Topical hypopigmenting agents for pigmentary disorders and their mechanisms of action.

Abstract

Melanin is produced in melanocytes and stored in melanosomes. In spite of its beneficial sun-protective effect, abnormal accumulation of melanin results in esthetic problems. Hydroquinone, competing with tyrosine, is a major ingredient in topical pharmacological agents. However, frequent adverse reactions are amongst its major limitation. To solve this problem, several alternatives such as arbutin, kojic acid, aloesin, and 4-n-butyl resorcinol have been developed. Herein, we classify hypopigmenting agents according to their mechanism of action; a) regulation of enzyme, which is subdivided into three categories, i) regulation of transcription and maturation of tyrosinase, ii) inhibition of tyrosinase activity, and iii) post-transcriptional control of tyrosinase; b) inhibition of melanosome transfer, and c) additional mechanisms such as regulation of the melanocyte environment and antioxidant agents.

Full text

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Topical Hypopigmenting Agents for Pigmentary Disorders and Their Mechanisms of Action
Vol. 24, No. 1, 2012
1
Received November 15, 2011, Accepted for publication November 15,
2011
*These authors contributed equally to this work.
†This study was supported by a grant (A100157) of the Good Health
R&D Project, Ministry of Health and Welfare, R.O.K.
Corresponding author: Kyoung-Chan Park, M.D., Department of Derma-
tology, Seoul National University Bundang Hospital, 166 Gumi-ro,
Bundang-gu, Seongnam 463-707, Korea. Tel: 82-31-787-7311, Fax:
82-2-3675-1187, E-mail: gcpark@snu.ac.kr
This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Ann DermatolVol. 24, No 1, 2012http://dx.doi.org/10.5021/ad.2012.24.1.1
REVIEW ARTICLE
Topical Hypopigmenting Agents for Pigmentary
Disorders and Their Mechanisms of Action
Hyojin Kim, M.D.*, Hye-Ryung Choi, Ph.D.*, Dong-Seok Kim, Ph.D.1, Kyoung-Chan Park, M.D.
Department of Dermatology, Seoul National University Bundang Hospital, Seongnam, 1Department of Biochemistry, Chung-Ang
University College of Medicine, Seoul, Korea
Melanin is produced in melanocytes and stored in
melanosomes. In spite of its beneficial sun- protective effect,
abnormal accumulation of melanin results in esthetic
problems. Hydroquinone, competing with tyrosine, is a
major ingredient in topical pharmacological agents. How-
ever, frequent adverse reactions are amongst its major
limitation. To solve this problem, several alternatives such as
arbutin, kojic acid, aloesin, and 4-n-butyl resorcinol have
been developed. Herein, we classify hypopigmenting agents
according to their mechanism of action; a) regulation of
enzyme, which is subdivided into three categories, i)
regulation of transcription and maturation of tyrosinase, ii)
inhibition of tyrosinase activity, and iii) post-transcriptional
control of tyrosinase; b) inhibition of melanosome transfer,
and c) additional mechanisms such as regulation of the
melanocyte environment and antioxidant agents. (Ann
Dermatol 24(1) 1∼∼6, 2012)
-Keywords-
Hydroquinone, Hypopigmentation, Melanin
INTRODUCTION
Melanin plays an important role in protecting the skin
from ultraviolet light. It also determines skin color and
influences phenotypic appearances. However, abnormal
accumulation of melanin may lead to esthetic problems.
Hydroquinone (HQ) is a major ingredient in topical phar-
macological agents that are used for hyperpigmentary
disorders. However, HQ is frequently associated with a
high rate of adverse effects1,2.
Therefore, several topical hypopigmenting agents have
been developed and widely used (Table 1). This review
summarizes the different approaches that have been
implemented to achieve hypopigmentation and classify
them on the basis of their mechanisms.
TOPICAL HYPOPIGMENTING AGENTS HQ
IN MELASMA
Treatment of melasma is difficult and a number of agents
have been used for this intractable condition. To date, the
most effective treatment is a triple-combination cream that
contains 4% HQ, 0.05% tretinoin and 0.01% fluocinolone
acetonide3. HQ is the most commonly used tyrosinase
inhibitor. Oxidation products of HQ result in oxidative
damage of membrane lipids and proteins, including tyro-
sinase, and depletion of glutathione4. However, HQ is not
commonly used in cosmetics because of long-term compli-
cations5. Tretinoin is used as an anti-wrinkle agent. How-
ever, it is also reported that topical retinoid is effective in
the treatment of pigmentary disorders or can be combined
with other topical agents6. The third ingredient featured in
triple combination products is corticosteroids. Steroids are
effective in the suppression of cytokines such as endothelin-
1 and granulocyte macrophage colony-stimulating factor
(GM-CSF), which mediate ultraviolet (UV)-induced res-

