Penciclovir cream 1% (10 mg/g penciclovir, Fenivir, Novartis Consumer Health, Switzerland) was provided in house and acyclovir cream 5% (50 mg/g acyclovir; Zovirax, GlaxoSmithKline, United Kingdom) was purchased from a Swiss pharmacy.
Penciclovir was provided in house and acyclovir was bought from Sigma Chemicals (Switzerland).
After securing the appropriate consent the ethical committee of the International Institute for the Advancement of Medicine (IIAM; United States) reviewed and approved our application for request of human tissue to be used in this study.
Full thickness human abdominal skin from 6 donors, which was taken at autopsy, was provided cryopreserved by IIAM. The skin was maintained frozen at -80°C. Prior to use, the skin was thawed and the subcutaneous tissue was carefully removed. The skin was dermatomed to 500 μm using a Wagner dermatome (model GB-231 Aesculap, Germany), which enabled us to obtain split thickness tissue constituted of the stratum corneum (10–20 μm), the epidermis (100 μm) and part of the dermis (1200 μm) [14, 15].
Test of skin integrity
The permeation of tritiated water was evaluated to determine the skin integrity as described by Bronaugh . Briefly, tritiated water (2.7 μCi/ml) was applied to the skin surface. After 30 min, the radiolabelled water was removed from the skin with cotton-wool tips. The receptor phase (2 ml) was taken in order to measure the amount of tritiated water (%) which permeated across the skin, using a liquid scintillation counter. Formulations were tested on skin samples having similar tritiated water permeation values. Less than 1% of the applied dose of tritiated water permeated through the skin.
Skin permeation is the diffusion of the drug across the skin layer into the receptor phase which represents blood vessels. It was measured using a static Franz diffusion cell of 1.75 cm2 for each skin sample exposed to the product to be tested at 5 mg/cm2 simulating in use conditions and in accordance to the test guideline OECD 428 .
Thawed human dermatomed skin samples were mounted horizontally on the Franz cells, dermis side down. The Franz cells were connected to a circulating water bath at 37°C, which yielded a tissue temperature of 32°C, comparable to the physiological temperature of the skin surface. The receptor phase of PBS pH 7.4 (phosphate buffered saline; 7.58 g/L Na2HPO4, 1.62 g/L NaH2PO4 and 4.4 g/L NaCl) contained within each diffusion cell (approximately 8 ml) was mixed using a magnetic stirring device to ensure appropriate homogenization of the released drug in the acceptor phase throughout the experiment. Samples of the receptor phase were collected at 24 h.
The skin penetration was determined by measuring the amount of drug residing in the various skin layers.
At the end of the 24 h after application skin samples were washed off with soapy water and cotton-wool tips. The top layer of the stratum corneum was removed with adhesive tape (3 M Scotch n° 550) and analyzed separately. Additional layers of the stratum corneum were removed by up to 12 consecutive adhesive tape strips.
Following collection of all strips, the first and the second strips were put into separate vials containing 10 ml of water. Strips 3 up to 7 were pooled in the same vial containing 20 ml of water. The remaining stripped skin was minced and placed into a vial containing 15 ml water. Mixtures were stirred overnight to ensure adequate extraction of the drug from the tape and the skin.
Penciclovir and acyclovir content in the various samples was measured by high performance liquid chromatography (Agilent HP 1100) on a Waters Spherisorb® 5 μm ODS2 (4.6 × 250 mm Analytical Cartridge PSS839540) phase column at 35°C using a 1 ml flow rate of the mobile phase of a mixture of methanol and 0.1 M pH 6.0 ammonium acetate buffer 1:10 (v:v) with UV detection at 254 nm. The sample was directly injected at a volume of 50 μl. Drug concentrations were determined from penciclovir or acyclovir standard curves between 10 and 50 ng/ml generated with the pure compounds solubilized in PBS at pH 7.4, which was the solvent used in the receptor phase. Maximal soluble concentration in this solvent was 0.9 mg/ml for penciclovir and 0.7 mg/ml for acyclovir. The retention time was respectively 6.7 min for penciclovir and 5.1 min for acyclovir. The limit of quantitation (LOQ) was 7 ng/ml for both drugs.
The characteristics of the penciclovir and acyclovir chemical structures were evaluated with an in house web based CHEMINFORMATICS system using the CORINA program. Generation and display of molecular surface properties enabled us to reveal those parts of the molecules which are involved in hydrophobic or hydrophilic interactions .
Western blot analysis
The skin sample from one donor was washed off with soapy water and cotton-wool tips 24 h after the cream application. The top layer of the stratum corneum was removed with adhesive tape (3 M Scotch n° 550) and analyzed separately. Additional layers of the stratum corneum were removed by up to 12 consecutive adhesive tape strips. Following collection of all strips, the first and the second strips were put into separate vials. Then pools of 3 to 4 strips were put in the same vial and the last strip was placed separately in another vial. A volume of 250 μl/tape strip of ice-cold lysis buffer (20 mM Tris-HCl; 2 mM EGTA; 2 mM EDTA; 30 mM NaF; 30 mM Na4O7P2; 2 mM Na3VO4; 1 mM [4-(2-Aminoethyl)benzenesulfonylfluoride] (AEBSF); 10 μg/ml leupeptin; 4 μg/ml aprotinin; 1% Triton X-100; pH 7.4) was added in the vial, which was left for 10 min on ice and then vortexed. Supernatants were collected and stored at -20°C overnight. Protein concentrations were measured by the BCA protein assay (Pierce).
Ten μl of protein samples corresponding to 3 μg of protein from tape strips lysat were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). Membranes were blocked by incubation with Trisbuffered saline (50 mM Tris, 150 mM NaCl) containing 0.2% (v/v) Nonidet P-40 and 5% (w/v) nonfat dry milk for 30 min at room temperature. Membranes were probed overnight at 4°C with a monoclonal mouse anti-human Keratin 5 (Millipore) antibody (1:20,000) and then with secondary horseradish peroxidase-conjugated goat anti-human IgG (1:20,000) (Transduction Laboratories, Lexington, KY) for 1 h at room temperature. The membranes were washed three times with Tris-buffered saline containing 0.2% (v/v) Nonidet P-40, and the antigen-antibody complexes were detected by the Super Signal Substrate method (Pierce). Identified protein bands were detected using a video densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Unpaired Student t-test were performed to define a P value between the pencyclovir cream and the acyclovir cream. A P value of less than 0.05 was considered statistically significant.