Dear Sagar,

The following publication describes in detail the in vitro diffusion penetration of a drug into nail preparations. I have copied important text from the methods and material section for quick view:

Sample Preparation for Analysis

The assay of CPO in the nail was performed by first dissolving the nails in 0.5 mL of 1 M sodium hydroxide (NaOH) solution. The nail solutions were filtered through Restek® 0.45 μm syringe filters (cellulose acetate membrane). These solutions were diluted sufficiently to reduce the strength of 1 M NaOH to 0.1 M before derivatization. For the derivatization of CPO, 25 μL of dimethyl sulfate was added to 0.2 mL of the dilute nail solution and vortexed for 5 min. These solutions were maintained at 37°C for 20 min. Twenty-five microliters of triethylamine was added and vortexed for 1 min to terminate the reaction. The solutions were then filtered through Restek® 0.22 μm syringe filters (cellulose acetate membrane) and analyzed using the HPLC method. In order to quantify the CPO in the buffer, a similar pre-column derivatization process was used. To 0.2 mL of the samples, 50 μL of 0.1 M NaOH and 25 μL of dimethyl sulfate were added and vortexed for 5 min. The solutions were maintained at 37°C for 20 min prior to the addition of 25 μL of triethylamine. The solutions were vortexed, filtered through Restek® 0.22 μm syringe filters (cellulose acetate membrane) and analyzed using HPLC.

High Pressure Liquid Chromatography

CPO content in the samples was assayed using an Agilent1100 series HPLC with dual wavelength detector, using a modified, pre-column derivatization method (20–22). Acetonitrile/water (50:50 v/v) was used as the mobile phase with a 150 × 4.6-mm Eclipse Plus C18 column (Agilent). The flow rate was 1 mL/min and the injection volume was 50 μL. The wavelength of detection for derivatized CPO was 298 nm.

Recovery of CPO from Human Nails

Solutions of CPO in pH 7.4 PBS with concentrations 0.0253, 0.0505, 0.202, 0.809, 3.24, and 6.47 mg/mL were prepared. Approximately 20 mg of human nail clippings was added to each concentration of CPO solution. Each concentration was studied in triplicate. The nail clippings were treated with 200 μL of each of these solutions for 3 h at 32 ± 1°C. To dissolve the nails, 200 μL of 1 M NaOH was added to each sample. The nail solutions were diluted using pH 7.4 PBS to reduce the concentration of 1 M NaOH to 0.1 M and the sample preparation method was followed to quantify the concentration of CPO.

The Recovery of CPO from Epidermis and Dermis

A stock solution containing 1.6 mg/mL of CPO in pH 7.4 PBS was prepared. Solutions with the concentrations 0.1, 0.2, 0.4, 0.8, and 1.2 mg/mL were prepared using the stock solution. Fifty microliters of each of these solutions, including the stock, contains 5.2, 10.4, 20, 40, 60, and 80 μg of CPO, respectively. Each of the above concentrations was studied in triplicate. The skin sections with 9 mm diameter were punched from a piece of human cadaver skin, using a cork borer. The epidermis and the dermis were separated for each section, using the modified heat separation method (23,24). Both the epidermis and the dermis, for each skin section, were separately treated with 50 μL of the drug solutions for 4 h in microcentrifuge tubes. After the drug treatment, 1 mL of the mobile phase was added to each of the microcentrifuge tubes and equilibrated for 24 h at 37°C for drug extraction. The extracting medium was filtered through Restek® 0.22 μm syringe filters, derivatized, and analyzed using HPLC.

Determination of the Saturation Solubility of CPO in the PEs

An excess amount of CPO was added to 0.5 mL of each PE at the concentration levels shown in Table I. The drug was allowed to equilibrate with the PEs for a period of 72 h at 32 ± 1°C. A saturated solution of CPO in pH 7.4 PBS served as the control solution. At the end of 72 h, the solutions were filtered through a 0.22-μm syringe filter. The samples were derivatized and analyzed using the HPLC method mentioned previously. The enhancement in solubility, EFsol, of CPO in presence of the PEs was calculated using the equation below.

Where [CPO]PE is saturation solubility of CPO in presence of PEs and [CPO]pH 7.4 PBS is the saturation solubility of CPO in pH 7.4 PBS (control).

