A. Clinical assessment of TA treatment efficacy
3. Modified melasma area and severity index (mMASI)
The mMASI score was decreased from 4.34 ± 2.03 (0 week) to 3.72 ± 1.80 (4 week, p <
0.05), to 3.02 ± 1.61 (8 week, p < 0.05 vs. 0 week and 4 week), and to 2.88 ± 1.51 (12 week, p < 0.05 vs. 0 week and 4 week, p = 0.17 vs. 8 week) (Fig. 3).
Fig. 3. Change of mMASI after topical TA treatment. There was gradual decrease of mMASI score during 12-week use of topical TA (* means p < 0.05 compared to 0 week).
- 9 - 4. Chromameter
The mean L* value of both lesional and perilesional skin was significantly increased 4 weeks after treatment compared with baseline, and the values were maintained in lesional skin and continuously increased in perilesional skin through the rest follow-up period (lesional, 0 week vs. 4 week, 59.44 ± 1.78 vs. 61.02 ± 1.81, p < 0.05; perilesional, 0 week vs.
4 week, 62.44 ± 1.46 vs. 63.25 ± 1.98, p < 0.05). Only marked increase between 0 week and 4 week were observed compared with other periods (Fig. 4).
Fig. 4. Change of L* value after topical TA treatment. The mean L* value of both lesional and perilesional skin was significantly increased 4 weeks after treatment compared with baseline, and the values were maintained in lesional skin and continuously increased in perilesional skin through the rest follow-up period (* means p < 0.05 compared to 0 week).
The mean a* value of both lesional and perilesional skin was significantly decreased 4 weeks after treatment compared with baseline, and the values were maintained in lesional and perilesional skin through the rest follow-up period (lesional, 0 week vs. 4 week, 13.32 ± 1.65 vs. 12.39 ± 1.43, p < 0.05; perilesional, 0 week vs. 4 week, 12.15 ± 1.54 vs. 11.35 ± 1.92, p < 0.05). Only marked decrease between 0 week and 4 week were observed compared with other periods (Fig. 5).
- 10 -
Fig. 5. Change of a* value after topical TA treatment. The mean a* value of both lesional and perilesional skin was significantly decreased 4 weeks after treatment compared with baseline, and the decreased values were sustained both in lesional and perilesional skin through the rest follow-up period (* means p < 0.05 compared to 0 week).
5. Subjective satisfaction score
The subjective satisfaction score was significantly increased 4 weeks after treatment compared with baseline, and the values were slightly increased through the rest follow-up period (0 week vs. 4 week, 1.94 ± 0.43 vs. 2.27 ± 0.55, p < 0.05). Only marked increase between 0 week and 4 week were observed compared with other periods (Fig. 6).
Fig. 6. Change of subjective satisfaction score after topical TA treatment. The subjective satisfaction score was significantly increased 4 weeks after treatment compared with baseline, and the values were slightly increased through the rest follow-up period (* means p < 0.05 compared to 0 week).
- 11 - B. Histological analysis
1. Pigmentation
Pigmentation was visualized with Fontana-Masson staining. The epidermal pigment in the lesional skin significantly decreased on 12 week compared with 0 week (PA/EA, 0 week vs.
12 week, 0.32 ± 0.07 vs. 0.25 ± 0.04, p < 0.05). Also, the epidermal pigment of perilesional normal skin showed the similar tendency (PA/EA, 0 week vs. 12 week, 0.18 ± 0.05 vs. 0.13
± 0.05, p < 0.05) (Fig. 7).
Fig. 7. Fontana-Masson staining to visualize epidermal pigmentation. There was significant decrease of epidermal pigmentation in both lesional and perilesional normal skin after 12-week use of topical TA (original magnification, x 200).
