Preparation of Rabbit Skin

For skin irritation studies, healthy male albino rabbits (2–2.5 kg) were used. For ex vivo permeation studies, proper NOC was taken from the Research Ethical Review Board of Gomal Center of Pharmaceutical Sciences, Faculty of Pharmacy, Gomal University, Dera Ismail Khan. The rabbits were allowed to roam freely and take food (standard) and water (ad libitum) at their own will. After administering an overdose of ketamine and xylazine injections, the rabbits were sacrificed. Hairs from the abdominal region of each rabbit were surgically removed. The skin was then dipped in warm water at 60 ◦C for 45 s for adhering fats. Excised skin was placed in distilled water, washed at –20 ◦C and stored until further use [30].

For the determination of drug (methotrexate) permeation from the formulated patches across rabbit skin, a Franz diffusion apparatus was used for this study. Before starting the experiment, excised skin that was kept at −20 ◦C was hydrated for at least 1 h. Then, between the donor and receptor compartments, the skin was placed. The stratum corneum (SC) side of the skin was placed facing the donor compartment of the Franz cell apparatus. Then, a 1 cm2 piece of the formulated patch was placed over the rabbit skin. The drugreleasing surface of the formulated patch faced the SC of the rabbit skin. In the receptor compartment, phosphate buffer (pH 7.4) was used. The temperature of the receptor fluid was maintained at 37 ± 0.5 ◦C by means of water circulating in the water jackets around the receptor compartment. The receptor fluids were stirred by means of magnetic beads. At predetermined time intervals of 0.5, 1, 1.5, 2, 4, 8, 12, 16, 20 and 24 h, a 2 mL sample was taken from the receptor fluid. In order to maintain sink conditions, fresh receptor fluid of an equal volume was added to the receptor compartment. For the determination of drug content, these samples were analyzed spectrophotometrically at a 303 nm wavelength [30].

#### *2.9. Drug Retention Study*

The drug retention study was carried out after the permeation experiment. Skin from the Franz cell apparatus was removed carefully and cleaned with phosphate buffer solution, then dried and cut into small pieces. The skin pieces were then stirred in phosphate buffer (pH = 7.4) overnight. Retained drug from the skin was extracted using methanol, and samples were centrifuged. The supernatant was filtered through a 0.45 μm cellulose acetate filter. The filtrate was analyzed with a UV–visible spectrophotometer at a 303 nm wavelength.

#### *2.10. Statistical Analysis*

One-way ANOVA was used as the statistical tool. A value of *p* < 0.05 was considered significant. All experiments were performed in triplicate, and the result was expressed as the mean value ± standard deviation.

#### **3. Results**

TDDS is user friendly, painless, and convenient, and it usually leads to enhanced patient compliance. Transdermal patches control drug delivery by employing different combinations of polymers. In the current study, various polymers were used to prepare methotrexate-loaded transdermal patches. The polymers used were ethyl cellulose (EC) and hydroxypropyl methyl cellulose (HPMC) at different ratios.
