Formulation: Gels containing 1% of astemizole/terfenadine, with varying concentrations of carbopol (0.6%, 0.8% and 1.0%) were prepared. The gels were tested for stability, content uniformity and in vitro release through excised mouse skin. Gels containing 0.8% carbopol 934 and 1% of drug in gel base were finally selected for wound healing studies, as these were found to be more stable and had good release rates than the others. The compositions of these gels are as follows:

The gels were kept in collapsible tubes and were placed in the stability chamber, which was maintained at 40°C and 75% relative humidity for 90 days. The gels were then observed for any change in the consistency and color. Besides, the drug content uniformity was evaluated as described below.

Five hundred milligram of the gel was taken and dissolved in a small amount of buffer pH 7.4. The volume was made up with buffer. The absorbance was measured at 284 and 258 nm respectively, for astemizole and terfenadine.

The rat skin soaked in buffer for 6-8 h was clamped carefully to one end of the dialysis cell (donor compartment). Fifty milliliter phosphate buffer saline (PBS) containing 20% ethanol was taken in a beaker (receptor compartment). Gel, of 1 g quantity, was spread uniformly on the membrane. The donor compartment was kept in contact with the receptor compartment. The temperature was maintained at 37 ± 0.1°C. At predetermined time intervals, 1 ml of the solution was pipetted out from the receptor compartment and immediately replaced with 1 ml of fresh PBS. The drug concentration of the acceptor fractions was determined against appropriate blank.

Healthy inbred male albino rats of Wistar strain, weighing 180-200 g were selected. They were individually housed and maintained on normal food and water ad libitum, under standard living conditions of room temperature 25°C and day and night reverse cycle. The surgical interventions, under ketamine anesthesia (10 mg/kg), were carried out on the shaven back of the animals. Under each wound model of study, the wound bearing animals were divided into four groups, each group containing eight animals (n = 8). The first group of animals was left untreated and served as control. The second, third and fourth groups of animals received the topical applications, viz. gel base (base control), astemizole gel and terfenadine gel respectively, at approximately 100 mg per wound, once daily for 10 days in the case of incision wounds and a maximum of 20 days in the case of animals with excision wounds, starting from the day of surgery.

Incision wound: Two para-vertebral straight incisions of 6 cm each were made through the entire thickness of skin on either side, at least one cm lateral to the vertebral column, as per the methods described by Ehrlich and Hunt.[5] The sutures were removed on the 7^th day of wounding. The wound breaking strengths were measured on the 10^th day of wounding by continuous, constant water flow technique, as described by Lee.[6]

Excision wound: Excision wounds were made as described by Morton and Malone,[7] by excising the full thickness circular skin (approximately 500 mm²) from the nape of neck, under ketamine anesthesia. Wound contraction was assessed by tracing the wound area on polythene paper first and subsequently on a mm sq paper, every alternate day. The wound closure rate was expressed as percentage of the original wound size of 500 mm² on selected days.

On day 15, an animal from each of the treated groups was sacrificed and the wound was excised and fixed in neutral buffered 10% formalin. Later, paraffin sections of 5 micron were made and stained with H & E stain for histopathological evaluation.

The number of days required for the scab to fall off without leaving a raw wound behind was considered the period of epithelization.

Healed scar on the day of epithelization was excised and used for determination of hydroxyproline content.[8]

The experimental protocols were approved by the Institutional Animal Ethics Committee of MAHE, Manipal.

Results were analyzed using the one-way ANOVA, followed by post hoc test, viz. Scheffe's test using the Windows based SPSS computer package.

Both the gel formulations of terfenadine and astemizole showed 99% drug content uniformity in their formulations [Table 1].

Gels of terfenadine and astemizole, containing 0.8% carbopol 934 and 1% of drug were found to be stable in the accelerated stability studies, as there was no change in the color and consistency. Besides, air bubbles and crystals did not appear during the studies.

In vitro release studies of astemizole and terfenadine through rat skin

The release of the drugs from the formulations was progressive and steady. At the seventh hour, the rates of release of astemizole and terfenadine from their gel formulations were 71.30% and 67.85% respectively [Table 2].

The mean strength of the healed wound scar in control group of animals was 266 g. While the base of the gels did not influence the strength of the scar, both astemizole and terfenadine gels significantly increased (P< 0.05) the breaking strength as compared to control. The rate of increase was between 26% and 39 % respectively for astemizole and terfenadine [Table 3].

In control group, wound contracted to the extent of 3.7, 37, 74 and 91% by days 2, 6, 10 and 14. The gel base treatment, however, enhanced the wound closer rate on day 6 and 10. Wounds of the animals, which received astemizole, showed a similar rate of contraction as that of gel base on day 6 and day 10. However, these wounds contracted significantly faster than the gel base treated wounds on day 14. On the other hand, terfenadine gel showed significantly (P< 0.05) greater wound contraction, as compared to gel base on days 2, 6, 10 and 14 [Table 4].

On day 15, in the control group, a small portion (less than 9%) of the original wound remained to be covered by the epidermis. Thus, these animals took a period of 18 days for complete epithelization. The gel base did not alter this. On the other hand, the gels of astemizole and terfenadine significantly (P < 0.05) promoted the epithelization process, thus shortening the period by two days [Table 4].

The collagen content in the healed scar of the control and the base applied wounds was around 45 mg/g of the tissue. The application of the gels of astemizole and terfenadine caused a significant (P < 0.05) increase in collagen content of the scar tissues [Table 4].

The wound sections from the animal in control group had a few notable histological observations such as more organized collagen, less number of inflammatory cells and blood vessels – markers of near complete healing. Besides, the growth of epithelial tongues was prominent in these sections [Figure 1]. The features of the sections of the gel base were almost similar to those of control [Figure 2]. On the other hand, the features of the sections of astemizole and terfenadine treated wounds were that of advanced healing, as compared to that of the control or gel base. In these sections, there was complete restoration of epithelial growth, similar to that seen in normal skin. Inflammatory cells appear almost negligible in the dermis [Figures 3 and 4].

The histological features of the sections of astemizole and terfenadine treated wounds confirm the pro-healing actions of these two gels.