INTRODUCTION:

Transdermal drug delivery systems are topically administered medicaments in the form of patches that are mainly used for non-invasive “intravenous infusion” of drugs for systemic effects at a predetermined and controlled rate.¹

Transdermal systems are designed to deliver the therapeutic agent at a controlled rate from the device to and through the skin into the systemic circulation. This route of administration avoids unwanted presystemic metabolism (first-pass effect) in the GI tract and the liver.

Patient satisfaction has been realized through decreased side effects, reduced dosing frequency, and improved plasma profiles as compared with conventional oral dosing or painless administration as compared with injection therapy. In the last two decades, among the greatest successes in CR drug delivery is the commercialization of transdermal dosage forms for the systemic treatment of a variety of diseases. ^2-7

To date, nearly 20 drugs alone or in combination have been launched into transdermal products worldwide. Additional drugs are in the late development phases (phase II to registration). Matrix based transdermal formulations have been developed for a vast number of drugs that include ephedrine, ketoprofen, metoprolol, labetolol hydrochloride, triprolidine, nitrendipine, lercanidipine, and propranolol. ^8-14

Ondansetron is a potent antagonist of Serotonin (5 HT₃) receptor which has been proved effective in prevention of chemotherapy and radiotherapy-induced nausea and vomiting. It can control diarrhoea and nausea in up to 100% of patients and occasionally ameliorate the flushing. In this work an attempt was made to formulate and evaluate TDDS for sustained release OSH by solvent casting method. Low molecular weight, good permeability, poor bioavailability (60%) and shorter half-life (5-6 h) of OSH made it a suitable drug candidate for the development of Transdermal patches. The main objective of formulating the Transdermal system was to prolong the drug release time, reduce the frequency of administration and to improve patient compliance.

MATERIALS AND METHODS:

Materials: Ondansetron hydrochloride was obtained as a generous gift from Sun Pharmaceuticals (Baroda, India). Eudragit RL100 and Eudragit RS100 were procured from Aurobindo Pharmaceuticals (Hyderabad, India). Di-butyl phthalate, menthol, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate phthalate were purchased from SD Fine Chemicals (Mumbai, India). All the polymers received were of pharmaceutical grade and were used as received. Other materials and solvents used were of analytical grade.

Methodology:

Preformulation study:

Solubility study: OSH has very low aqueous solubility and has not been reported in any official book, so determination of solubility is important. The solubility was determined in distilled water and Phosphate Buffered Saline (PBS) pH 7.4.

Saturated solution of OSH was prepared using 10 ml of distilled water/ PBS pH 7.4 in 25 ml volumetric flasks in triplicate. Precautions were taken so that the drug remained in medium in excess. Then by employing mechanical shaker, the flasks were shaken for 48 h and the sampling was done on 24^th& 48^th h. The sample withdrawn (1 ml after filtration) was diluted with appropriate medium and analyzed by using UV spectrophotometer (Systronic Pc-Based Double-Beam Spectrophotometer 2202, Ahmedabad, India) at 310 nm and 303.5 nm for PBS and distilled water respectively.¹⁵

Construction of standard graph: Standard graph of OSH was plotted in PBS pH 7.4 which was selected from solubility study. OSH was estimated spectrophotometrically at λ_max of 310 nm.

Preparation of Phosphate Buffer pH 7.4: Accurately measured 250 ml of 0.2 M potassium dihydrogen phosphate (KDHP) was taken in a 1000 ml of volumetric flask and added 195.5 ml of 0.2 M sodium hydroxide, and then water was added to make up the volume and adjusted pH 7.4 by using 0.2 M KDHP/sodium hydroxide.

Preparation of standard solution: Firstly, stock solution-1 of OSH was prepared by dissolving 10 mg of drug in 100 ml of PBS pH 7.4, so as to get a solution of 1 mg/ml concentration. Then stock solution -2 was prepared by taking 10 ml from the previous stock solution and dissolving in 100 ml of PBS pH 7.4, so as to get a solution of 100 mg/ml concentration. Accurately measured aliquot portions of standard drug solution, like 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, 1.2 ml, 1.4 ml and 1.6 ml were taken from stock solution-2 and were transferred in to 10 ml volumetric flasks and were diluted up to the mark with PBS pH 7.4. Absorbance of each solution was measured at λ_max of 310 nm against PBS pH 7.4 as the blank, by using UV-spectrophotometer. A graph of concentration of drug vs. absorbance was plotted.

Formulation of Transdermal Patches^16,
17

Preparation of blank patches: Polymers of single or in combination were accurately weighed and dissolved in respective solvent and then casted in a Petri-dish with mercury as the plain surface. The films were allowed to dry overnight at room temperature.

