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Vishal Sachdeva, Srujana Siddoju, Yi-Ying Yu, Hyun D. Kim, Phillip M. Friden, Ajay K. Banga
BY SHARAN KUMAR REDDY B M.Pharm I.P (IIsem) SPIPS VIDHYANAGAR, HANAMKONDA. SOURCE:INTERNATIONAL JOURNAL OF PHARMACEUTICS 388(2010)
Terbina¿ne (TBF) is a synthetic allylamine antifungal agent used for the treatment of super¿cial fungal infections of the skin and nail. The stratum corneum is the primary site of action for terbina¿ne in super¿cial cutaneous fungal infections. In order to treat fungal infections effectively, the drug must be present at the site of action at a concentration above the minimum inhibitory concentrations (MIC) during the entire treatment period.
Oral administration has been shown to be associated with drug-drug interactions (inhibition of CYP2D6,an important phase I metabolizing enzyme), hepatotoxicity, gastrointestinal and systemic side effects, lactose intolerance and other adverse effects. An improved topical drug delivery approach could overcome these limitations as it provides immediate access to the site of infection and reduces unwanted systemic drug exposure. However, a major limitation of topical delivery for skin infections is poor bioavailability.
CONTD«. Results have shown a bioavailability of less than 5% in humans even after a week¶s treatment period. as it is the only one that provides a physical driving force to move drugs through biological membranes. One of the more powerful enhancement techniques is iontophoresis. . This technique involves the use of small amounts of physiologically acceptable electric current (in the µA range) to drive charged or neutral drug molecules across the skin or nail.
. most likely due to its inability to deliver adequate drug levels into the deeper epidermis or dermis layers of the skin where the infection exists. it would be bene¿cial to investigate whether higher drug levels in deeper skin layers could be attained with the use of iontophoresis.CONTD«. Its use in the treatment of deep seated skin fungal infections (subcutaneous / cutaneous mycoses) is considered ineffective.. Hence.oral and topical terbina¿ne formulations are indicated only for super¿cial cutaneous fungal infections (dermatomycosis). Currently.
drug levels were determined in the stratum corneum (using tape stripping method).CONTD«. duration of current application and the presence or absence of formulation during the postiontophoretic period were also investigated.iontophoresis was used to deliver terbina¿ne hydrochloride into and across hairless rat skin in vitro. Subsequently. In this study. the underlying skin (using skin extraction method) and the receptor compartment. Studies to identify the rate limiting barrier for the penetration of terbina¿ne into the skin and predominant process driving the drug during iontophoresis were performed. The effect of current density. .
5mm diameter) and silver chloride Male hairless rats.Materials Terbina¿ne hydrochloride Sodium Borate Hexane Formic acid Acetonitrile (HPLC grade) Ammonium acetate Silver wire (0. 8±10 weeks old weighing 350± 400 g .
Skin isolation and preparation Abdominal skin was freshly excised and prepared for use in each in vitro study. These skin pieces were then mounted on the receptor compartment of the vertical Franz diffusion cells. Following skin isolation. subcutaneous fat (if present) was carefully removed. The skin was then cleaned using de-ionized water and cut into appropriate size. . Rats were euthanized by CO2 asphyxiation and gently laid in the area prepared for surgery.
The electrodes were freshly prepared on the day of the experiment. The cathode was custom made by coating a melt of silver chloride on a ¿ne silver wire.Preparation of electrodes A planar coil of silver wire was prepared manually and used as anode. . The coating procedure was continued until a uniform and suf¿cient coat of silver chloride was obtained.
Permeation experiments Vertical Franz diffusion cells. . and 10mM sodium chloride in de-ionized water.8). pH 5.5ml. pH 3. Receptor compartment : receptor buffer (5ml. Temperature: 37ÛC Terbina¿ne hydrochloride (4%.48) was then added to the donor chamber as donor solution. Samples obtained were protected from light and air until analyzed using HPLC. w/w) formulation (0. The anode (silver wire coil) was placed in the donor chamber and the cathode (custom made silver chloride electrode) was inserted into the receptor compartment through the sampling arm to perform anodal iontophoresis.30% propylene glycol. consisting of 10% ethanol.
. as evidenced by an increase in TEWL values to 8±10 times the base value (obtained before tape stripping).4mA/cm2)were performed for 1h across intact (with stratum corneum) and tape stripped (without stratum corneum) hairless rat skin. both passive permeation and anodal iontophoresis (0. Approximately 15 tape strips were able to remove the entire stratum corneum.Tape stripped skin studies In order to determine the primary barrier to the penetration of terbina¿ne into the skin. Trans-epidermal water loss (TEWL) measurements were recorded using a closed chamber evaporimeter as an indirect measure to ensure complete removal of stratum corneum by the tape stripping process.
removed from the Franz cells and the drug was extracted. skin samples were cleaned of excess formulation. . The amount of drug delivered into the underlying skin in the presence and absence of the stratum corneum was compared for both passive and iontophoretic groups.CONTD« Following 1h permeation study.