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Fig. 1. Schematic illustration of
possible strategies for inhibition of
melanogenesis. UV: ultraviolet.
Table 1. Classification of hypopigmenting agents. Compounds are divided on the basis of their reported mechanism of interference
with melanogenesis5
Before melanin synthesis
Regulation of tyrosinase transcription
TGF-β1, TNF-α, IL-1α, β, IL-6, lysophosphatidic acid, C2-ceramides, sphingosine-1-phosphate
Sphingosylphosphorylcholine, tretinoin
Inhibition of tyrosinase maturation
Glucosamine, tunicamycin, glycosphingolipid, calcium d-pantetheine- S-sulphonate
During melanin synthesis
Inhibition of tyrosinase activity
Hydroquinone, arbutin, kojic acid, 4-n-butylresorcinol, phenolic compounds, 4-hydroxy-anisole, methyl gentisate,
4-S-CAP & derivatives, ellagic acid, oxyresveratrol, resveratrol, aloesin, azelaic acid
After melanin synthesis
Posttranscriptional control of tyrosinase
Linoleic acid, α-linolenic acid, phospholipase D2
Inhibition of melanosome transfer
Niacinamide (Vitamin B3), serine protease inhibitors, lecthins and neoglycoproteins, RW-50353, soybean.milk extracts
Regulation of melanocytes environment
Corticosteroids, glabridin
Antioxidant agents
α-Tocopherol, ascorbic acid, ascorbic acid palmitate, D, L-α TF, VC-PMG, methimazole,
hydrocoumarins (6-hydroxy-3,4-dihydrocoumarins), thioctic acid (α-lipoic acid), phenol/catechol
TGF: transforming growth factor, TNF: tumor necrosis factor, IL: interleukin, TF: α-tocopherol ferulate, VC-PMG: magnecium-
Ascorbyl-2-phosphate.
ponses7,8. By using a triple-combination cream, combined
effects can be expected for the treatment of pigmentary
conditions.
The efficacy and safety of triple-combination creams have
been reported in several studies9. In one study, 26.1%
showed a complete clearing of melasma by week 810. In
another multicenter study, global evaluations revealed that
75% of patients showed “moderate or marked improve-
ment” or were “almost clear” or “clear” by week 8. These
results showed that a triple-combination cream is a rapidly
effective topical agent for the treatment of melasma11.
However, in one study, 57% of patients experienced at
least one treatment-related adverse reaction, although it
was claimed to be mild and transient in nature12. In parti-
cular, post-inflammatory hyperpigmentation should be
expected in darker-skinned patients13. A multicenter study
was also performed to compare the efficacy and safety of a
triple-combination cream in Asian patients. The results

