such as weight variation, folding endurance, moisture content, moisture uptake, drug content, tensile strength, elongation break test, FT-IR studies, in-vitro percent drug release, stability studies. The result suggested that formulation F3 exhibited optimum results in respect to physical evaluation and in-vitro drug release exhibited release in concentration independent manner (62.2±1.3% upto 8 hrs). All formulations followed zero-order kinetics and Higuchi’s model values showed ‘diffusion’ as predominant release mechanism. FT-IR analysis revealed that there are no potential chemical interactions between the drug and the polymers. Stability studies revealed promising result and the formulation (F3) was found to be stable at 40±2°C and 75±5% RH. The study indicated that transdermal delivery of Ondansetron HCl is a suitable way to increase therapeutic effectiveness.


Material and Methods A. Materials
Ondansetron HCl was supplied as a gift sample from Lupin Laboratory Park, Aurangabad, India. All other chemicals were purchased from Sigma-Aldrich, New Delhi, India. All other polymers and solvents used were of pharmaceutical and analytical grades respectively.

B. Preparation of Transdermal Patches
Transdermal patches of Ondansetron HCl were prepared by solvent casting method using various polymers such as Polyvinyl Alcohol (PVA), Hydroxypropyle Methyl cellulose (HPMC) K4M, Polyvinylpyrrolidone (PVP) K30 and Eudragit RL100. The calculated amounts of all polymers were mixed in methanol. After complete solubilization of polymers, a measured quantity of Ondansetron HCl was then mixed in polymeric solution followed by addition of eugenol (penetration enhancer). The slurry was thoroughly mixed with concurrent agitation to get semisolid consistency. At that point the arrangement was subjected to sonication to remove the air bubbles. After this, the content was transferred onto a glass surface utilizing 'O' shape ring (4cm in diameter) which was permitted to dry at room temperature for solvent evaporation. After 12 hrs, the dried patches were taken out and stored between sheets of wax in desiccators (14)(15). The composition of patches is given in Table  1. Ondansetron Hydrochloride (mg) 10

C. Preparation of Standard Curve of Ondansetron HCl
Ondansetron HCL (1 mg) was dissolved in phosphate buffer (pH=7.4) and volume was made up to 100 ml in volumetric jar. This stock solution (100 µg/ml) was further used to make dilutions of 2, 4, 6, 8, 10 µg/ml. Absorbance of each and every sample was estimated at 310 nm utilizing Shimadzu UV-1700 UV/VIS twofold shaft spectrophotometer and phosphate buffer (pH=7.4) was used as reference standard. The standard graph was plotted against concentration and slope was calculated (16).

D. Evaluation of Transdermal Patch a. Physical Characterization
The formulated transdermal patches were visually observed for surface smoothness and clarity. The thickness of the patches was evaluated at three different points using screw gauge. For each formulation, the study was done in triplicate. In weight variation study, three discs of 2×2 cm 2 were cut and weight using digital balance. The folding endurance was measured manually for prepared transdermal patches. A strip of 4×2 cm 2 size was cut evenly and folded repeatedly at the same point of strip until it broke. The drug content uniformity was performed by taking patch strip of 3.83 cm 2 which was dissolved in phosphate buffer of pH 7.4. Then chloroform and water were added to the mixture to make polymer soluble and remaining volume was made up with phosphate buffer upto 100 mL. Further dilutions were made and the absorbance of solution was taken at 310 nm followed by calculation of overall drug content present in formulation (17)(18)(19).
b. Moisture content (%) The patches were weighed freely and kept in a dessicator containing calcium chloride at 37°C for 24 hrs. The weight of patches was noted till constant weight observed (20,21). The percent of moisture content (MC) was calculated using a equation 1 given below: % MC = (X-Y) × 100 (1) Y Where, X = initial weight, Y = final weight c. Moisture uptake (%) Percent moisture uptake (MU) test was performed to identify physical integrity and stability of the prepared patches at high humidity. This can be measured by placing patch in dessicator containing 200 ml of potassium chloride saturated solution that suppose to develop humidity at 84% RH in dessicator. After 3 days, the patched were taken out and subjected to weight measurement (20,21). The percent moisture uptake can be calculated by equation 2 given below: % MU = (X-Y) × 100 (2) Y Where, X = initial weight, Y = final weight d. Tensile strength The tensile strength of the matrix type transdermal patches of Ondansetron HCl was measured by microprocessor force gauge attached with motor equipped with stand and cell. The smooth and clear patch strip of 20 mm in size were cut and placed between two clamps kept at a distance of 2 cm. The assembly of clamps is set in such a way that it should not cause any damage to the placed patch strip during experimentation. The lower clamp was set at fixed position while the upper clamp was allowed to move at speed of 2 mm/sec till the patch broke. The point of patch break just after elongation was noted down and tensile strength of patch was calculated using equation 3 given below: (22) Tensile Strength = Fb (3) A Where, Fb =Force at break (Kg), A= Initial Cross sectional area of sample (mm2) e. Percentage Elongation Break Test The percent elongation break for all transdermal patches was determined by observing the length just before the break point using the equation 4 given below: (23) % Elongation = (L 1 -L 2 ) × 100 (4) L 2 Where L 1 is the final length of each strip, and L 2 is the initial length of each strip. f.

