The Skin Permeation And Penetration

Published Date: 02 Nov 2017

Mairim Russo Serafini1; Cassia Britto Detoni2; Sílvia Stanisçuaski Guterres2, Gabriel Francisco da Silva3, Adriano Antunes de Souza Araújo1

1 Departamento de Fisiologia, Universidade Federal de Sergipe (UFS), São Cristóvão, SE, Brazil

2 Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre,RS, Brazil

3 Departamento de Engenharia Química, Universidade Federal de Sergipe (UFS), São Cristóvão, SE, Brazil

ABSTRACT

Usnic acid has been proposed as a potential topical treatment for microbial skin lesions, burn wounds as well as a sunscreen. An isocratic HPLC method was validated according to FDA’s Guidence for Industry: Bioanalytical Method Validation to determine skin penetration and permeation of usnic acid. The penetration and permeation of usnic acid was evaluated using Franz cells and porcine skin. The method was valid according to selectivity, linearity, precision, accuracy, and stability. Usnic acid was quatified in the skin surface (6.13 µg.cm2), stratum corneum (34.4 µg.cm2), viable epidermis (5.6 µg.cm2), dermis (28.2 µg.cm2) and receptor compartment (3.2 µg.cm2). These results help to understand the penetration profile of usnic acid and plan topical therapeutic approaches as well as plan new topical delivery systems to modulate this penetration profile.

Keywords: usnic acid; validation, HPLC, porcine skin

Introduction

Lichens are formed through symbiosis between a fungal and a photosynthetic partner such as algae or cyanobacteria. More than 17,000 species and over 800 lichen products are known. Components such as usnic acid (Figure 1) are utilized for perfumery and for medicinal purposes (Kohlhardt-Floehr et al., 2010).

Figure 1. Usnic acid (Ingolfsdottir, 2002).

Since its first isolation in 1844, usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione] has become the most extensively studied lichen metabolite and one of the few that is commercially available (Ingólfsdóttir, 2002). In humans, it is quite useful in improving burn healing and it can act as an anti-inflammatory, antimitotic, antineoplasic, antibacterial, antimycotic agent (Nunes et al., 2011; Vijayakumar et al., 2000; Cardarelli et al., 1997; Takai et al., 1979) and displays variable redox-active properties, acting an antioxidant e pro-oxidant agent, according to different system conditions and/or cellular environment (Rabelo et al., 2012).

Besides topical application for burn wounds, usnic acid has been proposed as a potential topical treatment of nosocomially-acquired S. aureus isolates as well as a sunscreen (Cocchietto et al., 2002). Even though several researches indicate that the topical application of usnic acid is promising for various treatments, no data on usnic acid permeation through the skin or into the skin are currently available. A few chromatographic methods have been used for the determination of usnic acid in human plasma samples (Venkataramana and Krishna, 1992) and Parmelia ssp (Carmiglia et al., 2001). None of them, however, focused on the skin as the biological matrix.

The purpose of this study was to develop and validate a chromatographic method for the routine determination of usnic acid, suitable conditions for the in vitro transdermal permeation experiment, and a method for the extraction of usnic acid from the skin.

Experimental

Chemicals

All solvents were HPLC grade. Acetonitrile and methanol were from Tedia (Rio de Janeiro – Brazil). Acetic acid and ether (analytical grade) were purchased from Labsynth (Diadema - Brazil). The lauryl sodium sulfate and hydroxylethyl cellulose were from Cosmetrade (Porto Alegre - Brazil). Ethanol, analytical grade, was from Nuclear (São Paulo, Brazil). The water used in all experiments was ultrapure.

HPLC conditions

The HPLC equipment consisted in a high pressure pump, UV detector and an autosampler - Series 200 (Perkin Elmer – USA) and Total Chrom Software®. The column used was a Luna C-18 5 µm (150 mm x 4.6 mm) (Phenomenex® - USA) and guard column C-18 (Phenomenex® –USA). The mobile phase used was methanol: water: acetic acid (80:15:5) with a flow rate of 1.0 mL.min-1. The detection wavelength was 350 nm and the injection volume 20 µL.

