Original Paper Dermatology 2015;230:269–275 DOI: 10.1159/000371386
Received: September 19, 2014 Accepted after revision: December 3, 2014 Published online: January 23, 2015
Fractional CO2 Laser Treatment to Enhance Skin Permeation of Tranexamic Acid with Minimal Skin Disruption Chien-Yu Hsiao a, c Hsin-Ching Sung d Sindy Hu b, e, f Chun-Hsun Huang b, c a Department of Nutrition and Health Sciences, b Department of Cosmetic Science and c Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, d Department of Anatomy and e Department of Medicine, College of Medicine, Chang Gung University, and f Aesthetic Medical Center, Department of Dermatology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
Abstract Background: Topical tranexamic acid has been used to treat melasma and as a skin whitener. Objective: The aim of this study was to compare the skin histology and permeation of tranexamic acid after fractional and conventional CO2 laser pretreatment. Methods: The effect of treatment with different strengths of fractional and conventional CO2 laser treatment was examined by scanning and transmission electron microscopy. Permeation of tranexamic acid through porcine skin was tested in vitro using a Franz diffusion chamber. Results: Four passes of fractional laser treatment caused less skin damage than conventional laser treatment at the same fluency. Fractional laser treatment caused at least 85% of the cumulative tranexamic aid permeation at the same fluency, and as fluency increased, the number of passes needed to achieve this goal decreased. Conclusion: Fractional laser treatment is as effective as conventional laser treatment in enhancing tranexamic acid delivery and causes less skin damage. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 1018–8665/15/2303–0269$39.50/0 E-Mail [email protected] www.karger.com/drm
Introduction
Tranexamic acid is a synthetic lysine analog that inhibits plasmin activity [1]. It was first used as a hemostatic agent and is now, at lower doses, used as a skin whitener and as an ingredient in some cosmetic creams. When used as an oral treatment, 500 mg/day for 6 months was reported to be effective in the treatment of melasma [1], and 750 mg/day for 8 weeks was reported to improve the effectiveness of laser treatment of melasma [2]. Eight weeks of topical treatment with or without the addition of oral treatment has also been reported to be effective as a treatment of melasma [3, 4]. A problem of the current topical and oral therapy with tranexamic acid is the long treatment period necessary. Tranexamic acid is said to act by inhibiting melanogenesis and disrupting melanocyte-keratinocyte interaction [2, 3]. To reach its site of action after topical application, it must penetrate the stratum corneum (SC), the major barrier to entry. If a way were found to increase its penetration, this might decrease the treatment period and therefore be clinically useful. Laser treatment is one way to increase the transdermal permeability to topical agents. At lower fluencies than Chun-Hsun Huang, PhD Department of Cosmetic Science Chang Gung University of Science and Technology Taoyuan 333 (Taiwan) E-Mail chuang @ gw.cgust.edu.tw
Downloaded by: Stockholms Universitet 198.143.54.1 - 8/14/2015 11:08:47 AM
Key Words Fractional CO2 laser · CO2 laser · Tranexamic acid · Skin permeation · Cosmetics
Materials and Methods
were then divided further as needed. TEM, SEM and diffusion assays were all performed on skin from the same pig. Eight pigs were used in all. Laser Experimental Protocol In order to determine the optimal balance between good permeation and minimal destruction, we started at 1 pass of the fractional laser machine at the lowest wattage (5 W) and compared the results with those using a conventional laser at the same wattage. We then increased the number of passes with the fractional laser at that wattage until 4 passes had been used. This protocol was then repeated with the next wattage. The wattages used were 5, 7, 9, and 11 W. Ultrastructure Examination by SEM Excised porcine skin samples with and without laser treatment were cut into appropriate-sized cubes and immediately fixed at 4 ° C in 3% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight, washed three times with 0.1 M cacodylate and 7% sucrose buffer for 15 min, post-fixed with 2% osmium tetroxide for 1 h, washed three times as before and immersed in 0.5% aqueous uranyl acetate for 30 min. The specimens were then dehydrated in graded concentrations of ethanol, transferred to isoamyl acetate and critical-point dried using liquid CO2. The dried specimens were affixed with gold-palladium in an ion coater and examined with a scanning electron microscope (S-5000; Hitachi, Tokyo, Japan).
Ultrastructure Examination by TEM Excised porcine skin samples with and without laser treatment were cut into appropriate-sized cubes and immediately fixed at 4 ° C in 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight, washed three times with 0.1 M cacodylate and 7% sucrose buffer for 15 min, post-fixed with 2% osmium tetroxide for 24 h and washed three times as before. The specimens were then dehydrated in graded concentrations of ethanol, embedded in an EPON-epoxy mixture and sectioned. Thin sections were double-stained with uranyl acetate and lead citrate and examined by TEM (H-7500; Hitachi).
