A Novel Formulation of Sildenafil-loaded Nanocarriers for Treatment of Alopecia Areata

The aim of the present work was to optimize sildenafil-loaded liposomes which could enhance percutaneous absorption and follicular penetration of sildenafil achieving improved therapeutic efficiency with minimal unnecessary side effects due to oral administration.

In addition, the study aimed to assess the efficacy of topical sildenafil-loaded liposomes formulation compared with topical minoxidil therapy for the treatment of patchy AA of the scalp through examination of its clinical effects along with dermoscopic findings.

Materials Lipoid S75 (phosphatidylcholine (70 %) with fat-free soybean phospholipids) was kindly donated by lipoid company (Germany). Sildenafil citrate was purchased from Fagron Iberica SAU. Acetonitrile HPLC reagent was purchased from Fisher Chemical. Ultrapure water was. Cholesterol was purchased from Fluka chemical co. (India). Ethanol (absolute) was purchased from El-Nasr Pharmaceuticals, (Egypt). All reagents and chemicals were purchased from (Sigma Aldrich).

Methodology Preparation of SIL-LIP using spraying technique SIL-LIP were formulated using spraying method. Pre-calculated amounts of Lipoid s75, cholesterol and sildenafil were dissolved in 2 ml of absolute ethanol and the prepared solution was transferred to a spraying apparatus [20]. In a closed system, the alcoholic solution of sildenafil and lipids was sprayed (100 ul were sprayed every 10 seconds) onto the surface of 2 ml distilled water containing sucrose (9% w/v) with stirring at 1250 rpm at 25º C. Stirring was maintained until complete evaporation of ethanol. To guarantee complete annealing of the formed liposomes, SIL-LIP were kept at 4°C overnight [21]. SIL-LIP were imaged using (JEM-1400,-Jeol,-Tokyo,-Japan) maintained at 80-kV. SIL-LIP was-left for ten minutes at 25°C before examination on carbon-coated-copper-grid.

Particle size measurement To gain more insight into size and size distribution, laser light diffraction was employed. the Prepared SIL-LIP were diluted with deionized water and was analyzed with Mastersizer 3000E Malvern Instruments, UK at 25°C. The result was the average of three repeated values.

Entrapment efficiency (E.E) of prepared liposomes

The EE% was determined by centrifugation of liposomal suspension at 1.000 rpm for 45 min at 4 °C to isolate the unentrapped sildenafil in the supernatant for indirect determination of EE% . The amount of sildenafil in the supernatant was determined by UV spectrophotometer (Spectronic Genesys®, Winspec Software, Spectronic, USA) at 228 nm . EE (%) was calculated as follows:

EE (%) = [(Qi -Qs)/Qt] × 100 where Qi is the initial amount of the sildenafil added during preparation and Qs is the sildenafil amount determined in the supernatant after centrifugation Clinical study A prospective randomized controlled study was conducted in the department of Dermatology, Assiut University Hospital including patients with patchy AA of scalp, aged between 10 and 40 years. Patients having alopecia totalis/universalis, or ophiasis, those with systemic and/or other dermatological diseases and those receiving treatment for AA in the previous 3 months were excluded.

This study was conducted in compliance with the guidelines of the Declaration of Helsinki and the study protocol was approved by the Research Ethics Committee of Faculty of Medicine, Assiut University (IRB no. 17300458). Informed consent was obtained from all patients before enrolments.

At the initial visit, full history taking and thorough clinical examination was done for all patients. The diagnosis was established based on both clinical and dermoscopic examination.

Treatment Protocol

Patients were randomly assigned to use either topical 5% minoxidil gel or topical 1% sildenafil-loaded liposomes twice daily for a period of 8 weeks.

Assessment criteria

Clinical evaluation was performed by the comparison of serial photographs of the lesions to the baseline ones. Severity of Alopecia Tool (SALT) score was calculated, at baseline, 4 weeks and 8 weeks and the average percentage of hair re-growth was determined using the SALT scoring system for hair re-growth and graded as A0: no change or further hair loss, A1: 1-24% hair re-growth, A2: 25-49% hair re-growth, A3: 50-74% hair regrowth, A4: 75-99% hair re-growth, and A5: 100% hair re-growth .

Dermoscopic examination was carried out before treatment and at 4 and 8 weeks, using the handheld dermoscope (DermLite DL4, 3Gen, Inc., San Juan Capistrano, CA 92675). Baseline and post-therapy digital photographs of dermoscopic fields were taken using an iPhone 6S smartphone camera. Dermoscopic features of AA were evaluated including yellow dots, black dots, broken hair, exclamation-mark hairs, short vellus hair and regrowing hair . Two dermoscopic fields were examined; one from the center and the other at 3 o'clock position of the alopecia patch. Trichoscopic parameters in each field were evaluated and quantified.

Data analysis was done using the SPSS software version 24 (SPSS Inc., Chicago, IL, USA). The Chi-square test and Mann-Whitney test were used for comparison of qualitative data and quantitative data, respectively. P < 0.05 was considered statistically significant.