COMPARATIVE STUDY OF LOCALIZED

INTRADERMAL MICROINJECTION OF TRANEXAMIC

ACID IN DIFFERENT DOSES FOR TREATMENT OF

MELASMA IN THAI PATIENTS

BY

MISS PRAPALPITCH WONGWICHARN

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE (DERMATOLOGY)

CHULABHORN INTERNATIONAL COLLEGE OF MEDICINE

THAMMASAT UNIVERSITY

ACADEMIC YEAR 2017

COPYRIGHT OF THAMMASAT UNIVERSITY

Ref. code: 25605929040458YKT

COMPARATIVE STUDY OF LOCALIZED

INTRADERMAL MICROINJECTION OF TRANEXAMIC

ACID IN DIFFERENT DOSES FOR TREATMENT OF

MELASMA IN THAI PATIENTS

BY

MISS PRAPALPITCH WONGWICHARN

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE (DERMATOLOGY)

CHULABHORN INTERNATIONAL COLLEGE OF MEDICINE

THAMMASAT UNIVERSITY

ACADEMIC YEAR 2017

COPYRIGHT OF THAMMASAT UNIVERSITY

Ref. code: 25605929040458YKT

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Thesis Title COMPARATIVE STUDY OF LOCALIZED

INTRADERMAL MICROINJECTION OF

TRANEXAMIC ACID IN DIFFERENT DOSES

FOR TREATMENT OF MELASMA IN THAI

PATIENTS

Author

Degree Master of science

Major Field/Faculty/University Dermatology

Chulabhorn International College of Medicine

Thammasat University

Thesis Advisor

Thesis Co-Advisor

goyggrrdnnatnatnPun Punyaphat Sirithanabadeekul, M.D.

-

Academic Years 2017

ABSTRACT

Introduction: Nowadays, there are numerous treatment modalities for

melasma. However, the results still vary and have not been able to reach satisfactory

outcomes. Intradermal microinjection of tranexamic acid (TA) has been reported as an

alternative treatment for melasma.

Objective: To evaluate the efficacy and safety of tranexamic acid (TA)

intradermal microinjection in treatment of melasma, between 50 mg/ml TA versus 4

mg/ml TA in reducing melanin level and modified Malasma Area Severity Index

scoring (mMASI score).

Method: Thirty four patients with melasma were enrolled a prospective,

randomized, triple-blinded, split-faced trial. TA 50 mg/ml was randomly intradermal

microinjection to one side of the face and 4 mg/ml TA was injected to the other side

every 2 weeks for 12 weeks and follow up 1-month after the last treatment. All

patients were instructed to applying the SPF 50 sunscreen to both side of the face in

every morning. Clinical images were taken monthly including melanin level and

hemoglobin level by using the skin analysis camera (Antera3D®), mMASI score

Miss Prapalpitch Wongwicharn

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which evaluated by two dermatologists, side effects to assess the clinical response.

Patients’ self-assessment score was assessed at 1-month follow up after the complete

treatment protocol.

Results: Although, the significant decreased in mMASI score and

melanin level was observed in both concentrations but the side treated with 50 mg/ml

TA could achieve the better result in shorter period comparing to the other side of

face which treated with 4 mg/ml TA. Furthermore, in the sided treated with 50 mg/ml,

there was 34 % improvement in mMASI score and 13 % improvement in melanin

level comparing to 25 % and 5 %, respectively in the side treated with 4 mg/ml. At 1-

month follow up, there was significant decrease in mMASI score and melanin level in

both sides of the face. No any serious adverse effect was observed. Majority of the

patients preferred 50 mg/ml TA intradermal microinjection than 4 mg/ml. 85% of the

patients scored as excellent improvement on the side treated with 50 mg/ml

comparing to 4 mg/ml which was scored 35% as excellent improvement.

Conclusion: Intralesional microinjection of 50 mg/ml TA showed a

statistically significant decrease in melasma without any serious side effects.

Moreover, 50 mg/ml of TA showed superior to 4 mg/ml in term of improvement in

melasma and also can reach the better result in the shorter period of time. Therefore,

50 mg/ml TA appeared to be a therapeutic option or adjunctive treatment for

melasma.

Keywords: Melasma, Melasma pathogenesis, Melasma treatment, tranexamic acid

(TA), intradermal microinjection

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ACKNOWLEDGEMENTS

First of all, I would like to extend my special thanks to Dr. Punyaphat

Sirithanabadeekul, M.D., my advisor, for her understanding, consultation, patience,

encouragement and especially for pushing me farther than I thought I could go. She is

such a very good role model for me. AS well my sincere gratefulness and respect

would be conveyed to Dr. Salinee Rojhirunsakool, M.D. and Dr. Suparuj Lueangarun,

M.D., the committee members, for such wonderful pieces of advice and kindly

suggestions to the modification of my research study towards the best and highest

quality. Moreover thank you so much to my family and Mr. Kevin Choe for their

valuable time for helping and giving me words of encouragement thoughout this

research study.

In particular, my appreciation is devoted to Tobacco Monopoly Hospital

for their willingness to me to use the study site, and distinctively the graciousness of

all staffs which always facilitates and comforts me well. Moreover, I owe my deepest

gratitude to all of my colleagues, especially Mr. Zaw Hla oo who help me survive the

stress time as always.

Finally, I would like to express my sincere gratitude to chulabhorn

international college of medicine, Thammasat University for letting me fulfill my

dream of being a student here and the opportunity to write an honors thesis.

Miss Prapalpitch Wongwicharn

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TABLE OF CONTENTS

Page

ABSTRACT (1)

ACKNOWLEDGEMENTS (3)

LIST OF TABLES (7)

LIST OF FIGURES (8)

LIST OF ABBREVIATIONS (10)

CHAPTER 1 INTRODUCTION 1

1.1 Background and rationale 1

1.2 Research question 2

1.3 Specific Objective 2

1.4 Hypothesis 3

1.5 Keywords 3

1.6 Ethic consideration 3

1.7 Limitation 4

1.8 Expected benefits and application 4

CHAPTER 2 REVIEW OF LITERATURE 6

2.1 Review of literature 6

CHAPTER 3 RESEARCH METHODOLOGY 26

3.1 Study design 26

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3.2 Study sample or study population 26

3.2.1 Target population 26

3.2.2 Sampling method 26

3.2.3 Discontinuation criteria 28

3.2.4 Sample size 28

3.3 Research grouping 28

3.4 Material and Methods 29

3.4.1 Informed consent process 29

3.4.2 History taking 29

3.4.3 Physical examination and evaluation 30

3.4.4 Intervention 30

3.4.5 Tranexamic acid preparation 31

3.4.6 Follow up 32

3.5 Outcome measurement 34

3.5.1Objectiveassessment 35

3.5.1.1 Skin analysis camera (Antera 3D®) 35

3.5.1.2 Side effects 36

3.5.2 Subjective assessment 36

3.5.2.1 modified MASI score (mMASI score) 36

3.5.2.2 Patients’ self-assessment of the 37

improvement of melasma

3.5.2.3 Dermatological Quality of Life Index 37

3.5.2.4 Pain score 38

3.6 Data analysis 38

CHAPTER 4 RESULTS AND DISCUSSION 39

4.1 Patients demographic data 39

4.2 Objective assessment 41

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4.2.1 Skin analysis camera (Antera 3D®) 41

4.2.1.1 Antera 3D® melanin level 41

4.2.1.2 Antera 3D® hemoglobin level 43

4.3 Subjective assessment 45

4.3.1 mMASIscore 45

4.3.2 Patients’self-assessment 45

4.3.3Side effect 49

4.3.4 Dermatologic Quality of Life Index 51

4.4 Discussion 52

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 60

5.1 Conclusion 60

5.2 Recommendation 61

5.3 Limitation 61

REFERENCES 62

APPENDICES 68

APPENDIX A 69

APPENDIX B 71

APPENDIX C 73

APPENDIX D 75

APPENDIX E 79

BIOGRAPHY 86

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LIST OF TABLES

Tables Page

1.1 Administration and time schedule 5

2.1 Classification of melasma based on the depth of melasma 8

2.2 Clinical trials of tranexamic acid in the treatment of melasmax 22

3.1 Inclusion and exclusion criteria 26

3.3 Outcome measurement 34

4.1 Patients demographic data 39

4.2 Antera 3D® Melanin level 42

4.3 Antera 3D® Hemogloin level 44

4.4 MASI score 46

4.5 Patients’ self-assessment score 47

4.6 Local Side effect 49

4.7 Systemic side effect 50

4.8 Dermatologic Quality of Life Index 52

4.9 The studies of the injection of tranexamic acid for the 56

treatment of melasma

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LIST OF FIGURES

Figures Page

2.1 Schematic view of melanogenesis induced by UV radiation in 7

direct and in direct pathway

2.2 Chemical structure of tranexamic acid 11

2.3 Pathogenesis of melasma and role of tranexamic acid 12

2.4 Pathology of normal skin and damaged skin stained with 14

Fontana- Masson

2.5 Pronounced telaniectatic erythema noticed on the melasma 15

lesion

2.6 Immunohistochemistry for factor VIIa-related antigen 15

enlarge and elongated blood vessels in the upper dermis

versus perilesional normal skin

2.7 Computer assisted morphometric analysis of factor VIIIa- 16

related Antigen stained section in vessel size, vessel density

and the related area covered by blood vessels

3.1 Demonstrate the injection site of tranexamic acid 29

3.2 Fitzpatrick skin type and characteristics 30

3.3 Interdermal injection technique 31

3.4 Tranexamic acid 32

3.5 Study methodology 33

3.6 Skin analysis camera (Antera3D®) 35

3.7 Pictures from the skin analysis camera 35

3.8 Modified MASI score and Calculation of mMASI score 37

3.9 Visual analogue scale 38

4.1 Previous treatment 40

4.2 Aggravating factor 40

4.3 Antera 3D® Melanin level 41

4.4 Antera 3D® hemoglobin level 43

4.5 mMASI score 45

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4.6 Patients’ self-assessment score 48

4.7 Local side effect 49

4.8 Dermatologic Quality of Life Index 51

5.1 Photograph and melanin level of subject 1 75

showing intradermal microinjection of 4 mg/ml TA

5.2 Photograph and melanin level of subject 1 75

showing intradermal microinjection of 50 mg/ml TA

5.3 Photograph and melanin level of subject 2 76

showing intradermal microinjection of 4 mg/ml TA

5.4 Photograph and melanin level of subject 2 76

showing intradermal microinjection of 50 mg/ml TA

5.5 Photograph and melanin level of subject 3 77

showing intradermal microinjection of 4 mg/ml TA

5.6 Photograph and melanin level of subject 3 77

showing intradermal microinjection of 50 mg/ml TA

5.7 Photograph and melanin level of subject 4 78

showing intradermal microinjection of 50 mg/ml TA

5.8 Photograph and melanin level of subject 4 78

showing intradermal microinjection of 4 mg/ml TA

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LIST OF ABBREVIATIONS

Symbols/Abbreviations Terms

α Alpha

β Beta

AA Arachidonic acid

AE Adverse effect

bFGF Basic fibroblast growth factor

CI Confidence interval

DIC Disseminated intravascular coagulation

DNA Deoxyribonucleic acid

EI Erythema index

ET-1 Endothelin-1

FbDP Fibrin degradation product

FGF

FA

fibroblast growth factor

Fluocinolone Acetonide

F/U Follow up

GA Glycolic acid

Hb level Hemoglobin level

HQ Hydroquinone

iNOS Nitric oxide synthase

IPL Intense pulse light

MSH Melanocyte-stimulating hormone

LK Leukotriene

Mg Milligram

MI Melanin index

Ml milliliter

ML Melanin level

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Symbols/Abbreviations

mMASI

NGF

Terms

Modified Melasma Area and Severity

Index

Nerve growth factor

OCP Oral contraceptive pill

PA Plasminogen activatior

PAI-1 Plasminogen activator inhibitor-1

PASI Psoriasis Area and Severity Index

PDL Pulsed dye laser

PGA Physician global assessment

PGE2 Prostaglandin E2

POMC Proopiomelanocortin

PIH Postinflammatory hyperpigmentation

PtGA Patient global assessment

QS Nd: YAG 1064 nm Q switched neodymium: Yttrium garnet-

1064 nm

Er: YAG 2940 nm Erbium: Yttrium aluminium gernet 2940

nm

RNA Ribonucleic acid

SCF Stem cell factor

Sc-uPA Sigle-chain Urokinase-type plasminogen

activator

SD Standard deviations

TA Tranexamic acid

TAT Thrombin-antithrombin III complexs

level

TCA Trichloroacetic acid

TRP-1 Tyrosinase-relaed protein 1

TRP-2 Tyrosinase-related protein 2

uPA Urokinase-type plasminogen activator

U Unit

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UV Ultraviolet

VAS Visual analog scale

VEGF Vascular endothelial growth factor

WHO World Health Organization

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CHAPTER 1

INTRODUCTION

1.1 Background and rationale

Melasma is a common cosmetic problem with facial hyperpigmentation

among Asians. It is a chronic acquired pigmentary disorder. The characteristics are

symmetrically distributed light to dark brown macules and patches in the sun

exposure areas, mostly on the face (e.g. forehead, cheeks, temple, upper-lips and

nose)(1). Three different types of melasma exist depending on the location of pigment

deposits: epidermal, dermal and mixed type(2). The exact prevalence of melasma is

still unknown. Women are more susceptible than men, particularly those at

reproductive ages and mainly affects darker skin types (Fitzpatrick skin type IV-

VI)(3). Although there is no definite pathogenesis, multiple studies have found that

common predisposing factors include genetic predisposition, ultraviolet radiation,

pregnancy, oral contraceptives, thyroid disease, cosmetic, nutrition and drugs(4).