2. Vascularity
Vascularity was visualized with CD31 expression for vessel and with D2-40 expression for lymphatics in dermis. The expression of CD31 in the lesional skin decreased on 12 week compared with 0 week (SA/DA, 0 week vs. 12 week, 0.009 ± 0.003 vs. 0.007 ± 0.002, p = 0.053). Also, its expression of perilesional normal skin showed the similar tendency (SA/DA, 0 week vs. 12 week, 0.006 ± 0.004 vs. 0.005 ± 0.003, p = 0.46), though statistically not
- 12 - significant (Fig. 8).
The expression of D2-40 in the lesional skin increased on 12 week compared with 0 week (SA/DA, 0 week vs. 12 week, 0.004 ± 0.003 vs. 0.005 ± 0.002, p = 0.56). However, its expression of perilesional normal skin decreased on 12 week compared with 0 week (SA/DA, 0 week vs. 12 week, 0.005 ± 0.004 vs. 0.004 ± 0.003, p = 0.62) (Fig. 9).
Fig. 8. CD31 expression of dermis after topical TA treatment. There was decrease of expression in the dermis of both lesional and perilesional normal skin after 12-week use of topical TA, though statistically not significant (original magnification, x 200).
- 13 -
Fig. 9. D2-40 expression of dermis after topical TA treatment. There was no significant change of D2-40 expression in the dermis of both lesional and perilesional normal skin after 12-week use of topical TA (original magnification, x 200).
3. Mast cell
Mast cell was visualized with c-kit expression in dermis. The expression of c-kit in the lesional skin increased on 12 week compared with 0 week (SA/DA, 0 week vs. 12 week, 0.003 ± 0.001 vs. 0.004 ± 0.004, p = 0.45). Also, its expression of perilesional normal skin showed the similar tendency (SA/DA, 0 week vs. 12 week, 0.004 ± 0.002 vs. 0.005 ± 0.003, p = 0.67), though statistically not significant (Fig. 10).
- 14 -
Fig. 10. C-kit expression of dermis after topical TA treatment. There was increase of c-kit expression in the dermis of both lesional and perilesional normal skin after 12-week use of topical TA, though statistically not significant (original magnification, x 200).
4. Other immunohistochemical results
The expression of ET-1 in the epidermis of lesional skin significantly decreased on 12 week compared with 0 week (SA/EA, 0 week vs. 12 week, 0.21 ± 0.09 vs. 0.13 ± 0.04, p <
0.05). Also, its expression of perilesional normal skin showed the similar tendency (SA/EA, 0 week vs. 12 week, 0.17 ± 0.08 vs. 0.07 ± 0.04, p < 0.05) (Fig. 11).
- 15 -
Fig. 11. ET-1 expression of epidermis after topical TA treatment. There was significant decrease of ET-1 expression in the epidermis of both lesional and perilesional normal skin after 12-week use of topical TA (original magnification, x 200).
The expression of α-MSH in the epidermis of lesional skin increased on 12 week compared with 0 week (SA/EA, 0 week vs. 12 week, 0.14 ± 0.06 vs. 0.15 ± 0.08, p = 0.88).
Also, its expression of perilesional normal skin showed the similar tendency (SA/EA, 0 week vs. 12 week, 0.11 ± 0.06 vs. 0.15 ± 0.07, p = 0.26) (Fig. 12).
- 16 -
Fig. 12. α-MSH expression of epidermis after topical TA treatment. There was increase of expression in the epidermis of both lesional and perilesional normal skin after 12-week use of topical TA, though statistically not significant (original magnification, x 200).
The expression of EP2 in the epidermis of lesional skin decreased on 12 week compared with 0 week (SA/EA, 0 week vs. 12 week, 0.006 ± 0.008 vs. 0.004 ± 0.003, p = 0.51).
However, its expression of perilesional normal skin increased on 12 week compared with 0 week (SA/EA, 0 week vs. 12 week, 0.004 ± 0.003 vs. 0.005 ± 0.004, p = 0.57) (Fig. 13).
- 17 -
Fig. 13. EP2 expression of epidermis after topical TA treatment. There was no significant change of expression in the epidermis of both lesional and perilesional normal skin after 12-week use of topical TA (original magnification, x 200).