In-vitro Permeation Studies
In-vitro drug release or permeation studies were conducted using Franz diffusion cell with a receptor compartment capacity of 60 mL. The drug content, from matrix type transdermal patch, was determined using cellulose acetate membrane with 0.45µ pore size. In the diffusion cell, the formulated transdermal patch was placed on the cellulose acetate membrane, covered with aluminum foil and mounted between donor and receptor compartment. After the assembly, the receptor compartment was filled with phosphate buffer (pH 7.4). The whole set up was fixed on hot plate magnetic stirrer maintaining the temperature 32±0.5°C of the receptor compartment followed by continuous and constant stirring using magnetic beads. The samples collection was done at different time intervals maintaining sink conditions. The drug content in sample was analyzed by using UV-VIS spectrophotometer (24,25).

g. Drug Release Kinetics of Ondansetron HCl Transdermal Patch
To investigate the mode of drug release from Ibuprofen loaded niosomes, the release data were fitted with the following mathematical models (26)(27)(28).
(i) Zero-order kinetics equation (5): Qt is the amount of drug released at time t, k 0 is the zero-order release rate constant, t is the time (ii) First-order kinetics equation (6): Where, Qt is the amount of drug released at time t, Q 0 is the initial amount of drug in the solution, k 1 is the firstorder release rate constant (iii) Higuchi model kinetics equation (7): Qt=kH.t1/2 (7) Where Qt is the amount of drug released at time t, k H is the Higuchi release rate constant

h. Drug-Excipient Interaction study by FT-IR Spectrometry
For interaction studies, FTIR spectrometry was performed on FTIR Model 8400-S Spectrometer, Shimadzu, Japan. Test samples were ground altogether with KBr control in mortar and pestle, in a weight proportion of 1:100 and after that mixed the blend followed by making of pellets in pellet press. After this, the pellet was kept in the holder and transmittance was measured in the frequency of wavelength ranged 400 and 2000 cm-1 at a determination of 2 cm-1 and sweep speed of 2 mm/sec. IR spectra of both pure drug and physical blend was then taken out and observed for any interactions of excipients with pure drug (29).
i. Stability Studies A stability study was performed as per ICH guidelines to observe the stability of Ondansetron HCl in F3 formulation. The samples, in triplicate, were packed in polyethylene coated aluminum sheet and kept inside stability chamber at 40±2° C and 75±5% RH for three months. After three months samples were collected and evaluated for drug content and percent drug release. Further, statistical analysis was done using paired t-test to calculate statistical difference at 0.05 level of significance followed by calculating similarity index of percent drug release of F3 formulation before and after storage period (30).

RESULTS A. Standard Curve of Ondansetron Hydrochloride
From already prepared stock solution, five dilutions were made by using phosphate buffer solution of pH 7.4 which has ultimate concentrations of 2 µg/ml, 4 µg/ml, 6 µg/ml, 8 µg/ml and 10 µg/ml. The absorbance was measured at λmax 310 nm by using UV-VIS spectrophotometer.  Table  2. In the observations, all the formulations exhibited weight variation ranging from 32±1.7 to 38±1.8 mg. The thickness of all the formulations ranged from 18±0.9 to 25±1.6.The folding endurance which represents the ability of patch to withstand the stress was found in the range of 69±1.4 to 81±1.2. The values for moisture content (%) and moisture uptake (%) in all the formulations were found to be less than 5%. The drug content for all formulations was found to be ranged from 96.28±0.45 to 99.31±0.24 %. The estimation value of tensile strength of all the formulations was found between 9.12±1.4 to 15.73±1.2. The elongation break test (%) values were between 7.6±1.32% to 23.8±0.92%. All the results of physical characterization parameters for each formulation are given in Table 2.  Table 3 and represented in Figure 2. All the formulations exhibited slow and controlled release upto 8 hours. The percent drug release was found to be in the range of 32.8 ±0.43% to 50.5 ±0.60%. The F6 formulation showed the highest percent drug release when compared to other formulations.