. 0. 45 and 60 min at a current density of 0. respectively.3mA/cm2. anodal iontophoresis was performed for 15. For the duration of current application study.2. Tape stripping and skin extraction was performed immediately after the study to determine the amount of drug delivered into the stratum corneum and underlying skin.Effect of current density and duration of current application The inÀuence of current density on the amount of drug delivered into the stratumcorneum and the underlying skin was investigated by conducting anodal iontophoresis for 1h at different current density values of 0.4mA/cm2. 30.3 and 0.
In order to investigate whether the presence of the drug formulation has an impact on permeation during the posttreatment period.Post-iontophoretic delivery studies To study drug permeation following iontophoretic delivery.4 mA/cm2 was followed by 23 h of passive permeation (post-iontophoretic period). At the end of 24 h ( 1 h treatment + 23 h post-treatment period). tape stripping and skin extraction studies were performed. studies were performed with (formulation not removed. FPR) or without (formulation completely removed. . FCR) the formulation in the donor compartment. FNR or formulation partially removed. 1 h anodal iontophoresis at 0.
Samples were directly analyzed using HPLC. The ¿rst two tape strips were discarded to avoid overestimation of the amount of terbina¿ne in the stratum corneum due to excess super¿cial formulation at the skin surface. .64 cm2) was tape stripped 15 times using 3 M Transpore tape .Tape stripping The treated area on each skin sample (0. Extraction of drug from the strips was performed at room temperature for 3±4 h while shaking on a roller shaker.
Following incubation. Borate buffer (pH 10. the mixture was cooled to room temperature and neutralized using 100 µl HCl (5N). 1. Hexane (6 ml) was added to each tube and the drug was extracted by shaking on a roller mixer for 60min.5 ml) was added and the tubes were vortexed. These skin pieces were transferred to tubes containing 1 ml of 5 M NaOH and incubated for 2 h at 60ºC. .Skin extraction The area of the tape stripped skin exposed to formulation was punch biopsied and cut into small pieces using a scalpel blade. The contents were then centrifuged for 10 min at 8000 ×g .
CONTD« The upper organic layer containing extracted drug was separated and fresh hexane (6 ml) was added to the original tubes to extract the remaining drug from the aqueous layer using the above procedure. The tubes were centrifuged at 8000 ×g for 10 min and the upper organic layer was completely evaporated. following ¿ltration through a 0. 50µl of the bottom layer was injected into the HPLC column for analysis.5 M) and isopropyl alcohol (85:15) was added to these tubes to back extract the drug from the organic layer by shaking for 30 min. Following the second extraction. . A solution (1. The organic layer isolated previously was completely evaporated using nitrogen Àushing to concentrate the drug. the tubes were centrifuged and placed in a -80ºC freezer to freeze the aqueous layer and obtain the entire organic layer. This organic layer was reconstituted into the tubes containing drug concentrate obtained earlier and vortexed.22µm ¿lter.5 ml) of sulfuric acid (0.
This value was taken into consideration while calculating the actual amount of drug present in the unknown skin samples. 25. Following equilibration.Recovery studies In order to determine the extraction ef¿ciency.0%. 125 and 250µg) were injected into the skin pieces with three replicates for each amount. .5±500 µg). The extraction ef¿ciency was found to be 75. 50µl of standard solutions having a known amount of drug were injected super¿cially into hairless rat skin samples (mean weight 250 mg) and allowed to equilibrate for 3 h. Four different amounts (5. The actual amount extracted was determined using standard curve (0. the drug was extracted from the skin.
MA. The mobile phase was acetonitrile. the retention time was ~6 min.Quantitative analysis Terbina¿ne hydrochloride was quanti¿ed by highperformance liquid chromatography using an Alliance system (Waters Corp. adjusted to pH 3.. . Isocratic elution was performed using RP-18 Phenomenex column . At a Àow rate of 1. v/v).5 ml/min and 30ºC column temperature. The run time employed was 13 min and the injection volume was 50µl. USA) with a photodiode array detector at 233 nm.75 using formic acid (60:30:10. water and 100 mM ammonium formate.
Amount of drug delivered to different skin layers and receptor compartment during permeation studies .
could be advantageous in ensuring continuous drug levels at the site of infection. which could translate into less frequent administrations and/or reduced chance for recurrence. .Conclusion Anodal iontophoresis enhanced the delivery of terbina¿ne hydrochloride. into the skin. such as those surrounding the nails. iontophoretic delivery of terbina¿ne into the soft skin tissues. a water soluble form of a hydrophobic drug. Rapid delivery to the target site was achieved with the use of this technique. Furthermore.
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