Laser Assemblies The fractional CO2 laser (150XJ; Sharplan Laser Inc., Yokneam, Israel) has a wavelength of 10,600 nm and uses an articulated arm to deliver the laser beam onto the skin surface. The handpiece is able to create microscopic treatment zones, i.e. microscopic columns of ablated skin. These ‘irradiation dots’ typically have a diameter of 150 μm and occupy an area of about 0.018 mm2. The dimensions of the treatment area of the handpiece were 12 × 12 mm. This area had 169 irradiation dots (13 × 13). The surface coverage of the microscopic treatment zones was 2% of the treatment area when one pulse of the laser was used (0.018 mm2 × 169/12 × 12 mm2). For the current study, the skin received 1–4 passes at fluencies from 5 to 11 W. The operator rotated the handpiece by different angles so that when more than 1 pass was used, the irradiation areas of the individual pulses did not overlap. The change to conventional laser treatment was achieved simply by changing the handpiece of the laser. The spot size of the conventional laser was 3 mm in diameter.
In vitro Permeation of Tranexamic Acid The diffusion cell used in this in vitro experiment was a Franz diffusion assembly. A piece of excised porcine dorsal skin was mounted in the apparatus with the SC facing the donor compartment. After laser pretreatment, the skin surface was wiped with a cotton wool swab several times. The receptor compartment (5.5 ml) was filled with a pH 7.4 citrate-phosphate buffer. The donor compartment (0.5 ml) contained 10% tranexamic acid (SigmaAldrich, St. Louis, Mo., USA) in a pH 7.4 citrate-phosphate buffer. The available area of the side-by-side cell was 0.7854 cm2. The receptor compartment was maintained at 37 ° C, and the contents were stirred with a magnetic bar at 600 rpm. At appropriate intervals, 300 μl aliquots of receptor medium were withdrawn and immediately replaced with an equal volume of fresh receptor solution. The length of the sampling period was 12 h. The amount of drug in the receptor medium was determined by high-performance liquid chromatography (HPLC).
Skin Samples Skin from eight pathogen-free pigs (1 week old) was supplied by the Animal Technology Institute Taiwan (Miaoli, Taiwan). The supplier euthanized the piglets by electrocution, harvested the dorsal skin, cut it to the appropriate size and shipped it directly to the authors’ laboratory. Porcine skin was used for scanning electron microscopy (SEM), transmission electron microscopy (TEM) and in vitro tranexamic acid permeation studies. For these studies, ten 2 × 2 cm2 portions of dorsal skin were removed from each pig (one for each laser condition) for laser treatment. These skin portions
270
Dermatology 2015;230:269–275 DOI: 10.1159/000371386
HPLC Analytical Method The samples were analyzed with an HPLC system consisting of a Dionex UltiMate 3000 pump, a Dionex UltiMate 3000 autosam-
Hsiao /Sung /Hu /Huang
Downloaded by: Stockholms Universitet 198.143.54.1 - 8/14/2015 11:08:47 AM
those used for skin resurfacing, laser treatment can ablate the SC and thus enhance delivery [5], but recovery from laser-induced skin damage can take several days [6]. Fractional laser therapy delivers the laser beam through a grid of micro-apertures in the area to be treated instead of delivering it to the entire area and, in this way, lessens the skin damage caused by conventional laser therapy and shortens the subsequent recovery period [6–8]. Fractional laser therapy has been shown to increase the topical delivery of a number of substances [5–8], including derivatives of ascorbic acid, another compound used as a skin whitener [5, 6]. However, in order for fractional laser treatment to be useful clinically, one needs information to help the clinician determine the fluency and number of passes that will result in a combination of optimal permeation and minimal skin damage. This relationship has not yet been explored for tranexamic acid. In the current study, we used microscopy to compare skin damage caused by conventional and fractional laser treatment at different fluencies and the Franz diffusion chamber to examine the influence of fluency and pass number on tranexamic acid permeation through porcine skin.
a
b
c
Fig. 1. SEM observations (magnification ×500) of porcine skin. a Without laser treatment. b With fractional laser treatment at 7 W, 4 passes. c Conventional laser treatment at 7 W.