Many studies have found that the affected skin will result in an increase in vascularity

and angiogenic factors in the epidermis. These may play a role in pathogenesis (5)(6).

UV radiation is the most aggravating factor for melasma (7).

There are many treatment modalities for melasma including whitening

agents such as hydroquinione, retinoic acid, azelaic acid, kojic acid, Chemical

peeling. Laser and light therapy are now effectively used in the treatment of

melasma. It is helpful in refractory cases. However, many researchers reported

adverse effects such as mottled hypopigmentation, irritation, acneiform eruption and

rebound hyperpigmentation(8).

Although many treatment modalities have been tried, no treatment has

been proven to be consistently satisfactory. Treating melasma remains a challenge for

dermatologists, as no gold standard exists and recurrence is common.

There are studies which revealed that tranexamic acid, a plasmin inhibitor,

is commonly used as a hemostatic agent due to its anti-fibrinolysis action. Tranexamic

acid also has recently obtained in popularity in melasma treatment. It can reduce

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hyperpigmentation effect. Interestingly, there are many studies about topical and oral

tranexamic acid in melasma treatment but the outcome of the topical treatment is still

not satisfactory while the oral tranexamic acid is quite dangerous due to the systemic

side effect of the drugs when given in high dosage. The most noteworthy side effects

include Thromboembolism, myocardial infarction and pulmonary embolism. While

the thrombotic risk may be considered low, there are factors such as old age, high

dosage, prolonged usage of TA and concomitant doses of other prothrombotic drugs

which can still lead to an increased risk (9).

There was a clinical trial study using localized intradermal microinjection

of tranexamic acid (TA). The result of this study showed a significant decrease in

pigmentation. The intralesional microinjection shows little systemic absorption and no

systemic side effects but the drawback of this study is the frequency of the treatment

(once/week)(7). In this study, we would like to evaluate the efficacy and side effects

of intradermal microinjection of 50 mg/ml TA versus 4 mg/ml, the same

concentration of TA of previous study.

This study would help the patients to save the cost and time for going to

visits to the dermatologists to do the treatment while lower frequency the intradermal

microinjections made patients feel more comfortable with the same or more effective

outcome of the treatment comparing to the previous study(10)

1.2 Research question

Our research question is whether the 50 mg/ml TA intradermal

microinjection could be more effective and superior to 4 mg/ml TA in term of

reducing melanin level and modified Melasma Area and Severity Index (mMASI

score).

1.3 Specific Objective

Primary objective

- To evaluate the efficacy of tranexamic acid (TA) intradermal

microinjection between 50 mg/ml and 4 mg/ml to reduce the melanin level of

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melasma by using skin analysis camera (Antera3D®)

Secondary objectives

- To demonstrate the efficacy of 50 mg/ml versus 4 mg/ml

tranexamic acid intradermal microinjection in treatment of melasma which is

measured by modified MASI score and hemoglobin

- To evaluate the local side effects of intradermal microinjection of

tranexamic acid in both concentrations.

- To evaluate the systemic side effects of intradermal microinjection

of tranexamic acid

- To analyzed Patients’ self-improvement score.

1.4 Hypothesis

Fifty mg/ml TA could be more effective and superior than 4 mg/ml in

melasma treatment including decrease melanin level, mMASI score. Moreover 50

mg/ml TA could achieve the better result within shorter period of time comparing to 4

mg/ml TA

1.5 Keywords

Melasma, Melasma pathogenesis, Melasma treatment, tranexamic acid

(TA), intradermal microinjection

1.6 Ethic consideration

- The study protocol was approved by Thammasat University’s Ethical

review Committee

- The objectives, methods and expected benefits of this study together

with general information about Melasma were briefed to all patients. The possible

adverse events were also clearly explained to all patients.

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- Patients were also informed that they can withdraw from the study

without any bias from the physicians to continue the standard treatment.

- The patient's data would be strictly confidential.

1.7 Limitation

The limitations of this study are the sunlight and patients’ activities,

patient compliance and sunscreen applying habit.

1.8 Expected benefits and application

Melasma is a common hyperpigmention disorder. It causes suffering to

patients as it can lead to deep psychological and social stress. The treatment is very

challenging. There are numerous melasma treatment modalities such as topical agents,

chemical peeling, laser and light therapy. However, the results still vary and have not

been able to reach satisfactory outcomes. Thus, this study was expected that 50 mg/ml

TA intradermal microinjection could be a potentially effective treatment modality for

melasma.

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Table 1.1 Administration and time schedule

Schedule

of Events

2017 2018

JAN-

MAY

JUN JUL AUG-DEC JAN-

MARCH

APRIL-

MAY

JUN-

JUL

Research

proposal

Research

ethics

Data

collection

Data analysis

Manuscript

Preparation

&Presentation

Thesis

paperwork

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CHAPTER 2

REVIEW OF LITERATURE

Melasma is a common localized, chronic acquired hyperpigmented skin

disorder. It appears on sun-exposed areas such as the face, neck and occasionally

forearms with symmetrical light to dark brown macule to patches (9). For

epidemiology, it is commonly found in darker skin women (Fitzpatrick skin type IV-

VI). The reported prevalence of melasma varies from 8.8% in Latino women in the

southern United States to 40% of women in Southeast Asia (5). Even though its

pathogenesis remains a mystery, the cell in human skin which play an important role

in regulating pigmentation and hemostasis of the epidermis is epidermal melanin unit

which compose of melanocytes and keratinocytes. Some studies confirmed that

increased numbers and activity of melanocyte as well as increased in melanin

production (melanogenesis) play a role in the hyperpigmentation in melasma. As for

the number and quality of melanins, they depend on number of melanocytes, degree

of melanogenesis, dendriticity and surrounding factor such as surrounding tissue

environment, blood supply and UV radiation, etc. Genes that encoded tyrosinase is a

key enzyme in melanin synthesis. Tyrosinase-related protein 1 (TRP-1) and

tyrosinase- related protein 2 (TRP-2) are the protein that stabilize and stimulate

melanocyte activity. Therefore, these enzymes relate in melanin production too

(11)(12). In addition, there are many studies showed that various vascular growth

factors, keratinocyte-derived melanogenic factors such as basic fibroblast growth

factor (bFGF), nerve growth factor (NGF), engothelin-1 (ET-1), the

proopiomelanocortin (POMC)- derived peptides such as melanocyte-stimulating

hormone (MSH) and adrenocorticotrophic hormone (ACTH) have been reported in

UV-induced melanogenesis via keratinocytes melanocytes interaction (13). Other

factor such as genetic factors, inducible nitric oxide synthase (iNOS) and Wnt

pathway modulator genes could also play a role in melasma pathogenesis (14).

Several etiological factors implicated in the pathogenesis including genetic

susceptibility which is reported in many studies to show high incidence in family

members. A study in Singapore reported that 10.2% of study subjects with melasma

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have a family history of melasma (15) while a survey study in Iran shows 54.7% of

pregnant women with melasma have a family history of melasma (16). Ultraviolet

(UV) light exposure induced melanocytosis (the presence of an excessive number of

melanocytes) and melanogenesis (the production of melanin) through directly and

indirectly pathway. For the directly pathway, UV light has directly effect on

melanocyte. This effect caused TYR, TYR-1 and TYR-2 up regulate in melanocytes

which lead to increase melanin production. Another pathway, indirectly pathway, the

UV irradiation has indirectly effects on keratinocytes releasing melanogenic factors

such as nitric oxide (NO), nerve growth factor, basic fibroblast growth factor (bFGF),

MSH, ACTH, ET-1stem cell factor (SCF), all of these are keratinocyte-derived

paracrine factors which have a role in melanogenic effect through keratinocyte

melanocytes interaction (13) (figure 2.1).

Figure 2.1 Schematic view of melanogenesis induced by UV radiation in direct and

indect pathway (13)

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While the relationship between hormones and melasma is still unknown

(5), numerous studies accepted that the onset of melasma begins with using oral

contraceptive pills. Actually oral contraceptive pills and pregnancy have been shown

to increase serum plasminogen activator (PA) which is produced by keratinocyte. The

keratinocyte-PA system can induce the melanogenesis which can explain the

pathogenesis of melasma. In the Resnick study, 29% of the 212 female patients in

obstetrics and gynecology clinics suffered from melasma due to the use of oral

contraceptive pills and 87% of this group of patients have melasma related

pregnancies (15). Another recent study by the Ortonne, et al., (6) global survey which

studied 324 women with melasma from dermatology clinics in many countries

revealed that 25% of this group had the onset of melasma triggered by oral

contraceptive use. The rates were higher in patients who did not have a family history

of melasma. Even if the definition of hormones associated with melasma is still

uncertain, the suggestion for this group of patients is to cease taking oral

contraceptive pills and avoid using such drugs in the future if possible (6). Other less

common risk factors included thyroids disease, cosmetic usage, and phototoxic drugs

(e.g., antiseizure medication) (1). Histopathology of melasma shows that melanin and

epidermal melanocyte were significantly increased in the epidermis as well as an

increase in the number of melanosomes.

There are three types of melasma consisting of epidermal, dermal and

mixed-type.

Table 2.1 Classification of melasma based on the depth of melanin

Type Clinical feature Histopathology Wood’s light

examination

Response

Epidermal Well-defined

with light

brownish color

Increase Melanin deposit in

basal and suprabasal of

epidermis

Enhancement of

color contrast

Good

Dermal Ill-defined

margin, blue-

gray/ashen color

Increase melanophage seen in

superficial and mid-dermis

No enhancement Poor

Mixed Dark brown Melanin deposition is found in

epidermis and dermis

Contrast Enhance

show in some area

Partial

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Table 2.1 shows the traditional classifications based on the depth of

melanin pigment in the histopathology. The wood’s lamp examination can distinguish

the type of melasma by characterizing the extent of involvement of melanin.

Nonetheless, this examination may not be exactly in determining how deep the

melanin deposition is in the skin. The association between the classification based on

wood’s light examination and histopathology is low (14).

Nowadays, there are several treatments for melasma including topical and

systemic treatment, chemical peeling, laser and light therapy. All of the treatment

modalities need to include the use a broad spectrum sunscreen and patients are to

avoid sun light.

Topical treatments such as whitening agents which contain hydroquinone

(HQ), a hydroxyphenolic chemical, inhibits the tyrosinase enzyme that prevents

DOPA from converting to melanin. Deoxyribonucleic acid (DNA) and Ribonucleic

acid (RNA) which synthesizes melanocyte, degrades the melanosomes and also

destructs the melanocytes also inhibited by HQ (17),(18). Hydroquinone is considered

as the gold standard in the treatment of melasma. Commercially available

hydroquinone concentrations vary from 2-4%. It is found to be more effective when

combined with other agents. These combinations include the Kligman’s modified

Kligman’s formula, Pathak’s formula and Westerhof’s formula(14). However there

are many side effects following the use of hydroquinone and combination topical

therapy as previously mentioned such as erythema, dryness, and desquamation,

burning, pruritus, allergic contact dermatitis, nail discoloration, postinflammatory

hypo and hyperpigmentation (PIH) (19),(20). Torok et al (21) conducted the study to

evaluate the efficacy of the combination therapy (HQ 4%, tretinoin 0.05%, FA 0.01%)

to facial melasma. The cream was applied daily at night. For the clinical assessment,

the Follow up held in every month until they achieved a satisfactory result. Fifty

seven percentages of the participants experienced related adverse effect (AE) for

example, erythema and desquamation on the site where the cream was applied. The

greater number of the course of the treatments the higher the incidence of application-

site AEs.

Chemical peeling works by eradicating melanin rather than inhibiting

melanocyte. Several agents such as glycolic acid (GA), salicylic acid, trichloroacetic

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acid (TCA), tretinoin, and resorcinol have been studied but GA peels remains the

most popular. Many studies have characterized them as easy to use, safe, low

occurrence of downtime and some post-peel hyperpigmentation. However,

dermatologists should be careful when prescribing these peeling agents in patients of

darker skin types to avoid incidences of PIH and worsening of melasma itself (22).

Several observations have been made in the study of laser therapy for the

treatment of pigmentary disorders: melanin possesses a wide absorption spectrum

which allows for a large range of laser and light sources; melanosomes have a short

thermal relaxation time; and wavelengths which are longer are able to penetrate

deeper to pinpoint dermal pigment even though the absorption of melanin is optimal

with shorter wavelengths (23).

The laser types currently used in the treatment of melasma are Q switched

neodymium: Yttrium aluminium garnet- 1064 nm (QS Nd: YAG 1064 nm), Erbium:

Yttrium aluminium gernet 2940 nm (Er: YAG 2940 nm), Q switched ruby ( 694 nm),

Intense pulsed light (IPL), Pulsed dye laser (PDL) 585 nm, Fractional laser 1550 nm

(14). The most popularly used laser is QS Nd: YAG with laser toning technique.

However, the laser treatment itself can damage surrounding tissues with an

inflammation process that can lead to post inflammatory hyperpigmentation (PIH).

This may be long lasting and even delayed at the onset (24).

Tranexamic acid (Trans-4-Aminomethylcyclohexane-carboxylic acid, TA)

(Figure 1.1) is a plasmin inhibitor. It is a synthetic derivative of the amino acid lysine

and works by reversibly blocking lysine binding sites on plasminogen molecules.