Method validation

The method was validated according to FDA’s Guidence for Industry: Bioanalytical Method Validation (2001). Selectivity was determined using six blank samples of porcine skin and of receptor medium. The lower limit of quantification was accepted to be the lowest standard concentration on the calibration curve, which had a response over 5 times the response obtained in the blank samples.

The calibration curve was obtained by spiking 2 g of porcine skin cut into small pieces with known concentrations of the usnic acid. The usnic acid concentration was varied by varying the volume of a standard solution in acetonitrile (0.5 mg.mL-1) and volume was completed to 10 mL, also with acetonitrile, before the extraction procedures. The calibration curve was composed of seven non-zero samples (1, 2.5, 5, 10, 15, 20 and 25 µg.mL-1). The equation that determines the relationship between concentration and response (area) was the mean of three replicates.

Separately, five replicates of the above explained procedure at the concentrations 6.5 µg.mL-1 (low), 15 µg.mL-1 (medium) and 25 µg.mL-1 (high) were prepared. The mean and coefficient of variation of the concentration obtained using the calibration curve equation was calculated to obtain the accuracy and precision, respectively.

Of the five proposed stability validations (freeze and thaw, short-term temperature, long-term, stock solution and post-preparative) only three met the experimental necessities of collecting, handily and storing conditions. Short-term temperature stability was evaluated by collecting three aliquots of a high (25 µg.mL-1) and low (2.5 µg.mL-1) concentration samples from the calibration curves and keeping them at room temperature for 24 hours. Stock solution stability concentration was measured as soon as it was prepared and 24 hour after. Post-preparative stability by running a calibration curve batch twice, with an interval of 24 hours.

Gel preparation

To perform topical application of usnic acid a hydroxyethylcellulose gel was prepared. Usnic acid (2%) was dispersed in ethanol (16%) and the volume was completed with water, subsequently 2% of hydroxyethyl cellulose was added to the dispersion. The formulation was preserved for 24 hours and homogenized manually before use.

Skin preparation

Porcine flank skin was donated from a regional slaughterhouse (Bento Gonçalves –RS; Brazil). Excess fat tissue, hipodermis and hairs were removed, maintaining the full-thickness skin. The skin was clean on the surface with a lauryl sodium sulfate 0.1% solution and on the dermis with an alcohol: ether (1:1) solution before storage at -20°C for a maximum of 3 months. Before each experiment the skin was cut in to round slices, all skin slices used had width between 1.8 mm and 2.2 mm.

Skin permeation and penetration assays

The penetration assay was preformed with an automatic Franz type diffusion cell (MicroettePlus Multi- Group® Hanson Research Corporation) during 12 hours. The receptor medium used to guaranty sink condition was 7 mL of an aqueous solution o DMSO 2% (v/v). The gel (50 mg) was manually applied on the diffusion area (1.77 cm2). The cells were occluded with a glass cover. The acceptor phase was submitted to constant stirring and a temperature of 35°C during the whole experiment. Samples of 1 mL were withdrawn from the acceptor phase and injected in the HPLC in determined intervals up to 12 hours. The collected aliquots were replaced with fresh solution.

At the end of 12 hours the excess gel on the surface of the skin was removed with one tape strip. The stratum corneum was obtained by the tape stripping technique, using 18 tape stripes (3M® –USA). Epidermis and dermis were separated using a warm (60°C) water bath for 45 seconds followed by removal with a spatula. Usnic acid was extracted with acetonitrile from all layer of the skin: skin surface (2 mL), stratum corneum (3 mL), epidermis (3 mL) and dermis (3 mL). The extraction was performed by 2 minutes of vortex flowed by 30 minutes of sonication. The samples were filtered into vials (0.45 μm Millipore filter) before HPLC analysis. All results are expressed as mean ± standard deviation (n=6).

Results and Discussion

Analytical methods, employed for the quantitative determination of drugs in biological samples, can influence the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. It is therefore essential to employ well-characterized and fully validated analytical methods to give reliable results and which can be interpreted with satisfaction (Shah et al., 1992; Potard et al., 1999). However, as far as we know, no quantitative method for the determination of usnic acid in skin has been reported in the literature. Therefore, a new method for the quantification of usnic acid in the skin by liquid chromatography was developed for a preliminary study on the transdermal penetration of usnic acid after its topical application.