The expression of VEGF in the epidermis of lesional skin decreased on 12 week compared with 0 week (SA/EA, 0 week vs. 12 week, 0.10 ± 0.06 vs. 0.06 ± 0.04, p = 0.15). Also, its expression of perilesional normal skin showed the similar tendency (SA/EA, 0 week vs. 12 week, 0.06 ± 0.05 vs. 0.03 ± 0.03, p = 0.23), though statistically not significant (Fig. 14).
- 18 -
Fig. 14. VEGF expression of epidermis after topical TA treatment. There was decrease of expression in the epidermis of both lesional and perilesional normal skin after 12-week use of topical TA, though statistically not significant (original magnification, x 200).
The expression of SCF in the epidermis of lesional skin significantly decreased on 12 week compared with 0 week (SA/EA, 0 week vs. 12 week, 0.30 ± 0.09 vs. 0.19 ± 0.10, p <
0.05). Also, its expression of perilesional normal skin showed the similar tendency (SA/EA, 0 week vs. 12 week, 0.14 ± 0.11 vs. 0.11 ± 0.07, p = 0.54), though statistically not significant (Fig. 15).
- 19 -
Fig. 15. SCF expression of epidermis after topical TA treatment. There was decrease of expression in the epidermis of both lesional and perilesional normal skin after 12-week use of topical TA, though statistically not significant in perilesional normal skin (original magnification, x 200).
- 20 -
IV. DISCUSSION
TA is widely considered as one of the treatment options for melasma. In 1979, its role in melasma was first demonstrated (Sadako, 1979). Afterwards, many clinical studies with oral TA treatment showed favorable responses in melasma. Other forms of TA administration for melasma also have been investigated. Weekly intradermal injections of TA (Lee et al., 2006), ionotophoresis of TA using chemical enhancer and constant electric current (Todo and Sugibayashi, 2012), and topical TA in liposome formulation was also introduced (Manosroi et al., 2002). Until now, only two clinical studies using topical TA has been reported (Kanechorn Na Ayuthaya et al., 2012; Na et al., 2013). However, the histopathologic analysis was not available in the study of Kanechom Na Ayuthaya et al. and the concurrent use of oral and topical TA in the study of Na et al. could not demonstrate the independent effect of topical TA in melasma. In our study, we investigated the independent effect of topical TA treatment on melasma with histopathologic analysis. Several findings were noted and discussed below.
First, with clinical assessment, time-sequential improvement of MSS and mMASI were noted, especially significant during the first 8 week. However, the additional improvement was minimal after 8 week. Consistent with the change of MSS and mMASI score, subjective satisfaction score showed the similar tendency during 12 weeks. With chromameter measurement, the significant improvement of the value of L* and a* in lesional skin was performed using oral TA, the results were relatively comparable to our results. It is explained that TA has a limitation of binding capacity in that TA reversibly blocks lysine binding sites on plasminogen molecules (Tse and Hui, 2013).
- 21 -
Contrary to our result, TA rather increased pigmentation of the perilesional skin from 0 week to 4 week, and to 8 week in previous report (Na et al., 2013). They suggested the darkening of perilesional skin is affected by seasonal changes, from spring to summer (Na et al., 2013). However, in our study, the L* and a* value indicated that the perilesional normal skin after treatment became brighter than those of baseline status, which showed a similar pattern to those of lesional skin. Considering our result, topical TA might effect on perilesional normal skin as well as lesional skin of melasma. Also, the aggravated erythema at 12 weeks on TA-treated site was reported previously (Kanechorn Na Ayuthaya et al., 2012), however, our result showed no worsening in erythema, although no further improvement was shown in the erythema index after 4 week. No other significant adverse effects, such as irritation were reported except mild dryness in some subjects.