Thus TA inhibits the plasminogen activator (PA) from converting plasminogen to

plasmin (25),(26). TA is used to prevent abnormal fibrinolysis to prevent abnormal

blood loss (22),(24)-(28). It has been widely used clinically for over 30 years to

prevent and provide therapy for hemorrhage and also primary and secondary

hyperfibrinolysis (29).

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Figure 2.2 Chemical structure of tranexamic acid(26)

TA has been used for the treatment of menorrhagia which has been

approved since the 1970s. The dosages for menorrhagia are 2.0-4.5 g/day during the

cycle (32). However no serious side effects were observed even when the dosages

were increased to 4-4.5 g/day. TA is also successful in treating prophylaxis of

hereditary angioedema for durations of 8 to 34 months without any serious side

effects or abnormal fibrinolytic activity (33). As an antifibrinolytic drug, TA can be

administered in both oral and intravenous form. The concentration of TA which has

antifibrinolytic effect is 5-10 µg/ml. The maximum concentration of oral TA is 13.83-

16.41 µg/ml (TA 1,300-3900 mg). The maximum concentration of TA in IV form is

81-86 mg after injection of TA 1 gram via intravenous route. Its oral bioavailability

ranges from 30-50 % with plasma protein binding of 3%. It can completely cross the

placenta. Its clearance via renal amount 95% and 90% of the drug is eliminated

mostly in 1 day and the half-life in adults is approximately 2.3 hours (29). Varying

dosages are recommended for various bleeding conditions. For systemic fibrinolysis,

10 mg/kg of TA is administered intravenously followed by 30-50 mg/kg of TA in oral

form every 3-4 hours will give adequate inhibitory plasma concentration. For local

fibrinolytic bleeding, 10-20 mg/kg of TA in oral form is to be taken three to four

times a day. In the case of menorrhagia, the recommended oral dosage is 1-1.5 g

every 6-8 hours a day for 3-4 days with the total oral dosage dose not exceeding 4

grams/day (32),(34). The data for the pharmacokinetics of topical TA applied on the

epidermis and TA in the tissue has not been reported yet.

It was first reported in 1979 by Nijo Sadako that TA has a role in the

treatment of melasma. The significant improvement of melasma severity was

accidentally found on the patient 2-3 weeks after using TA for treatment of chronic

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urticarial (26). After that he did the first study of TA in melasma treatment. The study

reported that among 12 patients with 30-69 years, most of them had a good clinical

response in melasma after taking 1.5 g TA plus with vitamin supplement ( B, C and

E) once daily for 20 weeks. However, the mechanism of TA affecting the severity of

melasma remains unknown (26).

However the exact pathogenesis of melasma is unknown. Many studies

reported that UV light exposure plays a major role in melasma pathogenesis. After

UV irradiation, the plasminogen activator synthesis resulted in an increase in

keratinocyte as well as plasmin activity (35)(36). Plasmin can also induce arachidonic

acid (AA) production by activated precursors of secretory phospholipid A2. Free AA

stimulates melanogenesis via its metabolite, Prostaglandin E2 (PGE2), and

leukotrienes (LK) (37),(38). In addition, the plasmin can induce AA to be released in

endothelial cells also. Increased plasmin elevates the α-MSH and bFGF which

induces melanocyte activity to synthesize melanin (9),(39). All of these are the

melanin production process in the skin (Figure 2.3).

Figure 2.3 Pathogenesis of melasma and role of tranexamic acid(9)

Other than the effect on melanocytes, Plasmin activation participates in

one of the processes of angiogenesis. It changes extracellular matrix-bound vascular

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endothelial growth factor (VEGF) into freely diffusible forms. TA, a plasmin

inhibitor, can suppress angiogenesis and the bFGF inducing neovascularization

(Figure 2.3).

Human keratinocyte contains both urokinase-type PA (uPA) and tissue-

type PA (tPA) but secretes only single-chain uPA (Sc-uPA). The Sc-uPA which

deposits in keratinocyte, results in upregulation of melanocytes, tyrosinase activity,

cell parameter and increased dendrite in a dose dependent manner. Moreover, Sc-uPA

can induce the growing of keratinocyte, differentiation, and migration. Hence, in-vitro

studies show that Sc-uPA-generated keratinocyte increases the activity of

melanocytes (40). The amount of Sc-uPA can be increased by plasmin activity.

Repeating UV exposure increases the number of mast cells and mast cell

tryptase. Tryptase deteriorates type IV collagen. This increases the number of mast

cells and typtase both of which might be a result of basement membrane weakness

which is observed in melasma. Mast cells also induced the solar elastosis

development in the skin, which is one of the characteristic features of melasma. In the

UV exposed skin, the elastin contents are related with the mast cell.(9)

Another important consideration in the pathogenesis of melasma is that

the use of contraceptive pills and pregnancy can induce serum PA increasingly, which

as mentioned earlier can stimulate the melanin production.

In addition to melasma histology, there is increased epidermal

pigmentation as a result of more active epidermal melanocytes in melasma when

compared with normal skin (Figure 2.4). From the electron microscopy, the enlarged

melanocytes with marked dendrite were observed. Furthermore, the indicators of

increased melanocytic activity are more expressed such as mitochondria, Golgi

apparatus, rough endoplasmic reticulum and ribosomes which result in increased

eumelanin synthesis. Besides this, various changes are also observed: the basement

membrane of the melasma is thinned and disrupted, prominent solar elastosis is

observed. The raising in the intensity of blood vessels and vascular endothelial growth

factor (VEGF) is noted also.

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Figure 2.4 Normal skin (A) and damage skin (B) stained with Fontana-Masson

(40x)(41)

As explained previously, TA is a lysine analogue. It prevents the binding

of plasminogen to the lysine binding on the keratinocyte so it can be implied that TA

inhibits UV-induced plasmin activity in keratinocytes. This results in less free AA

and reduced the ability to produce PGs, and consequently reduced melanogenesis in

melanocytes (26),(42). TA has no effect on non-sun exposed healthy skin (42). TA

can suppress angiogenesis by inhibiting the process of plasmin converting to VEGF

which could play a contributory role in its action on melasma. Plus, inhibition of the

Sc-uPA pathway may be another mechanism through which TA diminishes

hyperpigmentation. The structure of TA looks like the tyrosinase enzyme molecule

structure so TA can completely degrade the activity of tyrosinase enzyme.

As a result of physical examination, patients who suffered from melasma

notice the exhibited telangiectatic erythema within the melasma lesion (Figure 2.5). In

2006, kim, et al.(43), reported about the vascular characteristic of melasma and the

expression of VEGF, a major angiogenic factor of the skin. The study noticed that an

increase of vascularity in melasma showed not only in histopathology but also in the

dermoscopy and reflectance confocal microscopy. The results noted that the erythema

intensity was higher in melasma lesion because of both number and size of dermal

blood vessel were increase significantly by measuring with computer-asissted image

analysed of factor VIIIa-related antigen-stained sections (figure 2.6, 2.7). Thus, there

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was a relationship between the intensity of vessels and pigmentation in melasma.

Concerning for the VEGF, there was also increase significantly in melasma. As a

result of the increasing VEGF, this study suggested that VEGF could be an important

angiogenic factor for transformed vessels in melasma.

Figure 2.5 Pronounced telaniectatic erythema noticed on the malasma lesion (43)

Figure 2.6 Immunohistochemistry for factor VIIIa-related antigen revealed enlarged

and enlongated blood vessels in the upper dermis ( B), as compare with perilesional

normal skin (A) (43)

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Figure 2.7 Computer assisted morphometric analysis of factor VIIIa-related antigen

stained sections revealed a significant increase in vessel size (C), vessel density (D),

and the relative area covered by blood vessels (E ), as compared with perilesional

normal skin.(43)

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After many forms of TA, including oral, topical application, localized

intradermal microinjection and localized microneedling have been treated in melasma.

TA can be combined with other modalities such as intense pulse light or Nd-YAG

laser.

As mentioned previously, Nijo's study in 1797 in Japan made public the

positive effect of TA on melasma (26). Following that the effectiveness of TA for

melasma has been improved in numerous studies and in vivo studies. Maeda and

Tomitab (44) suggest that TA works by interfering in the process between

melanocytes and keratinocytes at the plasminogen/plasmin system. As a result of this

function of TA, it can prevent melanin production.

In 2007, Seong, et al.(12), used neonatal fore skin cultured melanocytes

to show the effects of TA on morphogenesis and melanogenesis and activity of

melanocytes after 20 mJ/cm2

UVB irradiation. There was a significant lower in

melanin synthesis, melanocytes activities and melanocyte viability after treated with

TA in a dose dependent manner. However, for the number and length of melanocyte

dendrites no any change was observed. In addition, not only decreased in melanin

production but also reduced expression of tyrosinase, TRP-1, TRP-2 mRNA which

play a role in melanogenesis in a dose dependent of TA manner

Topical TA in liposome formulation was developed in 2002. (45)

Kondou, et al. (53), demonstrated the efficacy of topical 2% TA emulsion in the

treatment of melasma. The 2% TA emulsion was applied for 5-18 weeks to 25

volunteers who had been diagnosed with melasma. The result showed improvement of

the pigmentation in 20 subjects (80%) without side effects. The improvement was

observed within 8 weeks. On the other hand, there is a split face study for topical 5%

TA versus vehicle for 12 weeks. The study was done in Thailand in 2012(46).

Twenty three melasma women were recruited in the study for 12 weeks. At the end of

the study, the result showed no significant difference between these two regimens.

Moreover, 5% TA caused more irritation to the applied area.

Zhu, et al. (26), in 2001, used 250 mg TA, 0.2 g vitamin C and 0.02 g

vitamin E orally three times a day to treat 128 melasma patients, compared to 30

control cases (only vitamin C and E in the same dosage) in one course of treatment

lasting 6-8 weeks. Authors found that increasing the treatment duration was more

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effective than increasing the dose of TA. Without changing the coagulation

parameters only a few cases reported side effects such as gastrointestinal upset.

Na et al.(47), in their study clarified the effects of oral TA form and

topical TA form on melasma lesions. The patients were prescribed Oral TA three

times daily along with topical TA which required application 2 times a day in the

morning and at night for 8 weeks. After finishing the study, the result showed

significant decrease in mean lesional melanin index (MI) scores. On the other hand,

for the perilesional skin the MI scores were increased. As for the erythema index

scores, no significant difference was observed between lesion and perilesion skin.

According to histological analysis, the intensity of Epidermal pigmentation, vessel

numbers and mast cell counts were markedly decreased. Thus, TA can decrease

epidermal pigmentation in melasma and also prevent melasma related dermal

changes, such as decreased vessel numbers and decreased numbers of mast cells. This

showed the efficacy of TA on melasma pathogenic mechanism.

In 2010, li, et al. (48), performed an experimental study of intradermal

TA on guinea pig skins, which had been exposed to UVB for 1 month. Intradermal

injection of TA was performed every day for another month. Finally they found out

that the melanin content at the basal layer of the epidermis was significantly lessened,

although melanocytes were not reduced. Thus, they suggested that TA had a benefit

on melanin expression but not the number of melanocytes.

Wu et al. (49), 74 Chinese women aged 21-52 years with symmetrical

facial melasma were recruited in the study. TA 250 mg tablet was prescribed orally 2

times/ day to the patients for 6 months. The melasma responded well with the TA but

surprisingly, for other pigmented lesions, such as freckles and senile lentigo they

remained unchanged. As for the result of this study, it might be due to the

differentiation of the pathogenic mechanisms. The improvement of the lesion was

seen at the end of 4 weeks whereas for some patients the improvement was noticed

only at the end of 8 weeks. Overall, 94.6 % of the patients observed noticeable fading

of melasma.

Cho et al. (50), they clarified the effectiveness of oral TA as an adjuvant

therapy in melasma treatment with laser and light-based devices in Fitzpatrick type III

and IV skin. Oral TA 500 mg/day was prescribed to the patients followed by

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receiving the IPL procedure simultaneously and three or four times of low fluence Q-

switched (Qs-Nd:YAG laser treatments). The same Oral TA dosage was found to

yield an improvement in clinical efficacy in light or laser based melasma treatment.

This was especially so during the period of relative high sun exposure. No serious

adverse effects were uncovered.

Bahrain’s study revealed that TA is as effective as the combined effect of

hydroquinone and dexamethasone in melasma treatment while being safer than the

standard treatment of melasma, hydroquinone (52).

In 2001, Angchaisuksiri, et al. (66), clarified the safety of using long-term

oral tranexamic acid in treatment of melasma. A prospective double-blind, placebo-

controlled study, they randomized patients in to 3 groups. Group A: placebo for 3

months then switched to 1,500 mg/ml of oral TA for 3 months. Group B: TA for 3

months then switched to placebo for 3 months and Group C: TA 1,500 mg/ml orally

in 6 months. The blood samples were collected from the melasma patients at month 0,

1, 4 and 6.They found that no any patients had clinical evidence of thromboembolism.

For Group A and B, plasminogen levels were significantly lower during the treatment

of TA period when compared with the placebo period. There are no change in

fibrinogen, fibrin degradation product (FbDP), plasmin inhibitor, plasminogen

activator inhibitor-1 (PAI-1), thrombin-antithrombin III complexes level (TAT). In

addition, complete blood count, thrombin time, Prothrombin time, Activated Partial

Thromboplastin time, Creatinine, Phosphokinase, liver and kidney function were in

normal range. They conclude that the systemic side effects such as systemic

fibrinolytic suppression and thrombogenic effect were not observed after taking the

long-term systemic treatment of TA in melasma. Therefore, Long-term oral TA

administration was safe. They also reported that anti-fibrinolyc mechanism of TA is

only happen in tissue.