a line plot with mean and standard deviation (SD) for each condition. Results of flux across porcine skin were summarized as mean ± SD for each condition, and these results were compared using one-way analysis of variance with a post hoc Duncan test. Statistical assessments were considered significant at p
Received: September 19, 2014 Accepted after revision: December 3, 2014 Published online: January 23, 2015
Fractional CO2 Laser Treatment to Enhance Skin Permeation of Tranexamic Acid with Minimal Skin Disruption Chien-Yu Hsiao a, c Hsin-Ching Sung d Sindy Hu b, e, f Chun-Hsun Huang b, c a Department of Nutrition and Health Sciences, b Department of Cosmetic Science and c Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, d Department of Anatomy and e Department of Medicine, College of Medicine, Chang Gung University, and f Aesthetic Medical Center, Department of Dermatology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
Abstract Background: Topical tranexamic acid has been used to treat melasma and as a skin whitener. Objective: The aim of this study was to compare the skin histology and permeation of tranexamic acid after fractional and conventional CO2 laser pretreatment. Methods: The effect of treatment with different strengths of fractional and conventional CO2 laser treatment was examined by scanning and transmission electron microscopy. Permeation of tranexamic acid through porcine skin was tested in vitro using a Franz diffusion chamber. Results: Four passes of fractional laser treatment caused less skin damage than conventional laser treatment at the same fluency. Fractional laser treatment caused at least 85% of the cumulative tranexamic aid permeation at the same fluency, and as fluency increased, the number of passes needed to achieve this goal decreased. Conclusion: Fractional laser treatment is as effective as conventional laser treatment in enhancing tranexamic acid delivery and causes less skin damage. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 1018–8665/15/2303–0269$39.50/0 E-Mail [email protected] www.karger.com/drm
Introduction
Tranexamic acid is a synthetic lysine analog that inhibits plasmin activity [1]. It was first used as a hemostatic agent and is now, at lower doses, used as a skin whitener and as an ingredient in some cosmetic creams. When used as an oral treatment, 500 mg/day for 6 months was reported to be effective in the treatment of melasma [1], and 750 mg/day for 8 weeks was reported to improve the effectiveness of laser treatment of melasma [2]. Eight weeks of topical treatment with or without the addition of oral treatment has also been reported to be effective as a treatment of melasma [3, 4]. A problem of the current topical and oral therapy with tranexamic acid is the long treatment period necessary. Tranexamic acid is said to act by inhibiting melanogenesis and disrupting melanocyte-keratinocyte interaction [2, 3]. To reach its site of action after topical application, it must penetrate the stratum corneum (SC), the major barrier to entry. If a way were found to increase its penetration, this might decrease the treatment period and therefore be clinically useful. Laser treatment is one way to increase the transdermal permeability to topical agents. At lower fluencies than Chun-Hsun Huang, PhD Department of Cosmetic Science Chang Gung University of Science and Technology Taoyuan 333 (Taiwan) E-Mail chuang @ gw.cgust.edu.tw
Downloaded by: Stockholms Universitet 198.143.54.1 - 8/14/2015 11:08:47 AM
Key Words Fractional CO2 laser · CO2 laser · Tranexamic acid · Skin permeation · Cosmetics
Materials and Methods
were then divided further as needed. TEM, SEM and diffusion assays were all performed on skin from the same pig. Eight pigs were used in all. Laser Experimental Protocol In order to determine the optimal balance between good permeation and minimal destruction, we started at 1 pass of the fractional laser machine at the lowest wattage (5 W) and compared the results with those using a conventional laser at the same wattage. We then increased the number of passes with the fractional laser at that wattage until 4 passes had been used. This protocol was then repeated with the next wattage. The wattages used were 5, 7, 9, and 11 W. Ultrastructure Examination by SEM Excised porcine skin samples with and without laser treatment were cut into appropriate-sized cubes and immediately fixed at 4 ° C in 3% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight, washed three times with 0.1 M cacodylate and 7% sucrose buffer for 15 min, post-fixed with 2% osmium tetroxide for 1 h, washed three times as before and immersed in 0.5% aqueous uranyl acetate for 30 min. The specimens were then dehydrated in graded concentrations of ethanol, transferred to isoamyl acetate and critical-point dried using liquid CO2. The dried specimens were affixed with gold-palladium in an ion coater and examined with a scanning electron microscope (S-5000; Hitachi, Tokyo, Japan).
Ultrastructure Examination by TEM Excised porcine skin samples with and without laser treatment were cut into appropriate-sized cubes and immediately fixed at 4 ° C in 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight, washed three times with 0.1 M cacodylate and 7% sucrose buffer for 15 min, post-fixed with 2% osmium tetroxide for 24 h and washed three times as before. The specimens were then dehydrated in graded concentrations of ethanol, embedded in an EPON-epoxy mixture and sectioned. Thin sections were double-stained with uranyl acetate and lead citrate and examined by TEM (H-7500; Hitachi).