Development of Transdermal Patches: Mercury substrate method was employed in preparing transdermal patches of OSH.

Table 1: Formulations of OSH Transdermal Patch

Formulation code	EC: PVP	RL: RS	PVA: PVP	HPMC K4M: PVP	SOLVENT
F1	8:2	-	-	-	CHLOROFORM
F2	7:3	-	-	-	CHLOROFORM
F3	6:4		-	-	CHLOROFORM
F4	5:5	-	-	-	CHLOROFORM
F5	4:6	-	-	-	CHLOROFORM
F6	-	8:2	-	-	ACETONE
F7	-	6:4	-	-	ACETONE
F8	-	5:5	-	-	ACETONE
F9	-	4:6	-	-	ACETONE
F10	-		8:2	-	WATER
F11	-	-	6:4	-	WATER
F12	-	-	5:5	-	WATER
F13	-	-	-	8:2	EDCM
F14	-	-	-	6:4	EDCM
F15	-	-	-	5:5	EDCM
F16				4:6	EDCM
F17				2:8	EDCM

EDCM= Ethanol: Dichloromethane

Mercury Substrate Method: The polymers, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate phthalate, Eudragit RL100 and Eudragit RS100, poly vinyl Pyrrolidone, poly vinyl alcohol were taken in a weighing bottle. About 10ml of solvent mixture of dichloromethane: methanol (6:4) / chloroform / acetone were added and shaked to prevent the formation of lumps and kept aside for swelling of polymers. After complete solubilization of polymers in mixture of solvent, required quantity of dibutyl phthalate was added to the mixture and stirred. Finally weighed quantity of OSH was dissolved in 5ml of solvent mixture, added to the polymer solution and mixed well. It was set-aside for some time to exclude any entrapped air and was then transferred into a previously cleaned Petri plate (70.00 cm²) and kept aside for solvent evaporation. The rate of solvent evaporation was controlled by inverting a glass funnel over the Petri plate. After 12h, the dried films were taken out and stored in a desiccator. The composition of the patches is given in Table 1.

Evaluation of Transdermal Patches:

Physical Methods:

Weight Variation: All the transdermal patches were visually inspected for color, clarity, flexibility & smoothness.

Thickness: Thickness of the patches was assessed at 3 different points using digital micrometer (Digital Caliper, Aerospace, India). For each formulation, three randomly selected patches were used.

Physical Appearance: Three disks of 2x2 cmwere cut and weighed on electronic balance (Shimadzu, Aux*220) for weight variation test. The test was done to check the uniformity of weight and thus check the batch- to- batch variation. ¹⁶

Flatness: Longitudinal strips were cut out from each patch, one the centre and two from either side. The length of each strip was measured and the variation in the length was measured by determining present constriction, considering 0% constriction equivalent to 100% flatness¹⁸.

Folding Endurance: The folding endurance of the prepared patch was measured manually. A strip of the film (4x3 cm) was cut evenly and repeatedly folded at the same place till it was broken. The thinner the patch more flexible it is.¹⁹

Moisture Uptake: The patches were placed in the desiccators containing 200 ml of saturated potassium chloride to get the humidity inside the desiccators at 84 % RH. After 3 days the films were taken and weighed, the percentage moisture absorption of the patch was found.¹⁹

% moisture absorbed= Final weight-Initial weight/ Initial weight * 100.

Moisture Content: The patches were weighed individually and kept in a desiccator containing fused calcium chloride at 40ºC for 24 h. The patches were reweighed until a constant weight was obtained. Moisture content was calculated in percentage based on the difference between the initial and constant final weights. An average of three readings was noted²⁰.

Swelling Study: Completely dried membranes with a specified area (3.83 cm²) were weighed and put in desiccators for 24 h. They were removed and exposed to relative humidity conditions of 75% (containing saturated solution of sodium chloride) in desiccators. Weight was taken on a single pan balance periodically until a constant weight was obtained. The swelling capacity of the membranes (in weight %) was calculated in terms of percentage increase in weight of membrane over the initial weight of the specimen. The experiments were carried out in triplicate and the average values were used for the calculation. The percentage degree of swelling (DS) was calculated as

DS%= Ws-Wd/Wd * 100

Where Ws and Wd indicate the weight of the swollen and dry membranes respectively