For intradermal TA injection, there is a pilot study done by Lee et al. (7),

in Korea. To evaluate the effectiveness of intradermal microinjection of TA to the

melasma, they used 4 mg/ml TA intradermal injection weekly for 12 weeks. The

MASI score showed a significant decrease since week 8. Furthermore, at the end of

the study, the MASI score improved 42.74% from the baseline. Addition to the lee, et

al study, the results of the study by Steiner, et al was in the same direction. The study

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evaluated the efficacy and safety of topical TA versus intradermal microinjection of

TA for melasma treatment. Group A: the patients received 4 mg/ml TA intradermal

microinjection weekly for 8 weeks versus Group B: 3% TA topical therapy.The mean

dose of TA injection in patients was 1.5 mg, which is lower than the usual dose used

for anti-fibrinolytic effect, and the concentration of topical cream at 3% has minimal

systemic absorption. There was statistically significant lower in MASI score after

week 12 in both groups but no significant different between these 2 regimens was

observed.

There was a comparative study about the therapeutic efficacy and safety

of TA between microinjection and microneedling in melasma treatments. The study

was done in India by Budamakuntla, et al. (1), in 2011. It was a prospective,

randomized, open-labelled study with 60 melasma patients divided into 2 groups

(1:1). Patients were administrated with localized microinjections of TA in one group,

and the other group with microneedling TA. The treatment was done at monthly

intervals (0, 4, 8 weeks) and follow ups for three months. The result of the study

showed that the MASI scores, Physician global assessment (PGA), and Patient global

assessment (PtGA) were significantly decreased from the baseline to the fourth, eight,

and twelfth week of treatment in both treatment arms. Although no significant

difference was observed in two group but the improvement scores of microneedling

group was slightly better than TA microinjection group. No any serious side effects

were noted.

As mentioned above, the efficacy of microinjection as well as

microneedling of TA seem to be of the same effectiveness for melasma treatment. As

for side effects, no serious side effects were reported apart from mild local

discomfort, burning sensation erythema, and wheal at the site of injection which

mostly appears as transient side effects and this concurs with Lee et al study (7)

Recently, the study of Saki et al(54) in 2017, it was a spilt-face controlled

trial study to compare the efficacy and safety between 20mg/ml TA intradermal

microinjection monthly for 3 months and topical HQ once at night for 3 months. The

result showed that both modalities could reduce melanin value by measuring a

colormeter, Dermacatch® (p value < 0.001). Monthly TA intradermal injection could

achieve the better result in term of reducing melanin value during the first 4 weeks

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when compared with topical HQ. By the way, after 20 weeks no significant difference

was observed between these 2 groups. This study concluded that the onset of action of

TA intradermal injection may be shorter when compared to topical HQ. Also the

longer treatment duration could achieve greater improvement.

The localized intradermal microinjection of tranexamic acid is a new

treatment modality for melasma. In the future, it may be a promising therapeutic

method due to its safety, effectiveness and reduced recurrent rate. Moreover, it is an

office-based procedure with relatively quick results and no significant serious side

effects, and almost downtime.

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Table 2.2 Clinical trials of tranexamic acid in the treatment of melasma

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CHAPTER 3

RESEARCH METHODOLOGY

3.1. Study design

Prospective randomized controlled trial triple-blinded split face study

3.2 Study sample or study population

EC approval from Chulabhorn International College of Medicine,

Thammasat University, Hospital.

This experimental were conducted at Thailand Tobacco Monopoly

Hospital between July 2017 to March 2018.

3.2.1 Target population

Female patients with symmetrical epidermal to mixed-type melasma on the face and

aged 26-60 who achieve the inclusion criteria were included in the study

3.2.2 Sampling method

Table3.1 Inclusion and exclusion criteria

Inclusion criteria Exclusion criteria

- Female patients with

symmetrical epidermal-mixed

type melasma,

- Women who are pregnant or breast

feeding

- Age 26-60 - Women who were taking

contraceptive pills on hormone

therapy during the past 1 year

- Ability and willingness to

comply with the requirement

of the protocol

- History of bleeding disorders or the

concomitant use of anticoagulants

such as warfarin, heparin

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- History of disseminated intravascular

coagulation (DIC) without severe

hemorrhage

- Women who have contraindication

for tranexamic acid such as acute

venous or arterial thrombosis, severe

renal impairment, history of

convulsion, acquired defective color

vision, history of deep vein

thrombosis, hypersensitivity to TA

- Women who are on any concurrent

treatment for melasma and did not

wash out at least a 1 month period

before the start of the study.

- History of drug allergy especially to

the study drug

- Known history or clinically relevant

allergy to components of sunscreen

or topical anaesthetic used in this trial

- Active dermatologic malignancy,

active wart, moderate to severe acne

vulgaris on face

- History skin infection: herpes

infections, impetigo

- Patient who has history receive

chemotherapy, radiotherapy

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3.2.3 Discontinuation criteria

3.2.3.1 Patient’s refusal to participate in the study

3.2.3.2 Patient who has serious adverse effect of TA injection

3.2.3.3 Unreliable and poor compliance patient

3.2.3.4 Patient who are in others type of treatment of melasma

3.2.4 Sample size

A total of 35 females who are aged between 26-60 years old with

melasma on the face are recruited for this study Reference from lee et al, 2006 (7) the

decrease in the mean MASI score is 5.65±2.54

Formula

Reference value (µ0) = 5.65

Mean (µ) = 4.65

Standard deviation (σ) = 2.2

Margin (δ) = 0

Alpha (α) = 0.05

Beta (β) = 0.2

Sample size includes subjects who do not continue follow up sessions: 30 + 5 = 35

3.3 Research grouping

Thirty-five females with bilateral epidermal to mixed- type melasma

were enrolled in a split-faced prospective trial. Thirty-five patients were injected with

tranexamic acid once for 2 weeks, one side of the face was injected with 4 mg/ml of

tranexamic acid and another side was injected with 50 mg/ml (figure 3.1).

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Intradermal injection

4 mg/ml of TA

Figure 3.1Demonstrate the injection site of tranexamic acid

3.4 Materials and Methods

3.4.1 Informed consent process

- Patients were fully informed of melasma general knowledge,

objective, expected benefit outcome, and adverse event of this study.

- The study participants provided informed consent prior to initiation

of any study-related standard conditions.

3.4.2 History taking

The collected data include

- Patients’ personal history; name, age, gender, phone number, skin

type, occupation, smoking and drinking habits, underlying disease, current

medication, menstruation, history of taking oral contraceptive pill (OCP), history

allergy in drug and food.

- Patients’ melasma history; age of melasma onset, duration of

melasma, probably cause of the melasma, history of previous treatment and response,

family history of melasma.

- Patients’ information for exclusion criteria such as pregnant or

breast feeding, taking contraceptive pills or on hormone therapy during the past

1 year, History of bleeding disorders or the concomitant use of anticoagulants such as

warfarin, heparin, Women who have contraindication for tranexamic acid (table 3.1)

Intradermal

injection 50 mg/ml of

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Figure 3.2 Fitzpatrick skin type and characteristics

3.4.3 Physical examination and evaluation

- Subjects will be asked to remove all make up and clean their faces

with make-up remover and water before doing the physical examination

- Five pictures will be taken before the procedure which consists of

both sides of the face at 45, 90 degree and the front of the face with a DSLR digital

camera (Nikon® D7200). The patient’s photographs will be taken again at week 4, 8,

12 and 1-month follow up by using the same camera setting, lighting and patient’s

position.

- Melasma evaluation were evaluated including melanin level,

Hemoglobin level by using skin analysis camera ( Antera 3D®) and mMASI score

which done by two dermatologists at baseline, week 4, 8, 12 and 1-month follow up.

3.4.4 Intervention

- Preoperative, a topical anesthetic cream (EMLA®; 2.5% lidocaine

and 2.5% prilocaine) was applied on the face for 45 minutes under occlusion before

treatment. After that it was washed off to achieve a completely dry skin surface.

- All subjects were treated with tranexamic acid which was repeated

every two weeks for 7 times. Two concentrations of TA were prepared under sterile

condition a 30- gauge, 4 ml/mg and 1 ml and 50 mg/ml. Injection of about 0.05 ml

were applied intradermally on the melasma lesion at a 1 cm interval (0.05 ml/1cm2)

(7). The injection site should form a wheal else it is probably too deep. (Figure 3.3)

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- Randomized the concentration of TA (4 and 50 mg/ml) for split

face study done by the staff who do not participate in this study without any study

participants, the researcher and the dermatologists knowing

- After treatment in every session the patient will be asked about any

pain for their pain score rating. The immediate side effects will be observed 30

minutes after treatment.

- Patients were advised to apply board-spectrum sunscreen with SPF

50 to both side of face in the morning, also avoid excessive sun exposure, and avoid

the use of any topical whitening agents on the lesion during the study period.

Figure 3.3.Intradermal injection technique

3.4.5 Tranexamic acid preparation

- Supplies: 1 ml syringes, 3ml normal saline, 21 gauge and 30 gauge

needle, tranexamic acid 1 ampule (250mg/5ml), (Transamin®, Daiichi Sankyo CO.,

LTD, Tokyo, Japan). (Figure 3.5)

- For 4 mg/ml TA concentration: TA is drawn in a 100 U/ml insulin

syringe about 4 mg and diluted with normal saline up to 1 ml to get a concentration of

4 mg/ml of tranexamic acid(1)

- For 50 mg/ml tranexamic acid concentration: draw TA 1 ml in

a 1 ml syringe without dilution.

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3.4.6 Follow up

- The follow up would be done at 1 month interval; week 4, week 8,

week 12 and week 16

- Patients were asked to score patients self-assessment at the end of

the study (week 16)

Figure 3.4 Tranexamic acid (Transamin®, Daiichi Sankyo CO., LTD, Tokyo, Japan).

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Figure3.5 Study Methodology

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3.5 Outcome measurement

Table 3.3 Outcome measurement

Assessment

Treatment

F/U

Treatment

w

wk 0

w

wk 2

w

wk 4

w

wk 6

w

wk 8

w

wk10

w

wk 12

w

wk 16

Photography

[ ]

[ ]

[ ]

[ ]

[ ]

Antera 3D®

- Melanin

level

- Hemoglobin

level

[ ]

[ ]

[ ]

[ ]

[ ]

mMASI score

[ ]

[ ]

[ ]

[ ]

[ ]

Side effects

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

Pain score

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

[

[ ]

Quality of life

index

[

[ ]

[ [ [ [ [ [

[ ]

Patients’ self-

assessment

[

[ ]

[ [ [ [ [ [ [

[ ]

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3.5.1 Objective assessment

3.5.1.1 Skin analysis camera (Antera3D®)

Figure 3.6 Skin analysis camera (Antera 3D®)

Figure3.7 Pictures from the skin analysis camera

Antera 3D® (Miravex Limited, Ireland) (Figure 3.7) is a

skin analysis camera and for image acquisiton. The light reflected from the skin’s

surface was converted into a digital image which shows topography, hemoglobin, and

melanin by using the complex algorithms software. It also has the ability to measure

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skin quality via the use of light emitting diodes and polarizers which illuminates and

captures light reflected an area of 36 cm2 the face. A useful feature of the interface

allows comparisons of skin color, texture, volumes, wrinkles, redness and

pigmentation before and after treatment.

In this study, the melanin level and redness would be measured.

3.5.1.2 Side effects

Side effects such as erythema, swelling, wheal and flare,

irritation were objectively assessed at all visits. Patients were asked to report adverse

effects such as pain which were evaluated by a visual analog scale (VAS), erythema,

irritation, pigmentation change (hypo and hyperpigmentation), purpura. For systemic

side effects, patients were asked about gastrointestinal disturbances, for example,

nausea, vomiting and diarrhea. They were asked about any unusual menstruation

characteristics such as amenorrhea, spotting or intermenstrual bleeding.

3.5.2 Subjective assessment

3.5.2.1 Modified MASI score (mMASI score)

Modified Melasma Area and severity Index (mMASI) was

proposed by Kimbrough-Green et al. in 1994. It has been designed from the pattern of

Psoriasis Area and Severity Index (PASI). For PASI, it uses the whole body to

evaluate the lesions of psoriasis. On the other hand, mMASI is the most widely

applied for assessment of melasma severity at the baseline and any changes during the

treatment(55). The mMASI scoring is determined by three variables based on the

percentage of area involvement (A) and was calculated from 0 to 6. For the area

involvement, the area of the face is splitted into 4 parts: the forehead, right malar, left

malar and chin are 30%, 30%, 30%, and 10%, respectively (figure 3.9). The intensity

of darkness (D) of melasma, D was calculated from the scale 0 (absent) - 4

(maximum). The mMASI was calculated by the following formula

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Figure 3.8 Modified MASI score and Calculation of Modified MASI score

“D” is represent to the darkness of the lesion and “A” is area. The

values 0.3, 0.3, 0.3, and 0.1 are the percentage of the facial area. The maximum score

is 24, with 0 being the minimum(7)

3.5.2.2 Patient’s self-assessment of the improvement of melasma

The improvement score was graded in to five levels

0= no change

1= slightly improvement (0-25%)

2= fair improvement (26-50%)

3= good improvement (51-75%)

4= excellent improvement (>75%)

3.5.2.3 Dermatological quality of life index (DLQI)

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3.5.2.4 Pain score

Pain score were evaluated as Figure 3.10

Figure3.9 Visual analogue scale

0= no pain

1-3= mild pain (annoying, mild interfering little with

activities of daily living)

4-6= moderate pain (interferes significantly with activities of

daily living)

7-10= severe pain (disabling; unable to perform activities of

daily living)

3.6 Data analysis

- All measured values will be expressed in the form of means and standard-

deviations (SD)

- A repeated measure ANOVA will be used to compared the change in

modified MASI between the baseline and final follow-up visits

- All statistical analyses will be performed out using SPSS

- Statistically significant in all case will be considered at the p-value <0.05

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CHAPTER 4

RESULTS AND DISCUSSION

The aim of this study is to evaluate the efficacy of tranexamic acid

intradermal microinjection between 4 mg/ml and 50 mg/ml for treatment of melasma.