Laser Assemblies The fractional CO2 laser (150XJ; Sharplan Laser Inc., Yokneam, Israel) has a wavelength of 10,600 nm and uses an articulated arm to deliver the laser beam onto the skin surface. The handpiece is able to create microscopic treatment zones, i.e. microscopic columns of ablated skin. These ‘irradiation dots’ typically have a diameter of 150 μm and occupy an area of about 0.018 mm2. The dimensions of the treatment area of the handpiece were 12 × 12 mm. This area had 169 irradiation dots (13 × 13). The surface coverage of the microscopic treatment zones was 2% of the treatment area when one pulse of the laser was used (0.018 mm2 × 169/12 × 12 mm2). For the current study, the skin received 1–4 passes at fluencies from 5 to 11 W. The operator rotated the handpiece by different angles so that when more than 1 pass was used, the irradiation areas of the individual pulses did not overlap. The change to conventional laser treatment was achieved simply by changing the handpiece of the laser. The spot size of the conventional laser was 3 mm in diameter.
In vitro Permeation of Tranexamic Acid The diffusion cell used in this in vitro experiment was a Franz diffusion assembly. A piece of excised porcine dorsal skin was mounted in the apparatus with the SC facing the donor compartment. After laser pretreatment, the skin surface was wiped with a cotton wool swab several times. The receptor compartment (5.5 ml) was filled with a pH 7.4 citrate-phosphate buffer. The donor compartment (0.5 ml) contained 10% tranexamic acid (SigmaAldrich, St. Louis, Mo., USA) in a pH 7.4 citrate-phosphate buffer. The available area of the side-by-side cell was 0.7854 cm2. The receptor compartment was maintained at 37 ° C, and the contents were stirred with a magnetic bar at 600 rpm. At appropriate intervals, 300 μl aliquots of receptor medium were withdrawn and immediately replaced with an equal volume of fresh receptor solution. The length of the sampling period was 12 h. The amount of drug in the receptor medium was determined by high-performance liquid chromatography (HPLC).
Skin Samples Skin from eight pathogen-free pigs (1 week old) was supplied by the Animal Technology Institute Taiwan (Miaoli, Taiwan). The supplier euthanized the piglets by electrocution, harvested the dorsal skin, cut it to the appropriate size and shipped it directly to the authors’ laboratory. Porcine skin was used for scanning electron microscopy (SEM), transmission electron microscopy (TEM) and in vitro tranexamic acid permeation studies. For these studies, ten 2 × 2 cm2 portions of dorsal skin were removed from each pig (one for each laser condition) for laser treatment. These skin portions
270
Dermatology 2015;230:269–275 DOI: 10.1159/000371386
HPLC Analytical Method The samples were analyzed with an HPLC system consisting of a Dionex UltiMate 3000 pump, a Dionex UltiMate 3000 autosam-
Hsiao /Sung /Hu /Huang
Downloaded by: Stockholms Universitet 198.143.54.1 - 8/14/2015 11:08:47 AM
those used for skin resurfacing, laser treatment can ablate the SC and thus enhance delivery [5], but recovery from laser-induced skin damage can take several days [6]. Fractional laser therapy delivers the laser beam through a grid of micro-apertures in the area to be treated instead of delivering it to the entire area and, in this way, lessens the skin damage caused by conventional laser therapy and shortens the subsequent recovery period [6–8]. Fractional laser therapy has been shown to increase the topical delivery of a number of substances [5–8], including derivatives of ascorbic acid, another compound used as a skin whitener [5, 6]. However, in order for fractional laser treatment to be useful clinically, one needs information to help the clinician determine the fluency and number of passes that will result in a combination of optimal permeation and minimal skin damage. This relationship has not yet been explored for tranexamic acid. In the current study, we used microscopy to compare skin damage caused by conventional and fractional laser treatment at different fluencies and the Franz diffusion chamber to examine the influence of fluency and pass number on tranexamic acid permeation through porcine skin.
a
b
c
Fig. 1. SEM observations (magnification ×500) of porcine skin. a Without laser treatment. b With fractional laser treatment at 7 W, 4 passes. c Conventional laser treatment at 7 W.
a line plot with mean and standard deviation (SD) for each condition. Results of flux across porcine skin were summarized as mean ± SD for each condition, and these results were compared using one-way analysis of variance with a post hoc Duncan test. Statistical assessments were considered significant at p