The In Vitro Nail Penetration of CPO

The PEs were screened for their ability to increase the concentration of the drug within the nail, using human nail clippings. The saturated solutions of CPO in the PEs and pH 7.4 PBS were prepared. Fourteen milligrams of previously washed and dried human nail clippings was weighed into the microcentrifuge tubes. The nail clippings were obtained from healthy volunteers (20–50 years) in the Department of Pharmaceutical Sciences, Temple University. The nail clippings were treated for 24 h with 0.5 mL of saturated solution of the drug in the enhancers at 32 ± 1°C. As a control, the nail clippings were treated with a saturated solution of CPO in pH 7.4 PBS. After 24 h of drug treatment, the nail clippings were washed three times with 5 mL of Nanopure water to remove any surface-adherent drug. The nail clippings were dried using tissue paper and were dissolved in 0.5 mL of 1 M sodium hydroxide solution at 37°C. The nail solutions were filtered using 0.45 μm syringe filters, derivatized, and analyzed using HPLC as described in the previous sections. This method is a modification of the screening method described in literature (18,25,26). The enhancement factor, EFnail, gives the improvement in CPO penetration into nail clippings relative to that in the control.

Where [CPO]PE and [CPO]pH 7.4 PBS are the concentrations of CPO within the nail clippings in the presence of PEs and pH 7.4 PBS (control), respectively.

The study is approved for use of human nail clippings by the Temple University Institutional Review Board (protocol number 13671).

The In Vitro Transungual Permeation of CPO

The nail penetration study helps to screen different PEs; however, the results have to be confirmed with an in vitro transungual permeation study. The in vitro transungual permeation of CPO was performed using only the PEs which showed promise in the nail penetration screen. The human cadaver toenails (Anatomy Gifts Registry) were washed with Nanopure water and blotted with tissue paper. The weights and thicknesses of the toenails were measured. The human cadaver toenails (Fig. 2a) were hydrated at 100% RH for 24 h, prior to mounting on Franz diffusion cell nail adapters (Fig. 2b). The nail adapters (3 mm orifice diameter) with toenails were placed between the donor and receiver compartments of the Franz cells (Fig. 2c). The receiver compartment was filled with 3 mL of pH 7.4 PBS containing 0.1% w/v gentamicin sulfate. Gentamicin sulfate was added to prevent microbial growth in the receiver compartment. The donor compartment was dosed daily with 21.4 μL of saturated solution of CPO in the potential enhancer from the nail penetration experiment. The temperature of 32 ± 1°C was maintained for the duration of 27 days. The study was performed under occlusion. On days 5, 10, 15, 18, 21, 24, and 27, 0.5 mL of the receiver compartment solution was sampled and analyzed for drug content. Additionally, on day 27, the nails were dismounted from the nail adapters and the drug content was quantified by separating the nail just below the orifice and the nail surrounding the orifice (Fig. 2d). The drug quantification was performed using the process described in the sample preparation.

a Human cadaver toenails. b Neoprene nail adapters with orifice diameter of 3 mm. c Franz diffusion cell showing the donor compartment, receiver compartment, and the nail adapter. d Area under the orifice of nail adapter and the peripheral nail ...

In Vitro Skin Permeation of CPO

The in vitro skin permeation of CPO was performed using the PermeGear Franz diffusion apparatus with the internal volume of 3 mL and orifice diameter of 9 mm. The human cadaver skin (AlloSource) was placed between the donor and the receiver compartments. The receiver compartment was filled with 3 mL of pH 7.4 PBS and the skin was allowed to equilibrate with the receiving medium for 60 min, prior to drug dosing. Twenty microliters of the saturated solution of the drug in the penetration enhancers was added to the donor compartment. The study was performed at 32 ± 1°C for a period of 24 h under occlusion. Each of the penetration enhancers were tested in triplicate and the data were compared with the control. The control cells were dosed with 20 μL of saturated solution of CPO in 10 mM pH 7.4 PBS. At 2, 4, 6, 8, 22, and 24 h, 0.5 mL of the receiver compartment solution was withdrawn and replaced with 0.5 mL of fresh buffer to maintain sink conditions. The drug content in the samples was analyzed using the HPLC method. The flux (J) for permeation (in micrograms per square centimeters per hour) was obtained from the slope of the linear potion of the plot of cumulative amount of CPO permeating per unit surface area of skin (in micrograms per square centimeter) against time (in hours). The permeability coefficient Kp was calculated from the ratio of the steady-state flux (J) and the concentration in the donor compartment (Cd). Since saturated solutions were used as donor solutions, Cd was the saturation solubility of CPO for all practical purposes of the calculation. The enhancement in flux for skin permeation (EFfinite flux) was calculated using the equation:

where FluxPE and FluxpH 7.4 PBS are the steady-state flux values for the permeation of CPO across the skin in presence of the PEs and pH 7.4 PBS (control), respectively.