The result of the study will be divided into 4 parts:

4.1 Patient demographic data

4.2 Objective assessments

4.3 Subjective assessment

4.4 Patients’ self-assessment and side effect

4.1 Patients demographic data

34 from 35 patients completed the study. One patient dropped out due to

inability to follow up. The data analysis were obtained from these patients.

Table 4.1 Patient demographic data (n=34)

Variables n %

1. Age

- Mean ± SD

- Min-Max

50.71 ± 6.52

32-60

2. Occupation

- Housewife

- Maid

- Office

- Ownbusiness

- Hospital staff

2

2

24

5

1

5.9%

5.9%

70.6%

14.7%

2.9%

3. Smoke 0 0%

4. Alcohol consumption 2 5.9%

5. Family history of melasma 19 55.9%

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Variables n %

6. Type of melasma

- Epidermal-type

- Mixed-type

4

30

11.8%

88.2%

7. Duration of melasma

- <5Y

- 5-10 Y

- >10 Y

12

9

13

35.3%

26.5%

38.2%

8. Skin type

- III

- IV

- V

1

23

10

2.9%

67.6%

29.4%

All patients belonged to the age group ranging from 30-60 years. The

average age of the patients was 50.71 year old. Most of them are officer ( n=24,

70.6%). They had Fitpatrick skin type III-V. 4 (11.8%) patients had epidermal type

and 30 (88.2%) had mixed-type of melasma. As for the duration of melasma, 12

patients (35.3%) were under 5 years. Nine patients (26.5%) were 5-10 years and

Figure4.1 Previous treatment Figure4.2 Aggravating factor

Aggravating factor

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thirteen (38.2%) were greater than 10 years. Nineteen (55.9%) patients had a history

of melasma in family. Sun exposure was a highest risk factor for these patients in 22

patients (64%). The percentage of the number of patients with other risk factor related

to occurrence of melasma such as pregnancy, use of contraceptive pill and cosmetic

was 15%, 9%, and 12% respectively. Regarding the prior treatment, 53% of the

patients had history of prior treatment. Majority of them used topical cream (29%)

followed by laser (18%) and others (6%) such as chemical peeling and PRP (Platelet-

Rich Plasma).

4.2 Objective assessment

4.2.1 Skin analysis camera (Antera 3D®)

4.2.1.1 Antera® melanin level

Figure 4.3 Melanin level results

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Table 4.2 Melanin level results

TA 50 mg/ml P-value1 TA 4 mg/ml P-value2

Mean

difference

(95%CI.)

P-value3

Before 0.695

(0.672, 0.718)

0.665

(0.641, 0.689)

-0.03

(0.009, 0.05)

.075

Week4 0.664

(0.643, 0.685)

0.657

(0.635, 0.679)

-0.006

(-0.009, 0.022)

0.669

Week 8 0.637

(0.616, 0.657)

0.65

(0.629, 0.671)

-0.014

(-0.029, 0.002)

0.346

Week 12 0.608

(0.59, 0.627)

0.639

(0.617, 0.66)

-0.031

(-0.045, -0.016)

.032*

Week 16 0.605

(0.586, 0.623)

0.631

(0.609, 0.653)

-0.026

(-0.043, -0.009)

.072

Difference

at week 4

-0.031

(-0.044, -0.019)

0.001* -0.008

(-0.017, 0.001)

0.085 -0.023

(-0.039, -0.008)

.003*

Difference

at week 8

-0.058

(-0.073, -0.044)

0.001* -0.015

(-0.025,-0.005)

0.005* -0.043

(-0.061, -0.026)

0.001*

Difference

at week 12

-0.087

(-0.101, -0.072)

0.001* -0.026

(-0.037, -0.016)

0.001* -0.06

(-0.078, -0.043)

0.001*

Difference

At week 16

-0.09

(-0.106, -0.075)

0.001* -0.034

(-0.046, -0.022)

0.001* -0.056

(-0.075, -0.037)

0.001*

Values presented as mean (95%CI.). P-value corresponds to Repeated ANOVA test.

P-value1: calculated probability the decrease of melanin level from before to week 4, 8, 12 and 16, respectively for the 50 mg/ml

side.

P-value2: calculated probability the decrease of melanin level from before to week 4, 8, 12 and 16, respectively for the 4 mg/ml

side.

P-value3: calculated probability the difference of melanin level between 4 and 50 mg/ml TA in each visit. Statistically

significant as P<0.05

Regarding the side treated with 50 mg/ml of TA, the melanin level at

baseline was 0.695 (95% CI: 0.672 - 0.718) and reduced to 0.664 (95% CI: 0.643-

0.685), 0.637 (95% CI: 0.616 -0.657), 0.608 (95% CI: 0.059, 0.627) and 0.605 (95%

CI: 0.586-0.623) at week 4, week 8, week 12, and week 16 respectively. As For

another side treated with TA 4 mg/ml, the melanin level started at o.665 and decrease

gradually to 0.657 (95% CI: 0.635 - 0.679), 0.650 (95% CI: 0.629 - 0.671), 0.639

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-0.05

-0.07*

-0.10* -0.09*

0

-0.04

-0.07* -0.10*

-0.06*

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

Baseline week 4 week 8 week 12 week 16

TA 50mg/ml TA 4 mg/ml

(95% CI: 0.617 - 0.66), 0.631 (95% CI: 0.609 -0.653) at week 4, week 8, week 12and

week 16 respectively.(Table 4.2)

The significant difference between these 2 doses of TA was observed at

week 12. However, the melanin level in both side of the face continuously decreased

till week 16th

even if there was not showed significant different between both

regimens at the end of the study (week 16).

The side treated with TA 50 mg/ml, the result showed significant

decrease in melanin level since week 4 (p-value <0.05), on the other hand, the

significant decrease was observed in week 8 for the side treated with 4 mg/ml TA (p-

value <0.05)

4.1.1.2 Antera® hemoglobin level

Figure 4.4 Antera® hemoglobin level

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Table 4.3 Antera® hemoglobin level (n=34)

TA 50 mg/ml P-value1 TA 4 mg/ml P-value2

Mean

difference

(95%CI.)

P-value3

Before 1.73

(1.67, 1.8)

1.72

(1.65, 1.8)

0.01

(-0.06, 0.08)

.723

Week4 1.69

(1.6, 1.78)

1.69

(1.61, 1.76)

0

(-0.07, 0.07)

.959

Week 8 1.66

(1.6, 1.73)

1.65

(1.58, 1.71)

0.02

(-0.05, 0.09)

.637

Week 12 1.63

(1.57, 1.7)

1.62

(1.56, 1.69)

0.01

(-0.06, 0.08)

.724

Week 16 1.65

(1.58, 1.71)

1.66

(1.59, 1.73)

-0.02

(-0.09, 0.06)

.652

Difference

at week 4

-0.05

(-0.11, 0.02) .135

-0.04

(-0.07, 0) .061

-0.01

(-0.08, 0.06)

.786

Difference

at week 8

-0.07

(-0.13, -0.02)

0

.010*

-0.07

(-0.12, -0.03) 0.003*

0

(-0.06, 0.07)

.887

Difference

at week 12

-0.1

(-0.16, -0.04)

0

.001*

-0.1

(-0.14, -0.05) <0.001*

0

(-0.07, 0.07)

.998

Difference

at week 16

-0.09

(-0.15, -0.03)

0

.007*

-0.06

(-0.1, -0.02) 0.009*

-0.03

(-0.08, 0.03)

.312

Values presented as mean (95%CI.). P-value corresponds to Repeated ANOVA test.

P-value1: calculated probability the decrease of hemoglobin level from before to week 4, 8, 12 and 16, respectively for the 50

mg/ml side.

P-value2: calculated probability the decrease of hemoglobin level from before to week 4, 8, 12 and 16, respectively for the 4

mg/ml side.

P-value3: calculated probability the difference of hemoglobin level between 4 and 50 mg/ml TA in each visit. Statistically

significant as P<0.05

The hemoglobin level (Hb level) of both side of the face showed there was

no significant difference. The side treated with 50 mg/ml TA, the reduction of the Hb

level gradually reduced from 1.73 (95% CI: 1.67, 1.8) to 1.69 (95% CI: 1.6- 1.78),

1.66 (95% CI: 1.6-1.73), 1.63 (95% CI: 1.57- 1.7) at 4th

week, 8th

week, 12th

week

respectively and slightly increased to 1.65 (95%CI; 1.58 -1.71) in 16th

week. On the

contrary, regarding the side treated with TA 4 mg/ml, the Hb level decreased slightly

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from 1.72 ( 95%CI: 1.655-1.8) to 1.69 (95%CI: 1.61-1.76), 1.65 (95%CI: 1.58-1.71),

1.62 (95%CI: 1.56-1.69) respectively and slightly raised up to 1.66 (1.56-1.73) in

week 16. The result showed no significant difference between 2 concentrations of TA

(P>0.05) in term of lowering Hb level. However, the significant reduction in Hb level

was observed at week 8 in both concentrations (p<0.05). For the week 16, even if the

Hb level was slightly increased in both concentrations, the scores were less than the

baseline. Moreover, the statistic revealed significance decrease between baseline and

week 16 (p< 0.05) (Table 4.3)

4.3 Subjective assessment

4.3.1 Modified MASI score (mMASI score)

Figure 4.5 mMASI score

0.00

-0.41*

-0.64*

-0.94* -0.98*

0.00

-0.26*

-0.34*

-0.62*

-0.84*

-1.20

-1.00

-0.80

-0.60

-0.40

-0.20

0.00Baseline week 4 week 8 week 12 week 16

TA 50 mg/ml

TA 4 mg/ml

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Table 4.4 mMASI score (N= 34)

Values presented as mean (95%CI.). P-value corresponds to Repeated ANOVA test.

P-value1: calculated probability the decrease mMASI score from before to week 4, 8, 12 and 16, respectively for the 50 mg/ml

side.

P-value2: calculated probability the decrease of mMASI score from before to week 4, 8, 12 and 16, respectively for the 4 mg/ml

side.

P-value3: calculated probability the difference of mMASI score between 4 and 50 mg/ml TA in each visit

The mMASI score at baseline and individual follow ups are shown in the

figure 4 and table4.3. In the side treated with TA 50 mg/ml, the mMASI was 2.88

(95%CI: 2.53-3.22) at the baseline followed by 2.46 (95%CI: 2.15-2.46), 2.24

(95%CI: 1.98-2.5), 1.94 (95%CI: 1.69-2.2) and 1.9 (95%CI: 1.63-2.17) at week 4,

week 8, week 12, and week 16 correspondingly. In the opposite side that treated with

TA 4 mg/ml, the mMASI revealed the slight decrease from 3.26 (95%CI: 2.9-3.63) at

TA 50

mg/ml

P- value1 TA 4 mg/ml P–value2

V Mean

difference

(95%CI.)

P-value3

Before 2.88

(2.53, 3.22)

3.26

(2.9, 3.63)

-0.388

(-0.88, 0.104)

0.12

Week4 2.46

(2.15, 2.78)

3.01

(2.66, 3.35)

-0.547

(-1.006, -0.088)

0

.02*

week 8 2.24

(1.98, 2.5)

2.92

(2.56, 3.28)

-0.679

(-1.119, -0.239)

0

.003*

Week 12 1.94

(1.69, 2.2)

2.65

(2.27, 3.03)

-0.71

(-1.15, -2.57 )

0

.003*

Week 16 1.9

(1.63, 2.17)

2.43

(2.04, 2.81)

-0.529

(-0.989, -0.07)

0

.025*

Difference

at week 4

-0.41

(-0.6, -0.23)

<

0.001*

-0.26

(-0.41, -0.1)

0

.002*

-0.159

(-0.398, 0.08)

0

.189

Difference

at week 8

-0.64

(-0.91, -0.36)

<

0.001*

-0.34

(-0.55, -0.14)

0

.001*

-0.291

(-0.625, 0.042)

0

.086

Difference

at week 12

-0.94

(-1.22, -0.65)

<

0.001*

-0.62

(-0.8, -0.44)

<

0.001*

-0.318

(-0.648, 0.013)

0

.059

Difference

at week 16

-0.98

(-1.27, -0.69)

<

0.001*

-0.84

(-1.03, -0.64)

<

0.001*

-0.141

(-0.485, 0.203)

0

.415

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the beginning of the treatment to 3.01 (95%CI:2.66-3.35), 2.92 (95%CI:2.56-3.28),

2.65 (95%CI:2.27-3.03), 2.43 (95%CI:2.04-2.81) at week 4, week 8, week 12, week

16, respectively.( Table 4.4)

According to the figure 4.5, lowering of the mMASI score at both sides of

the malar area treated with both concentrations of TA was noted since week 4

(p<0.05). Besides, there was a significant difference in the mMASI score between 50

mg/ml and 4 mg/ml in week 4 with the mean difference of -0.547 (95% CI:-1.006- -

0.088) (p<0.05).