The HPLC method for rapid and precise determination of transdermal permeation and dermal penetration of usnic acid have been optimized and validated in the present study.

Method validation

Usnic acid retention time in this method was 4.3 minutes (Figure 2), an adequate retention time for drug penetration and permeation assays. The method was selective, no response was observed for blank skins at 4.3 minutes, the retention time of usnic acid. The calibration curve was linear (r = 0.9994) and the deviation among the responses (areas) obtained by HPLC was under 20% for the concentration of 1.0 µg.mL-1 and lower than 15% for all other concentrations. The simplest model that adequately describes the concentration-response relationship was the linear equation: Y=119550.7 X + 31266.7.

<Figure 2>

Five replicates with the concentrations 6.5 µg.mL-1 (low), 15 µg.mL-1 (medium) and 25 µg.mL-1 (high) were prepared. The mean response values deviated within 14.9%, 4.6% and 1.4% respectively, indicating that the method is accurate and the coefficient of variation presented values of 2.7%, 2.0% and 1.8% respectively, indicating that the method is precise.

When evaluating the short-term temperature stability of a high (25 µg.mL-1) and low (2.5 µg.mL-1) concentration samples at room temperature for 24 hours, the variation observed were under 5%. Stock solution stability concentration was measured as soon as it was prepared and 24 hour after and no concentration decrease was observed. When submitted to the post-preparative stability assay the samples exhibited a concentration decrease under 5%.

The stability assays suggest that penetration/permeation experiment should not be interrupted or left over night due to a possible concentration decrease, even if small. The proposed method shows a promising applicability in determining usnic acid penetration and permeation from different cutaneous pharmaceutical forms. Penetration/permeation assays are very important in the research and development of cutaneous pharmaceutical forms and usnic acid possible forms of applications are precisely in the research and development phase.

Skin permeation and penetration assay

During the period of the assay it was only possible to quantify usnic acid in the receptor medium at the end of 12 hours. The penetration profile is presented in Figure 3. Usnic acid was capable of penetrating the skin and was quantified in the stratum corneum, viable epidermis and dermis. After 12 hours a small amount of usnic acid was still found on the surface of the skin (6.13 µg.cm2). Usnic acid penetrated greatly to the stratum corneum (34.4 µg.cm2), which most probably acted as a reservoir system. The viable epidermis did not act as a barrier for the molecule and accumulated a small amount (5.6 µg.cm2) of the substance. A larger amount of usnic acid was found in the dermis (28.2 µg.cm2), indicating that the active substance tends to accumulate in this layer and will probably lead to a systemic absorption. After 12 hours the transdermal permeation of usnic acid was very small (3.2 µg.cm2), but considering that the formulation used was very simple and did not intend to favor this rout the use of a transdermal application seems viable.

<Figure 3>

The in vitro experiment was unable to determine the permeation coefficient and flux due to a low permeation of the drug. Still, it is possible to mathematically predict these parameters based on the molecule properties. Usnic acid physico-chemical characteristics are suitable for cutaneous application, considering that, in general, molecules should have a molecular weight lower than 500 and a octanol/water partion coefficient (Ko/w) between 10-100 to be able to penetrate the skin. Usnic acid molecular weight is 344.3 and Ko/w= 75.9 (Log P = 1.88) rendering a permeation coefficient (Kp) of 3.12×10-4 cm/h, using the Potts and Guy method.

The theoretical calculation of the penetration coefficient in this case represents an overestimation of the experimentally observed penetration; a similar observation was made by Mestres and co-workers (2011). The penetration and permeation experiment favor a topical delivery of usnic acid instead of a transdermal delivery.

Conclusions

It was possible to develop and validate a simple, isocratic HPLC method useful in the determining skin penetration and permeation of usnic acid. The method was used to determine the accumulated amount of usnic acid in the different layers of the skin as well as the amount that permeated in vitro. These results help to understand the penetration profile of usnic acid and plan topical therapeutic approaches as well as plan new topical delivery systems to modulate this penetration profile.