Drug Content Determination: The patch of area 3.83 cm² was cut and dissolved in PBS pH 7.4. Then ethanol and dichloromethane were added to the mixture to make polymer soluble, and the remaining volume was made up with PBS pH 7.4 to 100 ml in 100 ml volumetric flask.1 ml was withdrawn from the solution and diluted to 10 ml. The absorbance of the solution was found at 310 nm and concentration was calculated. By correcting dilution factor, the drug content was calculated.²¹

Water Vapour Transmission Rate: Glass vials of 5 ml capacity were washed thoroughly and dried to a constant weight in an oven. About 1 g of fused calcium chloride was taken in the vials and the polymer films of 3.83 cm² were fixed over the brim with the help of an adhesive tape. Then the vials were weighed and stored in a humidity chamber of 80-90 % RH condition for a period of 24 h. The vials were removed and weighed at 24 h time intervals to note down the weight gain. The values are noted in table 4. Water vapour transmission rate is expressed as the number of grams of moisture gained/hr/cm².²²

Water Vapour Transmission Rate= Final weight-Initial weight/ Time*Area

Permeation Studies:

In vitro Permeation Studies using Dialysis Membrane: In vitro permeation of OSH from Transdermal patches through dialysis membrane (Hi-Media) with molecular weight cut off of 12000 was studied. The membrane was mounted over a Franz diffusion cell and a transdermal patch. The receiver compartment of the diffusion cell was filled with 15.0 ml of PBS pH 7.4 and the setup was placed over a magnetic stirrer with temperature maintained at 37⁰C. Samples of 3 ml were withdrawn and replenished immediately from the receiver compartment at 1, 2, 3, 4, 6 and 12h. They were stored in refrigerated condition till the analysis was performed. The content of drug in the samples was analyzed by UV-Visible spectrophotometer at 310 nm.

Ex vivo Rat Skin Permeation Studies:

Preparation of skin: A full thickness of skin was excised from dorsal site of dead rat and was washed with water. The fatty tissue layer was removed by using nails of fingers. The outer portion with hairs was applied with depilatory and allowed to dry. With the help of wet cotton the hairs were scrubbed and washed with normal saline solution. The skin was kept in normal saline solution and stored in refrigerator until further use. The skin was allowed to equilibrate with room temperature prior to use and was mounted between donor and receptor compartment of cell. It was clamped in such a way that the dermal side was in contact with receptor medium²³.

Method: PBS pH 7.4 was used as receptor solution. The volume of diffusion cell was 15 ml and stirred with magnetic beads. The temperature was maintained at 37 ± 1°C with the help of hot plate. The diffusion was carried out for 10 h and 3 ml sample was withdrawn at an interval of 1 h. The same volume of PBS pH 7.4 was added to receptor compartment to maintain sink conditions and the samples were analyzed at 310 nm.

Analysis of Permeation Data:

Determination of Flux: The flux (J) of OSH was calculated from the slope of the plot of cumulative amount of drug permeated per cm² of skin at steady state against the time using linear regression analysis. The steady state permeability coefficient (K_p) of the drug through rat epidermis was calculated by equation:

K_p =J / C

Where, J= flux (µg/cm²/hr) and C= concentration of drug in the patch

Kinetic Modeling of Drug Release: Various models were tested for explaining the kinetics of drug release.To analyze the mechanism of the drug release rate kinetics of the dosage form, the obtained data were fitted into zero-order, first order, Higuchi, and Korsmeyer-Peppas release model.^24-27

Stability study of Optimized Formulation: Stability studies were carried out at 45 °C and 75% RH for three months (climatic zone IV condition for accelerated testing) to assess their long-term (2 years) stability of Transdermal formulation. The protocols of stability studies were in compliance with the guidelines in the WHO document for stability testing of products intended for the global market. After 3 months samples were withdrawn and evaluated for physical properties and in vitro diffusion study. ²⁸

RESULTS AND DISCUSSION:

Preformulation study: Preformulation studies were primarily done to investigate the physical properties of drug.

Solubility Study: Ondansetron was best soluble in the PBS Buffer pH 7.4. The solubility results are shown in Table 2.