4.3.2 Patients’ self-assessment

Table 4.5 Patients’ self-assessment for TA 50 mg/ml and 4 mg/ml

Melasma

improvement grading

TA 50 mg/ml TA 4 mg/ml

No. % No. %

0

( no improvement) 0 0 0 0

1 (1-25%)

(slightly improvement) 0 0 0 0

2 (26-50)

( Fair improvement ) 1 2.94 3 8.82

3 (51-75%)

(good improvement ) 4 11.76 19 5.88

4 (76-100%)

( excellent improvement)

2

9 85.29 12 5.29

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Figure 4.6 Patients’ self-assessment for TA 50 mg/ml and 4 mg/ml

Figure 4.6 and table 4.5 demonstrated the results of the patients’ self-

assessment which was evaluated by the patients themselves at week 16. The patients’

self-assessment of melasma improvement was graded in five scales: more than 75%

lightening (excellent); 51 to 75% (good); 26 to 50%; (fair); 1 to 25% (minimal); and

0% (no improvement). In the side treated with 50 mg/ml, the result showed that 29

from 34 patients (85.29%) scored the outcome as excellent improvement followed by

4 patients (11.76%) scored as good improvement and 1 (2.94%) scored as fair

improvement. On the other side of face with the intervention of TA 4 mg/ml, more

than half of the patients graded as good improvement (55.88%), 35.29% graded as

excellent improvement and 8.82 % graded as fair improvement.

Majority of the patients could observe the fading of the melasma

noticeably on the 50 mg/ml of TA side (85%) in comparison to the side treated with

4mg/ml (35%).

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0.00

0.50

1.00

1.50

2.00

2.50

3.00

Pain score Irritation Erythema Bruise Swelling PIH

TA 50 mg/ml TA 4 mg/ml

4.3.3 Side effect

Figure 4.7 Local side effects

Table4.6 Local side effect

TA 50 mg/ml TA 4 mg/ml Mean difference

(95%CI.)

P-value2

Pain score 1.99 (1.67, 2.3) 2.3 (2.03, 2.57) -0.32 (-0.65,

0.02) 0.067

Irritation 0.05 (0, 0.12) 0.04 (0, 0.11) 0.01 (-0.02, 0.05) 0.499

Erythema 1.77 (1.49, 2.06) 1.1 (0.88, 1.32) 0.67 (0.48, 0.87) <0.001*

Bruise 0.45 (0.32, 0.59) 0.53 (0.38, 0.68) -0.08 (-0.2, 0.04) 0.186

Swelling 2.4 (2.15, 2.65) 1.25 (1.08, 1.42) 1.15 (0.9, 1.4) <0.001*

PIH 0.04 (0, 0.08) 0.12 (0.01, 0.24) -0.08 (-0.16, 0) 0.055

Values presented as mean (95%CI.). P-value corresponds to Paired t test.

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Table4.7 Systemic side effect

In this study, we evaluated the side effects in every session of the

treatment including local side effect and systemic side effect as demonstrated in the

figure 4.7 and table 4.6 as local side effect and systemic side effect showed in Table

4.7. We asked the patients immediately after each treatment about the localized side

effects such as pain, itching, erythema and edema. For the bruise and post

inflammatory hyperpigmentation, the patients were asked whether they observed the

side effects after the previous treatment.

The systemic side effects such as abnormal menstruation (spotting,

delayed menstrual cycle, etc.), nausea and vomiting, headache and back pain after the

treatment were evaluated.

Concerning the local side effects, the results showed that the average pain

score is 1.99 (95%CI; 1.67-2.3) for the side treated with TA 50 mg/ml and 2.3

(95%CI; 2.03-2.57) for the side treated with TA 4 mg/ml. The pain score was higher

in the side treated with TA 4 mg/ml. however, there was no significant difference in

both side. (p> 0.05). As for the results of side effects such as erythema and swelling,

higher average scores were observed in the side treated with 50mg/ml as compared to

another side with the scores of 1.77 (95%CI; 1.49-2.06) and 2.4 (95%CI; 2.15-2.65),

respectively. For the side treated with TA 4 mg/ml, the scores were 1.1 (95%CI; 0.88-

0.68) and 1.25 (95%CI; 1.08- 1.42) for the erythema and swelling side effect

respectively. Moreover, the significant difference between these concentrations in

terms of unwanted effects such as erythema and swelling was observed stating that

50mg/ml of TA can cause more erythema and edema. In addition, other localized side

Systemic side effect

Before Incidence (%)

Number (No.)

Abnormal menstruation 2 0.009 (2/218) 2

Nausea&Vomiting 0 0.004 (1/218) 1

Headache 0 0.014 (3/218) 3

Backpain 2 0.009 (2/218) 2

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13.97

4.59

Before week 16

DLQI

effects such as itching, bruise and PIH, no significant difference between 2 regimens

of TA was noted.

Regarding systemic side effects, two patients had experienced a delayed

menstruation period for 1-2 weeks but only for 1 cycle after the first session of

treatment. The patients were asked more information about gynecologic problems.

They had irregular menstruation before the treatment which may be due to pre-

menopause. One of them did the annual gynecologic checkup which found

endometrium thickening but no further treatment was required. Another patient has

not done the check up yet. For other systemic side effects as shown in the table 4.7,

one patient had nausea and vomiting after the treatment only at week 4. Three

patients had headache and two patients had back pain after the treatment but no any

treatments were needed. All of the patients could tolerate the side effects and

completed the study. Although the systemic side effects such as abnormal

menstruation are unlikely, we still recommended that menstruation history be

obtained carefully in all female patients before treatment and that patients should be

advised accordingly about the adverse effects which might happen during the

treatment.

4.3.4 Dermatologic Quality of Life Index (DLQI)

Figure 4.8 Dermatologic quality of life Index

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Table 4.8 Dermatologic quality of life index (DLQI)

Before After Difference at week 16 p-value2

DLQI 13.97 (12.66, 15.28) 4.59 (3.7, 5.48) -9.38 (-10.72, -8.05) <0.001*

Values presented as mean (95%CI.). P-value corresponds to Paired t test.

The Dermatologic quality of life index for this study was described in

figure 4.8 and table 4.8. According to the table 7, it showed that mean score of DLQI

was 13.97 (95%CI; 12.66-15.28) and reduced to 4.59 (95%CI; 3.7-5.48) at week 16.

The statistics showed significant decrease in DLQI score between before and after the

treatment (p<0.001).

4.4 Discussion

This was the study of comparative effect of localized intradermal

microinjection in different concentration of tranexamic acid 50 mg/ml versus 4 mg/ml

for melasma treatment in Thai patients. It is a prospective randomized triple blinded

split face trial. The outcomes were measured by both objective and subjective

assessments. Objective assessment included the melanin level (the primary outcome)

and Hemoglobin level from the skin analysis camera (Antera3D®). The subjective

assessment included mMASI score which was evaluated by 2 dermatologists, both

local and systemic side effects which were evaluated at every visit, patients’ self-

assessment which was analyzed at the end of the study and the DLQI before and after

the study.

Since malasma is an acquired hyperpigmented skin disorder and

commonly found in different places around the world with prevalence varying from

5.9% to 35 %, Thailand is not an exception(56). Melasma has serious impact on

personal self-esteem not only physical appearance but also emotional aspect.

Although there are many treatments modalities for melasma, but the results of the

treatment are inconsistent with variable side effect and high recurrent rate. Nowadays

no treatment modality can achieve the patient’s satisfaction. Therefore, melasma is a

challenging disorder for the dermatologists.

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Tranexamic acid (TA), trans-4-aminomethycyclohexanecarboxylic acid, is

a hydrophilic agent. It is a plasmin inhibitor. It has been widely used clinically in

abnormal bleeding such as menorrhagia, excessive bleeding in trauma cases or in

operation room by inhibiting fibrinolysis. TA was commonly administrated via oral

or intravenous route with the dose of 1 to 1.5 g three to four times a day and 4 g/day

for 5 days, respectively, for a high dosage for menorrhagia(53). Recently, there was a

study showed the involvement of mechanism of action of TA involve in interaction of

melanocyte and keratinocyte by inhibiting plasminogen/plasmin system. And

decrease in melanin production. (57). Both fibroblast growth factor and

prostaglandins are potent stimulate of melanocyte activity(40).TA can prevent the

conversion of plasminogen to plasmin lead to decrease in number of fibroblast

growth factor and prostaglandins. Thereby diminish melanocyte activity and decrease

melanin production consequence(26)(47). Therefore, TA has been used as an

alternative treatment for melasma. In addition, TA was claimed to decrease allergy

and inflammation especially due to its ability to inhibit plasmin.

Nowadays, TA has been used widely for melasma treatment for its

promising result. TA was available in topical form, oral form and intradermal

injection form.

Kim et al(58), did the meta-analysis and systemic review about efficacy

and safety of tranexamic acid in melasma in different forms such as topical, oral, and

localized microinjection. The study revealed that the most effective option in

decreasing MASI score was the oral TA, followed by that with localized intradermal

micro injection of TA and topical TA, correspondingly. But no statistical difference

between 3 subgroups (oral, microinjection and topical) was observed. Oral

tranexamic acid was reported in many studies to be effective for melasma.

Unfortunately, the side effects remained as concern such as gastrointestinal

discomfort, headache, hypomenorrhea and spotting menstruation. The most potential

risk is systemic thromboembolism(59). On the other hand, the result outcome of

topical TA was still debatable. According to the study by Ebrahimi, et al(52), this

double- blinded split-face trial evaluated the efficacy and safety of 3% topical TA

twice a day compared with combined solution of 3% HQ plus 0.01% dexamethasone

for 12 weeks. Even through both interventions are effective in melasma treatment in

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term of lowering MASI score. But no significant difference was observed between

them. On the other hand, Kanechorn Na Ayuthaya P, et al(46) in 2012, demonstrated

the similar efficacy between 5% TA and vehicle in treatment of melasma.

In order to avoid the side effects caused by systemic administration of TA,

In this study, we comparatively by evaluated the efficacy of localized intradermal

microinjection in different concentrations of tranexamic acid 50 mg/ml versus 4

mg/ml by objective and subjective assessments such as melanin level and hemoglobin

level by Antera 3D®, mMASI by two dermatologists, local and systemic side effects

and patients’s self-assessment.

In this study, most of the patients have mixed-type melasma especially

distributed in malar area. The epidemiological study showed that the most common

factor for melasma in this study is sunlight (UV radiation, 64%) followed by

pregnancy (19%) and oral contraceptive pill (12%). As for the role of UV irradiation

in melasma , It stimulates melanogenesis both directly to melanocyte and indirectly to

keratinocyte by paracrine interaction between keratinocyte and melanocytes(13).

Recently there were many studies have been reported the efficacy and side

effects of localized TA intradermal microinjection. There was a pilot study in 2006,

lee, et al. (7), in which they evaluated the effectiveness of 4 mg/ml of TA in

localized intradermal microinjection weekly in melasma treatment for 12 weeks. It

was a prospective opened label study. The significant decreased in MASI score was

noted in week 8 and week 12 from the baseline. In 2009, Steiner, et al.(53) Performed

a comparative study which evaluated the efficacy and safety between topical 3% TA

(twice a day) and 4 mg/ml TA intradermal microinjection administrated weekly for

12 weeks. Seventeen patients completed the study. The significant improvement in

both groups was shown at the end of the study but there was no significant different

between these two modalities. In 2013, a study by Budamakuntla et al.(1),

demonstrated the comparative study evaluated efficacy and safety of microinjection

of TA versus microneedling of TA in melasma treatment. The study was done in

Kamatake India. Thirty patients in group A administrated with 4 mg/ml localized

intradermal microinjection of TA and another 30 patients in group B were treated

with microneeding in the same concentration of TA. The treatments were performed

monthly interval at baseline, week 4, week 8, and 3 months follow up after the end of

Ref. code: 25605929040458YKT

55

the treatment. The significant decrease in MASI score was noted in both groups.

Although the MASI score in microneedling TA group showed superior to other

group, but there was no significant difference between both groups. Lately, there was

a split-face controlled trial study in 2017, by Saki, et al.(54), which compared the

efficacy of topical 2% HQ versus 20 mg/ml TA intradermal microinjection.

According to the methodology of the study, one of the face was treated with TA

intradermal injection one session per month for altogether 3 sessions and topical 2%

HQ on the other side of face once a night for three months. The results showed

monthly TA injection was superior to topical 2 % HQ in decreasing melanin value in

the first 4 weeks, however, there was no significant difference between both groups

after the end of the study. As for the result, the study assumed that prolonged

treatment duration could lead to better improvement in fading melasma. Moreover,

the study showed TA microinjection can achieve the good result within a short period

of time comparing to the topical HQ.