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showed that a triple-combination cream was also effective
in Asian patients with tolerable adverse reactions. Although
most adverse events were mild, almost 50% of patients
reported the incidence of related adverse events14. Thus, it
should be noted that triple-combination creams are
effective for melasma, but can have a high frequency of
adverse reactions.
CLASSIFICATION OF TOPICAL AGENTS FOR
PIGMENTARY CONDITIONS
At present, the most effective hypopigmenting agents are
tyrosinase inhibitors. However, melanogenesis can be
controlled by regulating (i) the transcription and activity of
enzymes such as tyrosinase, tyrosinase-related protein-1
(TRP-1), tyrosinase-related protein-2, and/or peroxidase;
(ii) the uptake and distribution of melanosome in keratino-
cytes and (iii) melanin and melanosome degradation and
turnover of “pigmented” keratinocytes5. However, it is
very clear that hypopigmenting agents can work by diffe-
rent combined mechanisms. Furthermore, melanogenesis
is controlled by additional factors via keratinocytes, fibro-
blasts and also by local and/or systemic conditions (Fig. 1).
Regulation of enzymes
1) Regulation of transcription and maturation of tyrosi-
nase
Transcription of genes encoding tyrosinase and TRP-1 is
under the control of the microphthalmia transcription
factor (MITF)15. Mitf is a critical transcription factor for
both melanocyte proliferation and melanogenesis. As Mitf
is regulated by the Wnt signaling pathway as well as
cAMP, and by both p38 signaling and the MAP kinase
pathway, any agents that can potentially regulate these
signaling pathways will also affect Mitf and melanogene-
sis. Although it is not clinically available, sustained extra-
cellular signal regulated kinase (ERK) activation by sphin-
gosine-1-phosphate (S1P) can lead to MITF phosphory-
lation and degradation, which in turn are responsible for
decreased melanin synthesis16. Transforming growth factor
(TGF-β1) also plays an inhibitory role in melanogenesis.
TGF-β1 induced a significant delay in ERK activation and
ERK-induced down-regulation of Mitf17. Furthermore,
lysophosphatidic acid and C2 ceramides are able to
induce Mitf degradation or decrease Mitf expression18-20.
These are examples that inhibit melanogenesis by trans-
criptional regulation of the tyrosinase gene. Tyrosinase is a
glycosylated protein. Therefore, glucosamine or tunicamy-
cin, which are specific inhibitors of lipid carrier-depen-
dent glycosylation, can induce hypopigmentation21. In
addition, calcium D-pantetheine-S-sulphonate (PaSSO3Ca)
causes an inhibition of melanogenic enzymes possibly
through the alteration of tyrosinase and TRP-1 glycosy-
lation without affecting their expression22.
2) Inhibition of tyrosinase activity
There are several tyrosinase inhibitors that have been used
to produce hypopigmenting topical agents or cosmetics.
Arbutin, a naturally occurring HQ beta-D-gluconopyrano-
side is commonly used23. Arbutin decreases tyrosinase
activity without affecting mRNA expression and inhibits
5,6-dihydroxyindole-2-carboxylic acid (DHICA) polyme-
rase activity24. Kojic acid (5-hydroxy-2-hydroxymethyl-4H-
pyran-4-one) was also a commonly used antibiotic agent
produced by species of Asperigillus and Penicillum25.
Among these, 4-n-butylresorcinol has been characterized
as a strong tyrosinase inhibitor26. We compared the
hypopigmenting effects of HQ and 4-n-butyl resorcinol
and showed that HQ (100μM) and 4-n-butyl resorcinol
(10μM) had similar tyrosinase inhibition activities. These
finding suggests that 4-n-butyl resorcinol is a more potent
tyrosinase inhibitor than HQ. Thus, the hypopigmenting
effect can increase if the concentration of 4-n-butyl resor-
cinol is increased. However, a cautious increase will be
necessary to ensure both efficacy and safety. Positive
hypopigmenting effects of 4-n-butyl resorcinol have pre-
viously been demonstrated27.
Generally, phenolic compounds are known as hypopig-
menting agents because of their ability to serve as alter-
native substrates for tyrosinase28.
3) Post-transcriptional control of tyrosinase
There are examples of agents that inhibit melanogenesis
by the increased degradation of tyrosinase proteins. Unsa-
turated linoleic acid decreases tyrosinase activity, whereas
saturated palmitic or stearic acids increases tyrosinase
activity. Also, topical application of linolenic, linoleic and
oleic acids produce a hypopigmenting effect on guinea
pig skin stimulated with UV light29. It is reported that
linoleic acid decreases the amount of tyrosinase through
increased tyrosinase ubiquitination and degradation by the
proteasome30,31. In addition, other agents like phospholipase
D2 decrease melanogenesis through the same ubiquitin-
mediated degradation of tyrosinase32.
Inhibition of melanosome transfer
Melanosomes are specialized organelles in which melanin
is synthesized and deposited. The addition of TGF-β1 to
cultured melanocytes produced less pigmented melano-
somes even when the cells were concomitantly treated
with αMSH to increase their fully melanized melano-