c. In-vitro drug release Kinetics
In-vitro drug release of Ondansetron HCl from all the prepared transdermal patches was found to be concentration independent and slow during duration of release study. The drug release kinetics was studied for zero order, first order and Higuchi Model. The correlation coefficient (r) values in the analysis of release data (K) of all formulations for each order are given in Table 3 and Figure 3, (a) zero order, (b) first order and (c) Higuchi's model kinetics, is shown. From the results, it is observed that drug release from all formulations followed zero order kinetics.

d. Drug-Excipient Interaction study by FT-IR Spectrometry
FTIR spectra are important record which gives sufficient information about the structure of a compound. Unlike UV spectrum which comprises of relatively few peaks, this technique provides a spectrum containing a large number of absorption bands from which structural information are revealed. FTIR spectra of the pure drug were obtained using FTIR spectrometer (FTIR-8400S spectrophotometer, Shimadzu, Japan). The interpretation of FTIR spectra of drug and blend is shown in Table 4. e. Stability Studies A stability studies were performed for three months at 40±2° C and 75±5% RH. After three months storage, the formulation (F3) was checked for drug content and percent drug release (shown in Table 5). The similarity index of F3 percent drug release profiles when compared before and after storage was found to be 85.91. The result from paired t-test (P<0.05) revealed that there is no significance difference in drug content and percent drug release values before and after storage.

Discussion
The present work focuses on the development of transdermal patches containing Ondansetron HCl by solvent casting method using combination of hydrophilic and hydrophobic polymers. The prepared patched were subjected to various evaluation parameters. Standard curve of pure drug was prepared using phosphate buffer pH 7.4 at 310 nm by UV-VIS spectrophotometer. Results of physical characterization revealed uniformity in all formulations in terms of thickness, weight variation and drug content whereas folding endurance observation represented formulations flexibility and integrity with general skin folding. The folding endurance was found to be highest in formulation F3, containing combination of PVA, EC and PVP in the ratio of 1:1:2. The moisture content (%) and Moisture uptake (%) of all formulations were found in acceptable limits i.e. below 5% (18). The low moisture content showed the more stability of prepared formulations during storage with respect to reduced brittleness and microbial contamination. The hydration of polymers is important parameter as concerned with the regulation of drug release mechanism from release matrix (controlled/sustained). From the film evaluation, the values of moisture uptake and % moisture content of all formulations were in the acceptable range. The study of hydration of polymers is an important finding that regulates the drug release from controlled/sustained release matrix. In-vitro drug release study showed that F3 was found to have more controlled release pattern than other formulations. The controlled drug release involves the diffusion mechanism as polymer matrix restricts the movement of drug molecules due to its three dimensional network. The drug release in all formulations followed zero-order kinetics that represented the release in concentration independent manner. The FT-IR interpretations of pure and blend (drug +polymers) explained no such significant interactions between them. Results from stability study of F3 revealed no significant change (P<0.05) in drug content and percent drug release before and after 3 months storage. In addition to that, the value of similarity index was found to be above 50 % which represents the similarity between release profiles before and after storage (31).
Conclusion In the present study, an effort was made to provide transdermal delivery of Ondansetron HCl with combination of hydrophilic and hydrophobic polymers. The transdermal patch was prepared using solvent casting method using polymers such as PVA, EC, PVP and HPMC in different ratios and eugenol as penetration enhancer.
Smooth surfaced, thin and transparent matrix patches were obtained and thickness of all the formulations remained almost similar with low SD values. Physical characterizations of patches were also done and the results found within limits. The in-vitro release study showed that all the formulations exhibited uniform and diffusion-controlled release and followed zero order kinetics. Based on combined results from physical parameters and in-vitro percent drug release and release kinetics, F3 showed the optimum performance. Stabilities studies was conducted for F3 formulations and found stable before and after the storage at 40±2°C and 75±5% RH. From the results, it can be concluded that ondansetron HCl, drug with shorter half life and undergoes first pass metabolism, is a suitable candidate for transdermal delivery system. In addition to that, Ondansetron HCl is considered to be safe in pregnant women and thus prescribed by the medical practitioner in the early stages of pregnancy and also to those patients with hyperemesis gravidarum condition. Hence, transdermal delivery of Ondansetron HCl will benefit more in the patients facing problems with oral delivery. Besides, it offers a proper way to manage hyperemesis gravidarum condition leading to more patient compliance. Further, this study open up the scope of pharmacokinetic studies and controlled clinical studies on humans in future.