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56

Table 4.9 Studies of the injection of tranexamic acid for the treatment of melasma

This clinical trial found out that although, there was significant decrease

in mMASI score by both of regimens since week 4 but superior efficacy of TA 50

mg/ml in reduction of MASI score was observed comparing to 4 mg/ml (34% versus

25 %). Furthermore, there was a significant difference decreasing in mMASI score

since week 4. According to the melanin level detected by Antera3D®, the results

showed the same outcome as mMASI score. It showed that the result of the side

treated with 50 mg/ml TA was significant since week 4 (p<0.05) and the melanin

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57

reduced almost 13 % from the baseline. On the other hand, for the 4 mg/ml TA side,

the significant decrease in melanin level started at week 8 and there is a reduction in

melanin level approximately 5 % from the baseline. Baseline melanin level and

mMASI score for both sides of the face showed no any significant. Hence, in this

study we suggested that the higher concentration of TA may have more effectiveness

and shorter onset of action in diminished the melanocyte activation and melanin

production. As state by Seong, et al.(12), in 2007, they evaluated the influence of TA

in different dosages on the melanocytes activity, viability, and melanogenesis from

cultured normal human melanocytes. The consequence showed that there was a

statistically significant decrease in melanocyte viability and melanin production by

TA in a dose dependent manner. According to table 10, Saki, et al(54) stated that the

melanin level after 20 mg/ml TA intradermal microinjection was 5.6 % and 6.44%

improvement at week 8 and at 3 month follow up, respectively. In our study, the

melanin level after 50 mg/ml TA injection was 8.35% and 13% improvement and for

4 mg/ml TA intradermal microinjection was 2.26 % and 5.11 % improvement at week

8 and 1 month follow up, respectively. It appears that TA for intradermal

microinjection was dose dependent. In addition to the dose dependent nature of TA,

longer durations of the treatment can achieve a better result in melanin reduction and

fading of the hyperpigmented lesion.

Recently there were studies demonstrated about vascularity increasing in

melasma pathogenesis. Kim, et al.,(43) reported about the vascular characteristics of

melasma in 2006. The result showed that the erythema intensity was higher in

melasma lesion when compared with peri-normal skin. Moreover, the result from the

computer-assisted image analyzed of factor VIIIa-related antigen-stained sections

also noted the significant increase in number and size of dermal blood vessel. The

study suggested that there could be angiogenesis in melasma lesion. Concerning the

vascular endothelial growth factor (VEGF), the area with melasma showed higher

expression from keratinocytes in comparison with peri-normal skin(60). There were

reports suggested that the effectiveness of TA can diminish the erythema in melasma

skin by diminishing number of vessels and number and activity of mast

cell(47)(61)(62). TA as a plasmin inhibitor, can not only inhibit the angiogenesis but

also arrest basic fibroblast growth factor (bFGF) inducing neovascularization

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58

(63)(64). In melasma lesion, the mast cells were proliferated in the dermis more than

normal skin. Chronic UV irradiation induced mast cell up-regulation. Mast cells can

induce vascular proliferation through secreting angiogenic factors for example VEGF,

FGF-2 and transforming growth factor-β(65). In our study, we measured the vascular

intensity in term the hemoglobin level by using Antera3D®, there was significant

decrease in Hb level at week 8 in both side of the face however but no significant

difference between 2 regimens were observed.

The mean dose of TA injection in our study was 150- 200 mg which is

lower than the usual dose for antifibrinolytic effect(53). Therefore, in this study, no

significant systemic side effect was observed. Three patients reported delayed

menstruation but after asking for more information, two of them have this experience

before the study due to pre-menopause. One of them had just stopped OCP for 2

months before the study.

Concerning for the local side effect between 2 concentrations, erythema

and swelling were significance observed on TA 50 mg/ml side. However, theses side

effects disappeared in 2 hours after the injection. For other local side effects, for

example pain, irritation, bruise and PIH, the result showed no significant difference

between 2 regimens. All side effects were minimal. All patients could tolerate and

completed the study.

Regarding the patients’ self-assessment and dermatological quality of life

index, it was found out that the majority of patients preferred the side treated with TA

50 mg/ml more than the other side (85% versus 35%) stating that they could observe

the fading of melasma faster and more noticeably on the 50 mg/ml of TA side when

compared to the side treated with 4 mg/ml. As for the dermatological quality of life

index, it was evaluated at the end of the study (week 16). There was a significant

difference between before and after the study. It showed that most of the patients

were satisfied with the result of intradermal microinjection of TA in melasma.

The fact that Thailand is located near the equator means that the sunlight

is very strong even though the study period was not conducted during the summer

season. Sunlight is the greatest triggering factor and can interfere with the treatment.

Another drawback of this present study was the improper usage of sunscreen. Most

patients applied the sunscreen in inadequate portions and only occasionally. Also, the

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59

short follow up period after the treatment might not detect the noticeable recurrence

of the hyperpigmented lesion. Future studies should keep an eye on the earlier

mentioned points to achieve a more accurate result.

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CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusion

According to this study, it was a prospective, randomized, triple blinded,

split-face trial study. We evaluated the efficacy and side effect of intradermal

microinjection of 50 mg/ml TA versus 4 mg/ml TA in melasma treatment. As for the

methodology, two concentrations of TA intradermal microinjection were randomized

in to different sides of the face. The injection administrated every 2 weeks for 12

weeks. The patients came to follow up at week 4, 8, 12, and 1-month after the end of

the treatment in order to evaluate the outcome of the treatment. According to the

results of this study, it was found out that the efficacy of localized TA intradermal

microinjection could improve the melasma without significant side effects. The side

treated with 50 mg/ml could decrease mMASI score and melanin level significantly.

Moreover, the side treated with 50 ml/ml could achieve better result in term of fading

of pigmentation within shorter period of time comparing to 4 mg/ml TA. Regarding

the recurrence of the pigmentation of melasma at the end of the follow up showed that

the mMASI score and melanin level had continuously decreased as shown in the table

4.4 and 4.2, respectively, on both side of the faces.

With regard to local side effects, although there was more redness and

swelling had been observe on the side treated with 50 mg/ml TA comparing to the

side treated with 4 mg/ml but the pain score, irritation, erythema, bruise, and PIH

were no significant difference between 2 concentrations. These kind of local side

effect were tolerable by all patients. About for the systemic side effects, we cannot

evaluate separately for 50 mg/ml TA and 4 mg/ml TA because of this was an intra-

individual study.

Last least, the patients’self-assessment score which evaluated by the

patients themselves at the end of the study showed that the majority of the patients

were satisfied with the outcome of 50 mg/ml TA intradermal microinjection for

melasma treatment. Even though there were some patients preferred 4 mg/ml TA.

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61

TA 50 mg/ml intradermal microinjection not only showed significant

decrease in mMASI score and melanin level but also showed the fading of

pigmentation was seen earlier when compared with the other side. Therefore, we can

assume that TA was a dose dependent for intradermal microinjection modality. Fifty

mg/ml TA appeared to be promising therapeutic option for melasma.

5.2 Recommendations

- A further study using TA with drug delivery system could be used in

order to achieve better results and with less pain.

- A further study with a longer follow up period after the treatment could

be done in order to observe any rebounding of hyperpigmentation clearly.

- A further study prospective double-blind split-face study for topical HQ

with 50 mg/ml TA intradermal microinjection versus 50 mg/ml TA intradermal

injection only should be performed in order to evaluate the efficacy and safety of TA

intradermal microinjection with HQ and whether it may achieve the better result.

5.3 Limitation

- The limitations of this study are the sunlight and patients’ activities,

patients’ compliance and sunscreen applying habit.