Finite Dose Skin Penetration of CPO

The skin used in the finite dose permeation study was further analyzed for drug content by separating the epidermis and the dermis using a modified heat separation method (23,24). Post the finite dose skin permeation experiment, the skin was dismounted from the Franz cell and washed with Nanopure water thrice on both the epidermal and the dermal sides to remove any surface drug. The skin was then blotted with tissue paper; the area exposed to the drug was separated from the peripheral skin and wrapped in aluminum foil. Each of the samples was heated in the oven at 45°C for 10 min. The epidermis was then peeled from the dermis using sharp forceps. The thickness of the epidermis and the dermis was measured using an electronic digital micrometer (Marathon Management). The two layers were then placed in separate glass vials containing 1 mL of the mobile phase each, for 24 h to extract CPO. The concentration of the drug in the extracting medium was quantified using HPLC. The drug levels were reported in micrograms per milliliter to determine if the minimum inhibitory concentration (MIC) was achieved in the epidermis and the dermis. The enhancement factors, EFepidermis and EFdermis, give the increase in accumulation of CPO in the epidermis and dermis relative to the control.

Where [CPO]PE and [CPO]pH 7.4 PBS are concentrations of CPO in the epidermis/dermis in presence of PEs and pH 7.4 PBS (control), respectively.

Analysis of Results

Relationship plots with EFnail, EFflux, EFepidermis, or EFdermis on the y-axis and EFsol on the x-axis were prepared to understand how the different enhancement factors were affected by change in solubility. These plots also helped to identify the mechanism of action of the PEs in the two different biological membranes—skin and the nail. Each of the relationship plots was divided into four quadrants (I to IV) by drawing lines parallel to the x- and y-axes, passing though enhancement factor 1 on each axes. The EF of 1 stands for the control sample used in the determination of the respective enhancement factor. This method of analysis is a modification of the reported method (25).

Statistical Analysis of Experimental Data

All the experiments were performed in triplicate and the data were reported as mean value (± standard deviation). A one-way ANOVA (Microsoft Excel 2007) was used to determine statistical significance, where the p value of less than 0.05 was considered as statistically significant.

To view the full publication, please use the following link:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666006/

Other studies on this topic:

To view other publications on this topic, please use the following links:

1-AAPS PharmSciTech. 2010 Jun; 11(2): 986–993.

Published online 2010 Jun 3. doi:  10.1208/s12249-010-9457-1

PMCID: PMC2902339

Study of In Vitro Drug Release and Percutaneous Absorption of Fluconazole from Topical Dosage Forms

Claudia Salerno, Adriana M. Carlucci, and Carlos Bregni

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

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Abstract

The present study aimed to evaluate different dosage forms, emulsions, emulgels, lipogels, and thickened microemulsion-based hydrogel, as fluconazole topical delivery systems with the purpose of determining a formulation with the capacity to deliver the whole active compound and maintain it within the skin so as to be considered a useful formulation either for topical mycosis treatment or as adjuvant in a combined therapy for Cutaneous Leishmaniasis. Propylene glycol and diethyleneglycol monoethyl ether were used for each dosage form as solvent for the drug and also as penetration enhancers. In vitro drug release after application of a clinically relevant dose of each formulation was evaluated and then microemulsions and lipogels were selected for the in vitro penetration and permeation study. Membranes of mixed cellulose esters and full-thickness pig ear skin were used for the in vitro studies. Candida albicans was used to test antifungal activity. A microemulsion containing diethyleneglycol monoethyl ether was found to be the optimum formulation as it was able to deliver the whole contained dose and enhance its skin penetration. Also this microemulsion showed the best performance in the antifungal activity test compared with the one containing propylene glycol. These results are according to previous reports of the advantages of microemulsions for topical administration and they are very promising for further clinical evaluation.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902339/

2- A review article:

Transungual Drug Delivery: An Overview

Vivek B. Rajendra, Anjana Baro, Abha Kumari, Dinesh L. Dhamecha,

Swaroop R. Lahoti, Santosh D. Shelke

ABSTRACT

The body normally hosts a variety of microorganisms, including bacteria and fungi.

Some of these are useful to the body and others may cause infections. Fungi can live on the dead tissues of the hairs, nails. Continuous exposal of nail to warm, moist environments usually develops nail infection. Nail plate is main route for penetration of drug. Varity of conventional formulation like gel, cream and also oral antifungal are available for treatment of nail infection. The nail lacquer is a new drug delivery system in treatment of nail infections. The major hurdle associated with developing nail lacquers treatment for nail disorders is to deliver the active

(antifungal) therapeutically effective concentrations to the site of infection, which is often under nail. However possible means to enhance nail penetration must be explored in greater depth before effective local treatments for fungal nail infections are developed. Lack of proper in vitro methods to measure the extent of drug permeation across the nail plate is the major difficulty in the development of transungual delivery. Penetration of topical antifungal through the nail plate

requires a vehicle that is specifically formulated for transungual delivery. Recent focus is emphasizing on development of a promising antifungal treatment in form of nail lacquer owing to its beneficial advantages.

http://www.japsonline.com/admin/php/uploads/368_pdf.pdf

Hoping this will be helpful,

Rafik