Table 2: Solubility data for OSH

Solubility medium	Time duration	Solubility (μg/ml)
Distilled water	24 hours	62.03±3.35
Distilled water	48 hours	78.63±1.25
Buffer pH 7.4	24 hours	82.14±1.49
Buffer pH 7.4	48 hours	96.34±1.92

Table 3: Standard graph of OSH in PBS pH 7.4

CONCENTRATION(µG/ML)	ABSORBANCE
0	0.00
2	0.129
4	0.231
6	0.359
8	0.482
10	0.591
12	0.697
14	0.837
16	0.982
Slope	0.06
R²	0.998

Fig.1: Standard curve of OSH

Fig2a: In-vitro release profile of F1-F7

Fig.2b: In-vitro release profile of F8-F12

Table 4: Physical evaluation data of OSH Transdermal patches. Results are the mean of triplicate observations ± SD

Formulation code	Weight variation (mg) ±SD	Thickness (mm) ±SD	Folding endurance ±SD	(%)Moisture uptake ±SD	(%) Moisture content ±SD	WVT Rate (g.cm²/day X10^-4±SD	Drug content (%)±SD	Swellability (%)±SD
F1	65.34±1.6	0.025±1.6	71±0.9	2.96±0.95	3.08±0.97	2.36±0.14	97.24±0.2	12.73±0.43
F2	65.87±1.6	0.025±1.6	72±1	3.27±0.62	3.11±0.83	2.48±0.15	97.36±0.2	13.25±0.36
F3	66.12±1.8	0.024±1.6	71±0.9	3.89±0.86	3.28±0.75	2.62±0.16	97.45±0.2	14.28±0.38
F4	66.45±1.8	0.026±1.6	72±1	4.85±0.91	3.32±.058	2.93±0.16	98.41±0.3	16.34±0.42
F5	65.34±1.6	0.026±1.6	72±0.9	4.55±1.14	3.98±1.17	3.07±0.17	98.58±0.3	18.94±0.48
F6	66.39±1.8	0.025±1.6	71±1	4.75±1.08	4.63±0.67	3.14±0.17	98.34±0.3	20.67±0.46
F7	65.48±1.6	0.025±1.6	71±0.9	4.27±1.17	4.92±1.38	3.35±0.18	101.17±0.3	22.01±0.38
F8	67.28±1.7	0.045±1.8	77±1	4.93±0.6	3.12±0.3	3.66±0.13	99.38±0.4	38.59±0.61
F9	67.91±1.7	0.045±1.7	77±1	4.68±0.6	3.26±0.3	3.82±0.12	96.75±0.4	35.48±0.45
F10	68.08±1.7	0.047±1.9	80±2	4.86±0.8	3.53±0.6	3.91±0.13	96.81±0.4	32.87±0.46
F11	68.36±1.8	0.046±1.8	79±2	4.53±0.8	3.34±0.3	4.15±0.11	96.84±0.5	30.13±.055
F12	68.94±1.8	0.046±1.8	77±1	4.37±0.7	3.47±0.3	4.28±0.13	96.48±0.5	28.63±0.54
F13	64.86±1.8	0.045±1.5	78±2	4.48±0.5	4.39±0.5	4.12±0.26	98.28±0.7	42.15±0.62
F14	64.53±1.5	0.036±1.3	78±2	4.65±0.4	4.62±0.5	4.16±0.28	98.46±0.7	44.86±0.64
F15	64.21±1.4	0.037±1.4	77±2	4.83±0.6	4.92±0.8	4.28±0.24	98.74±0.3	46.38±0.39
F16	64.83±1.5	0.037±1.3	77±1	4.96±0.4	4.87±0.6	4.38±0.21	98.83±0.7	48.34±0.42
F17	64.46±1.4	0.036±1.3	79±2	5.03±0.5	5.01±0.7	4.48±0.21	100.15±0.8	48.92±0.64

WVT=Water Vapour Transmission; SD=Standard Deviation.

Table 5a: In vitro drug release from F-1 to F-7

Cumulative % drug released
Time	F1	F2	F3	F4	F5	F6	F7
0	0.00	0.00±	0.00	0.00	0.00	0.00	0.00
1	6.81±0.45	7.06±0.54	7.91±0.71	8.50±0.86	8.94±0.94	9.31±0.96	9.75±1.01
2	13.80±0.46	14.29±0.56	16.33±0.74	16.89±0.89	17.32±0.97	17.77±0.98	18.45±1.07
3	20.98±0.49	22.74±0.58	23.63±0.76	24.30±0.92	24.93±0.99	25.92±1.01	26.34±1.11
4	28.49±0.51	30.61±0.61	32.23±0.77	33.49±1.02	34.37±1.01	35.34±1.05	36.44±1.15
5	34.58±0.53	37.16±0.63	38.19±0.82	41.56±1.05	42.53±1.05	43.85±1.12	45.11±1.18
6	41.73±0.55	44.38±0.66	47.01±0.85	50.68±1.06	53.89±1.08	55.33±1.16	57.16±1.23
12	76.31±0.57	79.76±0.69	84.07±0.87	88.47±1.07	90.38±1.13	92.98±1.18	96.79±1.29