*2.5. Stability Studies*

Stability studies were carried out for a period of 180 days for all formulated patches. The incubator was maintained at 37 ± 0.05 ◦C and 75 ± 5 RH, and the patches were kept for stability studies. After a regular interval of 30 days, drug content and physical appearance were evaluated for the formulated patches. Drug content determination was carried out according to the procedure described in Section 2.4.1.

#### *2.6. Skin Irritation Studies*

For skin irritation studies, healthy male albino rabbits (2–2.5 kg) were used, and proper NOC was taken from the Research Ethical Review Board of Gomal Center of Pharmaceutical Sciences, Faculty of Pharmacy, Gomal University, Dera Ismail Khan, KPK, Pakistan. The rabbits used in this study were given standard food at least three days before administration of the formulations. The standard food was prepared according to a published recipe composed of 10% white fish meat, 18% middlings, 20% grass meal and 40% bran [29]. Water was also allowed ad libitum. All rabbits were housed in a temperature (25 ± 2 ◦C)- and relative-humidity-controlled (50 ± 10%) room. The sparse hairs on the abdomen of each rabbit were carefully shaved a day before the scheduled experiment with an electrical clipper without damaging the stratum corneum. The application area was swept with dry cotton. The Draize patch test was used for skin irritation studies. Five groups, each containing 3 rabbits, were selected for skin irritation (hypersensitivity) reactions. Group 1 served as the nontreated group, and Group II served as the control group with USP adhesive tape. Group III was served with methotrexate-loaded patches, Group IV was served with 0.8% *v*/*v* aqueous solution of formalin (which is a standard irritant) and Group V was served with blank patches. The skin irritation study was carried out for a period of 1 week. A visual scoring scale was used for the identification of skin irritation grades. Skin irritation was graded as follows: "0" indicated no skin irritation, "1" indicated slight skin irritation, "2" indicated well-defined skin irritation, "3" indicated moderate-type skin irritation and "4" indicated scar formation on the skin [29].

#### *2.7. In Vitro Drug Release Studies*

In Vitro drug release studies were carried out using a Franz diffusion cell apparatus (model γ9-CB (71026), PermeGear, Hellertown, PA, USA). A Tuffryn membrane was used as a synthetic membrane for drug release from the formulated patch of methotrexate with EC and HPMC at different concentrations. Between the donor and receptors compartments, the Tuffryn membrane was placed. A 1 cm2 area of the formulated patch was placed over the Tuffryn membrane. The formulated patch piece was placed in such a manner that the drug-releasing surface faced the Tuffryn membrane. Phosphate buffer (pH = 5.5) was used in the receptor compartment. The receptor compartment is surrounded by water jackets in which water circulates. The receptor fluid temperature was maintained at 32 ± 0.5 ◦C. Magnetic beads were used for the stirring of receptor fluids. 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].