Secondly, with histological analysis, we focused on three main factors i.e., pigmentation, vascularity, and mast cell. Consistent with our clinical assessment and previous report, the epidermal pigmentation significantly reduced in both lesional and perilesional normal skin. It has been known that TA could suppress the process of angiogenesis as well as induce the melanogenesis in melanocytes (Kal et al., 2004; Na et al., 2013). It was reported that TA also inhibits the neovascularization induced by basic fibroblast growth factor (bFGF) (Kal et al., 2004). The positive correlation between the induction of pigmentation and increase of dermal blood vessels in lesional skin of melasma was suggested (Kim et al., 2007), and the significant decrease of vessel numbers as well as the reduction of pigmentation during TA treatment was also reported (Na et al., 2013). In this study, the decreased CD31-stained blood vessel in dermis was observed in lesional and perilesional normal skin after topical TA treatment, though not statistically significant. In terms of mast cells, which were more prominent in the elastotic areas of melasma skin (Hernandez-Barrera et al., 2008), significantly decreased number of mast cells was observed after 8-week use of oral and topical TA (Na et al., 2013). However, there was little difference in the number of c-kit-stained mast cell after topical TA treatment in our study. Taken together, topical TA treatment could reduce the epidermal pigmentation, although their effect on dermal environment showed limited, compared with oral TA.
Thirdly, our data showed the significant decrease of ET-1 expression after treatment in epidermis of both lesional and perilesional normal skin. It has been reported that paracrine
- 22 -
linkages among keratinocytes, fibroblasts, and melanocytes play important roles in regulating epidermal pigmentation. In response to various stimuli, human keratinocytes secrete various cytokines that serve as mitogens or melanogens for human melanocytes, including ET-1, α-MSH, and SCF (Imokawa et al., 1992; Morita et al., 1994). Among them, ET-1 can trigger melanogenesis via the MITF-regulated GPNMB pathway and plays an indispensable role in epidermal pigmentation in hyperpigmentary disorders (Zhang et al., 2013). We demonstrated the significant decrease of ET-1 expression in the epidermis of both lesional and perilesional normal skin. Also, the expression of SCF, one of the cytokines secreted by human keratinocytes and human fibroblasts (Imokawa et al., 1998), was significantly decreased after treatment in the epidermis of lesional skin. However, there was no significant difference in the expression of α-MSH after treatment in both lesional and perilesional normal skin. Therefore, our data is speculated that both ET-1 and SCF might play an important role in action mechanism of TA on melasma. TA is known to suppress the production of prostaglandins, and subsequent UV-induced melanogenesis through the suppression of the epidermal plasmin activity. Lesser free arachidonic acids and depleted production of prostaglandins result in the reduction of tyrosinase activity in melanocytes (Maeda and Naganuma, 1998; Maeda and Tomita, 2007). However, there was no significant difference in the expression of EP2 after treatment in both lesional and perilesional normal skin. Also, human melanocytes may respond to angiogenic factors because normal human melanocytes express functional VEGF receptors (Kim et al., 2005), and the expression of VEGF was significantly increased in melasma (Kim et al., 2007). In contrary, the expression of VEGF after treatment showed minimal difference. It is suggested that dermal structures were not affected by topical TA possibly because of their low penetration into the dermis.
- 23 -
V. CONCLUSION
With topical TA treatment for 12 weeks, we demonstrated that the significant improvement of MSS and mMASI scores and subjective satisfaction score, and confirmed by chromameter. With histological analysis, the epidermal pigmentation was decreased significantly in the lesional skin, and even in perilesional normal skin of melasma. With immunohistochemical analysis, only ET-1 and SCF expression of epidermis were reduced significantly after TA treatment. However, dermal vascularity, number of mast cells, and VEGF expression of epidermis showed minimal changes. These findings suggest that the expected mechanism of topical TA might be responsible for modulating epidermal environment rather than dermal environment.