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62

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APPENDICES

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APPENDIX A

MELANIN LEVEL ASSESSMENT

TA 50 mg/ml TA 4 mg/ml

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16 1

1 0.72 0.725 0.689 0.623 0.622 1 0.666 0.7 0.658 0.664 0.645

1

2 0.773 0.698 0.624 0.594 0.59

2

2 0.69 0.676 0.69 0.662 0.601

1

3 0.715 0.678 0.634 0.615 0.585

3

3 0.67 0.655 0.654 0.625 0.638

1

4 0.824 0.714 0.684 0.628 0.637

4

4 0.652 0.686 0.652 0.624 0.609

1

5 0.728 0.704 0.694 0.629 0.626

5

5 0.75 0.749 0.703 0.686 0.696

1

6 0.527 0.513 0.512 0.501 0.55

6

6 0.55 0.541 0.538 0.535 0.535

1

7 0.692 0.639 0.609 0.601 0.617

7

7 0.732 0.696 0.677 0.682 0.674

1

8 0.756 0.68 0.669 0.647 0.619

8

8 0.702 0.7 0.658 0.643 0.65

1

9 0.623 0.58 0.505 0.487 0.498

9

9 0.573 0.568 0.553 0.545

0

.537

1

10 0.831 0.802 0.75 0.694 0.684

1

10 0.768 0.739 0.737 0.726 0.734

2

11 0.699 0.717 0.677 0.678 0.688

1

11 0.758 0.699 0.669 0.657 0.662

2

12 0.704 0.688 0.626 0.624 0.647

1

12 0.7 0.69 0.684 0.731 0.729

2

13 0.71 0.626 0.572 0.568 0.581

1

13 0.65 0.63 0.623 0.624 0.579

2

14 0.69 0.666 0.603 0.576 0.564

1

14 0.67

0

.667 0.665 0.63 0.642

2

15 0.707 0.68 0.654 0.63 0.628

1

15 0.689 0.673 0.668 0.647 0.639

2

16 0.583 0.535 0.53 0.487 0.501

1

16 0.486 0.466 0.435 0.449 0.447

1

17 0.769 0.665 0.682 0.663 0.67

1

17 0.724 0.709 0.716 0.707 0.69

18 0.65 0.609 0.615 0.59 0.564

1

18 0.706 0.662 0.662 0.66 0.655

Ref. code: 25605929040458YKT

70

TA 50 mg/ml TA 4 mg/ml

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

1

19 0.718 0.677 0.661 0.634 0.615

1

19 0.697 0.702 0.683 0.655 0.627

2

20 0.693 0.681 0.645 0.615 0.593

2

20 0.514 0.596 0.588 0.578 0.587

2

21 0.699 0.66 0.652 0.608 0.606

2

21 0.625 0.61 0.613 0.604 0.612

2

22 0.687 0.689 0.658 0.616 0.638

2

22 0.622 0.627 0.638 0.604 0.607

2

23 0.716 0.712 0.706 0.664 0.678

2

23 0.693 0.682 0.688 0.682 0.683

2

24 0.672 0.654 0.621 0.616 0.618

2

24 0.614 0.613 0.622 0.619 0.592

2

25 0.661 0.678 0.659 0.618 0.603

2

25 0.754 0.732 0.734 0.753 0.763

2

26 0.544 0.527 0.501 0.476 0.463

2

26 0.547 0.553 0.578 0.544 0.535

2

27 0.69 0.651 0.659 0.637 0.647

2

27 0.667 0.7 0.646 0.641 0.661

2

28

-

-

-

--

-

-

-

-

-

-

2

28 - - - - -

2

29 0.623 0.623 0.6 0.565 0.521

2

29 0.654 0.628 0.659 0.612 0.592

3

30 0.718 0.693 0.659 0.655 0.649

3

30 0.646 0.634 0.659 0.653 0.638

3

31 0.733 0.652 0.625 0.616 0.61

3

31 0.684 0.68 0.677 0.642 0.594

3

32 0.685 0.731 0.685 0.638 0.627

3

32 0.694 0.673 0.648 0.638 0.641

3

33 0.607 0.614 0.62 0.59 0.545

3

33 0.656 0.657 0.68 0.668 0.643

3

34 0.694 0.688 0.683 0.637 0.634

3

34 0.649 0.598 0.613 0.604 0.592

35 0.771 0.713 0.678 0.66

0

.64

3

35 0.761 0.753 0.736 0.725 0.72

Ref. code: 25605929040458YKT

71

APPENDIX B

Hemoglobin level

TA 50 mg/ml TA 4 mg/ml

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

1

1

1

1.961

1

1.807

1

1.557

1

1.546

1

1.1593

1

1

1

1.919

1

1.828

1

1.758

1

1.693

1

1.919

2

2

1

1.829

1

1.693

1

1.762

1

1.727

1

1.689

2

2

1

1.555

1

1.683

1

1.575

1

1.638

1

1.555

3

3

1

1.664

1

1.703

1

1.641

1

1.728

1

1.542

2

3

1

1.723

1

1.64

1

1.599

1

1.658

1

1.723

4

4

1

1.632

1

1.779

1

1.685

1

1.51

1

1.628

2

4

1

1.595

1

1.648

1

1.655

1

1.493

1

1.595

5

5

1

1.436

1

1.431

1

1.438

1

1.412

1

1.458

2

5

1

1.442

1

1.483

1

1.446

1

1.412

1

1.442

6

6

1

1.742

1

1.439

1

1.472

1

1.337

1

1.511

2

6

1

1.655

1

1.335

1

1.377

1

1.384

1

1.655

7

7

1

1.742

1

1.731

1

1.708

1

1.673

1

1.751

2

7

1

1.76

1

1.783

1

1.736

1

1.738

1

1.76

8

8

1

1.722

1

1.557

1

1.419

1

1.609

1

1.566

2

8

1

1.695

1

1.613

1

1.649

1

1.625

1

1.695

0

9

1

1.703

1

1.614

1

1.672

1

1.585

1

1.777

2

9

1

1.846

1

1.792

1

1.591

1

1.654

1

1.846

1

10

2

2

1

1.966

1

1.654

1

1.677

1

1.82

1

10

1

2.143

1

2.065

1

1.819

1

1.937

1

2.143

1

11

1

1.651

2

2.373

1

2.005

1

2.084

1

1.775

2

11

2

2.308

1

1.993

1

1.92

1

1.813

1

2.308

1

12

1

1.33

2

1.245

2

1.335

2

1.337

2

1.264

3

12

1

1.629

1

1.667

1

1.85

1

1.849

1

1.629

1

13

1

1.869

1

1.574

1

1.651

1

1.585

1

1.707

3

13

1

1.678

1

1.623

1

1.659

1

1.492

1

1.678

1

14

1

1.99

2

2.031

1

1.862

1

1.782

1

1.681

3

14

1

1.722

1

1.738

1

1.658

1

1.578

1

1.722

1

15

1

1.79

1

1.743

1

1.789

1

1.587

1

1.779

3

15

1

1.734

1

1.692

1

1.792

1

1.704

1

1.734

1

16

1

1.547

1

1.546

1

1.628

1

1.417

1

1.395

3

16

1

1.569

1

1.642

1

1.563

1

1.534

1

1.569

1

17

1

1.607

1

1.506

1

1.475

1

1.429

1

1.45

3

17

1

1.541

1

1.472

1

1.421

1

1.424

1

1.541

Ref. code: 25605929040458YKT

72

TA 50 mg/ml TA 4 mg/ml

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

1

18

1

1.631

1

1.479

1

1.595

1

1.519

1

1.684

1

18

1

1.776

1

1.749

1

1.69

1

1.596

1

1.776

2

19

2

2.188

2

2.148

2

2.025

1

1.981

1

1.936

2

19

2

2.252

2

2

2.144

2

2.282

2

2.252

3

20

1

1.469

1

1.449

1

1.496

1

1.483

1

1.452

2

20

1

1.435

2

1

1.335

1

1.294

1

1.435

4

21

1

1.875

1

1.583

1

1.747

1

1.609

1

1.628

2

21

1

1.806

1

1.584

1

1.579

1

1.619

1

1.806

5

22

1

1.447

1

1.227

1

1.303

1

1.366

1

1.4

2

22

1

1.618

1

1.589

1

1.57

1

1.552

1

1.618

6

23

1

1.919

1

1.837

2

2.002

2

2.089

1

1.869

2

23

1

1.871

1

1.906

1

1.885

1

1.804

1

1.871

7

24

1

1.674

1

1.735

1

1.792

1

1.786

1

1.675

2

24

1

1.52

1

1.542

1

1.633

1

1.621

1

1.52

8

25

2

2.146

2

2.127

1

1.93

1

1.861

1

1.023

2

25

1

1.909

1

1.866

1

1.72

1

1.631

1

1.909

0

26

1

1.651

1

1.511

1

1.51

1

1.62

1

1.561

2

26

1

1.625

1

1.559

1

1.6

1

1.665

1

1.625

1

27

1

1.7

1

1.633

1

1.623

1

1.625

1

1.62

2

27

1

1.592

1

1.5

1.502

1.479

1.592

1

28

1

- - - - -

2

28

0

- - 0

1

29

1

1.749

1

1.645

1

1.613

1

1.672

1

1.726

3

29

1

1.439

1

1.32

1

1.218

1

1.363

1

1.376

1

30

1

1.8

1

1.813

1

1.762

1

1.699

1

1.748

3

30

1

1.745

1

1.917

1

1.844

1

1.629

1

1.793

1

31

1

1.59

1

1.795

1

1.833

1

1.79

1

1.824

3

31

1

1.459

1

1.488

1

1.577

1

1.483

1

1.551

1

32

1

1.777

1

1.645

1

1.71

1

1.631

1

1.591

3

32

1

1.53

1

1.55

1

1.551

1

1.485

1

1.461

1

33

1

1.918

1

1.932

1

1.752

1

1.768

1

1.855

3

33

1

1.834

1

1.707

1

1.626

1

1.673

1

1.747

1

34

1

1.693

1

1.729

1

1.671

1

1.634

1

1.981

3

34

1

1.775

1

1.632

1

1.504

1

1.589

1

1.876

3

35

1

1.497

1

1.365

1.387

1.365

1.414

3

35

1

1.871

1

1.826

1

1.905

1

1.731

1

1.908

Ref. code: 25605929040458YKT

73

APPENDIX C

modified MASI score

TA 50 mg/ml TA 4 mg/ml

Subj wk0 Wk4 Wk8 Wk12 Wk16 Subj Wk0 Wk4 Wk8 Wk12 Wk16

1 1.8 1.2 1.2 0.9 1.2 1 1.8 1.8 1.8 1.2 0.6

2 2.4 2.1 2.1 1.8 1.2 2 2.4 2.1 2.4 1.8 0.6

3 3.6 2.7 1.2 1.2 0.6 3 3.6 3 3 2.1 2.7

4 2.7 2.7 2.7 2.1 2.4 4 1.8 2.4 1.8 1.2 0.6

5 2.7 2.4 2.1 1.2 0.9 5 1.8 1.8 1.2 0.9 1.2

6 1.2 1.2 1.8 0.9 1.2 6 3 2.7 2.4 2.4 2.4

7 2.7 2.7 2.4 2.1 2.7 7 2.7 2.4 2.1 2.4 1.8

8 0.9 0.6 0.3 0.3 0.3 8 1.8 1.8 0.9 0.6 0.9

9 3.6 3 3.6 2.7 2.4 9 4.5 4.5 4.2 3.6 3

10 3 2.4 1.8 1.2 1.2 10 4.5 3 2.7 2.7 3

11 2.7 1.8 2.7 1.8 1.8 11 2.7 2.4 2.1 1.8 2.1

12 1.2 1.8 2.4 2.4 1.8 12 1.8 1.8 1.8 2.1 2.1

13 2.7 2.7 2.4 1.8 2.4 13 2.7 2.4 2.4 2.4 1.8

14 4.5 3.6 2.7 2.4 2.4 14 4.5 4.5 3.6 3.6 3.6

15 3.6 3 2.7 2.4 2.4 15 4.5 4.5 3.9 3.9 3

16 1.8 0.9 1.2 1.8 1.8 16 2.1 1.8 1.2 1.8 1.8

17 3 2.7 2.1 2.1 2.4 17 3.6 3 3.6 3 2.7

Ref. code: 25605929040458YKT

74

TA 50 mg/ml TA 4 mg/ml

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

S

Subj.

W

wk0

W

wk4

Wk8

Wk12

Wk16

18 2.1 1.8 2.4 2.1 1.8 18 2.7 2.4 2.4 2.4 1.8

19 4.5 3.6 3 2.7 2.7 19 4.8 4.8 4.5 4.2 3

20 3.6 3 2.7 2.4 2.1 20 3.6 3.6 3.6 3 3.6

21 2.4 3 2.4 2.4 2.1 21 2.4 2.1 2.1 1.8 2.1

22 3.6 3.6 2.7 2.4 2.1 22 3.6 3.9 4.5 3 3

23 3.6 2.7 3 2.7 2.4 23 4.5 3.6 3.6 3.6 3.6

24 1.8 2.7 2.4 2.4 2.1 24 3.6 3.6 4.2 4.2 3.6

25 4.5 4.5 3.6 3.6 3.6 25 4.5 3.6 3.6 4.5 4.8

26 1.8 1.8 2.4 2.1 1.8 26 1.8 2.1 2.7 1.8 1.2

27 3.6 3 2.7 2.4 3 27 4.5 4.8 4.2 4.2 4.5

28 - - - - 28 - 0

29 1.8 1.2 0.6 0.6 0.6 29 1.8 1.2 1.8 0.9 0.6

30 3.6 2.4 2.1 1.2 1.8 30 4.5 3.6 4.8 4.8 3.6

31 2.7 1.2 1.2 1.2 1.2 31 3.6 3.6 3.6 2.7 2.4

32 4.5 3.6 2.7 2.7 2.4 32 4.5 3.6 3 2.7 2.7

33 3 2.7 2.4 2.1 2.1 33 3.6 3.6 3.6 3 2.7

34 3.6 3.6 2.7 2.7 3 34 3.6 2.7 3 3 2.7

35 3 1.8 1.8 1.2 0.6 35 3.6 3.6 3 2.7 2.7

Ref. code: 25605929040458YKT

75

APPENDIX D

DIGITAL IMAGE OF SUBJECTS

Figure5.1 Photograph and melanin level of subject 1 showing intradermal

microinjection of 4 mg/ml TA

micron injection of 4 mg/ml TA

Figure 5.2 Photograph and melanin level of subject 1 showing intradermal

micron injection of 50 mg/ml TA

Ref. code: 25605929040458YKT

76

Figure 5.3 Photograph and melanin level of subject 2 showing intradermal micro

injection of 4 mg/ml TA

Figure 5.4 Photograph and melanin level of subject 2 showing intradermal micro

injectioninjection of 50 mg/ml

Ref. code: 25605929040458YKT

77

Figure 5.5 Photograph and melanin level of subject 3 showing intradermal micron

injection of 4 mg/ml TA

Figure 5.6 Photograph and melanin level of subject 3 showing intradermal micron

injection of 50 mg/ml TA

Ref. code: 25605929040458YKT

78

Figure 5.7 Photograph and melanin level of subject 4 showing intradermal micron

injection of 50 mg/ml TA

Figure 5.8 Photograph and melanin level of subject 4 showing intradermal micron

injection of 4 mg/ml TA

Ref. code: 25605929040458YKT

79

APPENDICES E

PATIENT RECORD FORM

แบบบนทกขอมลท ำกำรวจย

เรอง การศกษาเปรยบเทยบประสทธภาพของการรกษาฝาโดยการใชยา tranexamic acid

ฉดเขาใตผวหนง

เลขทแบบบนทกขอมล………………………

ขอมลทวไปของผปวย ( patient demographic information)

เฉพาะเจาหนาท

1. วน/เดอน/ป

2. ชอ/นามสกล

3. ทอย /เบอรโทรศพท

4. เพศ………….

5. อาย………….

6. อาชพ ……… 1. ขาราชการและวสาหกจ ……..2. พนกงาน

……… 3. แมบาน ………4. นกเรยน/นกศกษา

……… 5. กจการสวนตว ………6. คาขาย

……… 7. อน ๆ

7. สบบหร สบ................. ............ ไมสบ

8. ดมแอลกอฮอล ดม................. ไมดม............

9. โรคประจ าตว............................................

10. ยาททานประจ า รวมถงวตามน...............................................

11. ประจ าเดอน .................มาตามปกต หมดประจ าเดอน...............

Ref. code: 25605929040458YKT

80

12. แพยา/แพอาหาร.....................................................................

13. ระยะเวลาทเปนฝา ……………………………….

14. ประวตคนในครอบครวทเปนฝา ………1. ม ………2. ไมม

15. ปจจยกระตนทท าเกดฝา

……… 1. ตงครรภ ……..2. การไดรบฮอรโมน

……… 3. การไดรบแสงแดด ………4. การใชเครองส าอางค

………5. ยากนชก ………6. ยาทมปฎกรยากบแสง

16. ผลของฝาทกระทบความกงวลในจตใจ 1. ไมรสก 2. รสกเสยความมนใจเลกนอย

3. รสกเสยความมนใจปานกลาง 4.รสกเสยความมนใจมาก 5. รสกเสยความ

มนใจมากทสด

17. ประวตการรกษาทเคยไดรบมากอน ………..1. เคย ………..2. ไมเคย

18. จ าแนกชนดของสผวตาม skin type 1.………2. ………3.………4. ………5…….

Ref. code: 25605929040458YKT

81

แบบสอบถำม

Pain score

ขวำ

ขวำ

ซำย

0 = ไมปวด/เจบ

1-3 = เจบเลกนอย

4-6 = เจบปานกลาง (ยงสามารถท ากจวตรประจ าวนไดตามปกต)

7-10 = เจบมาก (ไมสามารถท ากจวตรประจ าวนได)

Ref. code: 25605929040458YKT

82

ผลขำงเคยง

ขวำ

ประเมนผลขางเคยง: โดยผปวย ……..1. ม ……..2. ไมม

ผลขางเคยง

ไมมเลย

นอยมาก

(2)

คอนขางนอย

(3)

ปานกลาง

(4)

คอนขางมาก

(5)

มากทสด

(6)

แสบรอน,คน

(irritation)

แดง

อาการช า

บรเวณทฉด เขม

ขน

Ref. code: 25605929040458YKT

83

ซำย

ประเมนผลขางเคยง: โดยผปวย ……..1. ม ……..2. ไมม

ผลขางเคยง

ไมมเลย

นอยมาก

(2)

คอนขางนอย

(3)

ปานกลาง

(4)

คอนขางมาก

(5)

มากทสด

(6)

แสบรอน,คน

(irritation)

แดง

อาการช า

บรเวณทฉด

เขมขน

Ref. code: 25605929040458YKT

84

ผลขางเคยงภายใน ม ไมม

ประจ าเดอนมาผดปกต

คลนไสอาเจยน

ปวดศรษะ

ปวดหลง

Ref. code: 25605929040458YKT

85

ความพง

พอใจจากก

ารรกษาโดยรวม

ไม

เปลยนแปลง

(0)

ดขนเลกนอย

(1)

Slightly

improvement

(0-25%)

พอใจ

(2)

Fair

improvement

(26-50%)

พอใจมาก

(3)

Good

improvement

(51-75%)

พอใจมากทสด

(4)

Excellent

improvement

(>75%)

Ref. code: 25605929040458YKT

86

BIOGRAPHY

Name

Miss Prapalpitch Wongwicharn

Date of Birth August 31, 1988

Educational Attainment 2016-present: Master of Science (dermatology)

Chulabhorn International College of Medicine,

Thammasat University

2008-2013: Doctor of medicine, Mahidol

University

Work Experience

Publication

Award

2014-2016: Medical Doctor, Practitioner Level

U-thong Hospital

2013-2014: Chao Phraya Yom Marat Hospital

None

None

Ref. code: 25605929040458YKT