- 24 -
REFERENCES
1. Cho HH, Choi M, Cho S, Lee JH: Role of oral tranexamic acid in melasma patients treated with IPL and low fluence QS Nd:YAG laser. J Dermatolog Treat 24: 292-296, 2013
2. Hernandez-Barrera R, Torres-Alvarez B, Castanedo-Cazares JP, Oros-Ovalle C, Moncada B: Solar elastosis and presence of mast cells as key features in the pathogenesis of melasma. Clin Exp Dermatol 33: 305-308, 2008
3. Higashi N: Clinical study of tranexamic acid cream on melasma: A multi-center double blind trial. Skin Res 6: 649-652, 2007
4. Imokawa G, Yada Y, Miyagishi M: Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem 267: 24675-24680, 1992 5. Imokawa G, Yada Y, Morisaki N, Kimura M: Biological characterization of human
fibroblast-derived mitogenic factors for human melanocytes. Biochem J 330 ( Pt 3):
1235-1239, 1998
6. Kal HB, Struikmans H, Gebbink MF, Voest EE: Response of rat prostate and lung tumors to ionizing radiation combined with the angiogenesis inhibitor AMCA.
Strahlenther Onkol 180: 798-804, 2004
7. Kanechorn Na Ayuthaya P, Niumphradit N, Manosroi A, Nakakes A: Topical 5%
tranexamic acid for the treatment of melasma in Asians: a double-blind randomized controlled clinical trial. J Cosmet Laser Ther 14: 150-154, 2012
8. Kang HY, Hwang JS, Lee JY, Ahn JH, Kim JY, Lee ES, Kang WH: The dermal stem cell factor and c-kit are overexpressed in melasma. Br J Dermatol 154: 1094-1099, 2006
9. Kang WH, Yoon KH, Lee ES, Kim J, Lee KB, Yim H, Sohn S, Im S: Melasma:
histopathological characteristics in 56 Korean patients. Br J Dermatol 146: 228-237, 2002
10. Karn D, Kc S, Amatya A, Razouria EA, Timalsina M: Oral tranexamic acid for the treatment of melasma. Kathmandu Univ Med J (KUMJ) 10: 40-43, 2012
11. Kim EH, Kim YC, Lee ES, Kang HY: The vascular characteristics of melasma. J
- 25 - Dermatol Sci 46: 111-116, 2007
12. Kim EJ, Park HY, Yaar M, Gilchrest BA: Modulation of vascular endothelial growth factor receptors in melanocytes. Exp Dermatol 14: 625-633, 2005
13. Lee JH, Park JG, Lim SH, Kim JY, Ahn KY, Kim MY, Park YM: Localized intradermal microinjection of tranexamic acid for treatment of melasma in Asian patients: a preliminary clinical trial. Dermatol Surg 32: 626-631, 2006
14. Li Y, Sun Q, He Z, Fu L, He C, Yan Y: Treatment of melasma with oral administration of compound tranexamic acid: a preliminary clinical trial. J Eur Acad Dermatol Venereol 28: 393-394, 2014
15. Maeda K: The mechanism of ultraviolet B radiation-induced skin pigmentation and the effect of topical t-AMCHA on the pigmentation. Flavour Fragr J 18: 42-49, 2003
16. Maeda K, Naganuma M: Topical trans-4-aminomethylcyclohexanecarboxylic acid prevents ultraviolet radiation-induced pigmentation. J Photochem Photobiol B 47:
136-141, 1998
17. Manosroi A, Podjanasoonthon K, Manosroi J: Development of novel topical tranexamic acid liposome formulations. Int J Pharm 235: 61-70, 2002
18. Morita E, Lee DG, Sugiyama M, Yamamoto S: Expression of c-kit ligand in human keratinocytes. Arch Dermatol Res 286: 273-277, 1994
19. Na JI, Choi SY, Yang SH, Choi HR, Kang HY, Park KC: Effect of tranexamic acid on melasma: a clinical trial with histological evaluation. J Eur Acad Dermatol Venereol 27: 1035-1039, 2013
20. Pandya AG, Hynan LS, Bhore R, Riley FC, Guevara IL, Grimes P, Nordlund JJ, Rendon M, Taylor S, Gottschalk RW, Agim NG, Ortonne JP: Reliability assessment and validation of the Melasma Area and Severity Index (MASI) and a new modified MASI scoring method. J Am Acad Dermatol 64: 78-83, 83 e71-72, 2011
21. Sadako N: Treatment of melasma with tranexamid acid. The Clin Rep 13: 3129-3131, 1979
22. Sarkar R, Arora P, Garg VK, Sonthalia S, Gokhale N: Melasma update. Indian Dermatol Online J 5: 426-435, 2014
23. Shin JU, Park J, Oh SH, Lee JH: Oral tranexamic acid enhances the efficacy of
low-- 26 low--
fluence 1064-nm quality-switched neodymium-doped yttrium aluminum garnet laser treatment for melasma in Koreans: a randomized, prospective trial. Dermatol Surg 39: 435-442, 2013
24. Taylor SC, Torok H, Jones T, Lowe N, Rich P, Tschen E, Menter A, Baumann L, Wieder JJ, Jarratt MM, Pariser D, Martin D, Weiss J, Shavin J, Ramirez N: Efficacy and safety of a new triple-combination agent for the treatment of facial melasma.
Cutis 72: 67-72, 2003
25. Torres-Alvarez B, Mesa-Garza IG, Castanedo-Cazares JP, Fuentes-Ahumada C, Oros-Ovalle C, Navarrete-Solis J, Moncada B: Histochemical and immunohistochemical study in melasma: evidence of damage in the basal membrane.
Am J Dermatopathol 33: 291-295, 2011
26. Tse TW, Hui E: Tranexamic acid: an important adjuvant in the treatment of melasma.
J Cosmet Dermatol 12: 57-66, 2013
27. Wu S, Shi H, Wu H, Yan S, Guo J, Sun Y, Pan L: Treatment of melasma with oral administration of tranexamic acid. Aesthetic Plast Surg 36: 964-970, 2012
28. Zhang P, Liu W, Yuan X, Li D, Gu W, Gao T: Endothelin-1 enhances the melanogenesis via MITF-GPNMB pathway. BMB Rep 46: 364-369, 2013
- 27 -
SUPPLEMENT
Appendix 1. Verhoeff-Van Gieson staining (SA/DA)
Normal Lesion
0 week 0.18±0.06 0.30±0.18
12 week 0.29±0.10* 0.29±0.12
* p < 0.05 compared to 0 week
Appendix 2. MMP2 expression of dermis after topical TA treatment
Normal Lesion
0 week 0.0058±0.0056 0.0090±0.0069
12 week 0.0073±0.0053 0.0097±0.0088
- 28 -
Appendix 3. Factor XIIIa expression of dermis after topical TA treatment
Normal Lesion
0 week 0.0072±0.0024 0.0063±0.0027
12 week 0.0077±0.0023 0.0094±0.0041
Appendix 4. Vimentin expression of dermis after topical TA treatment
Normal Lesion
0 week 0.0394±0.0185 0.0371±0.0182
12 week 0.0344±0.0131 0.0427±0.0190
Appendix 5. BMP4 expression of epidermis after topical TA treatment
- 29 -
Normal Lesion
0 week 0.0117±0.0199 0.0043±0.0052
12 week 0.0042±0.0045 0.0123±0.0112*
* p < 0.05 compared to 0 week
Appendix 6. BMP4 expression of dermis after topical TA treatment
Normal Lesion
0 week 0.0016±0.0027 0.0054±0.0075
12 week 0.0009±0.0013 0.0010±0.0006
Appendix 7. BMP6 expression of epidermis after topical TA treatment
Normal Lesion
0 week 0.0216±0.0163 0.0375±0.0304
12 week 0.0207±0.0197 0.0180±0.0315
- 30 -
Appendix 8. BMP6 expression of dermis after topical TA treatment
Normal Lesion
0 week 0.0002±0.0002 0.0002±0.0003
12 week 0.0025±0.0077 0.0003±0.0003
- 31 - severity scale (MSS), modified melasma area and severity index (mMASI), 그리고 색도계 를 통해 객관적으로 이루어졌으며 피험자 만족도 설문도 기록되었다. 10명의 피