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somes33. It is also reported that ERK activation by S1P can
lead to hypopigmentation16. Interestingly, decreased mela-
nization of melanosomes was also found in S1P treated
melanocytes (unpublished data). Moreover, S1P-treated
cells showed undifferentiated early stage melanosomes,
whereas control cells showed internal fibrils and dense
pigments in melanosomes. These findings suggest that inhi-
bition of melanosome formation can be a good strategy to
control melanogenesis. However, there are no agents that
are currently available to meet this requirement.
Melanosome formation is an important step in melanog-
nesis and melanosomes need to be transferred to keratino-
cytes from melanocytes for the completion of this step.
Thus, the inhibition of melanosome transfer can produce a
hypopigmenting effect. Theoretically, the inhibition of
serine protease can result in an impaired activation of
protease-activated receptor 2 on the keratinocyte leading
to the accumulation of melanosomes within the melano-
cyte34. Clinically, niacinamide (Vitamin B3), which is
commonly used to manufacture cosmetics, has been
found to inhibit melanosome transfer to keratinocytes both
in vitro and in vivo35.
Additional mechanisms
1) Regulation of melanocytes environment
Endothelin 1 (ET-1), which is produced by keratinocytes
after exposure to inflammatory stimuli or UV exposure,
stimulates melanogenesis. ET-1 has strong stimulatory
effects both on DNA synthesis and melanization in human
melanocytes. Thus, topical application of M. chamomilla
extract inhibits ultraviolet B (UVB)-induced pigmentation
by inhibiting ET-1 effects36.
Clinically, it is well-known that topical corticosteroids
have strong anti-inflammatory effects. They have been
used for the treatment of melasma to decrease irritation
caused by hypo-pigmenting agents37, and work by the
suppression of cytokines through the inhibition of nuclear
factor kappa B (NF-κB) activation. Topical steroids can be
effective by the suppression of cytokines such as
endothelin-1 and GM-CSF, which mediate UV-induced
pigmentation7,8.
There are several ingredients with anti-inflammatory
activity. Glabridin, the main component of hydrophobic
fraction of licorice extracts decreases tyrosinase activity in
B16 melanoma cells and inhibits UVB-induced skin pig-
mentation as well as erythema. The capability to inhibit
cyclooxygenase activity and superoxide anion production
implies that this anti-inflammatory effect requires an inter-
ference with the arachidonic acid cascade. Consequently,
protection against oxidative stress plays a key role in
controlling melanogenesis38.
2) Antioxidant agents
In general, antioxidants exhibit hypopigmenting effects by
interacting with o-quinones, thus avoiding the oxidative
polymerization of melanin intermediates, or with copper
at the active site of tyrosinase. In addition, antioxidant
agents can regulate the signaling process by scavenging
ROS in the skin39. For example, ascorbic acid can interfere
with melanization by interaction with copper ions at
tyrosinase and reduction of dopaquinone and DHICA
oxidation40,41. α-Tocopherol and its derivatives can also
regulate melanogenesis. The antioxidant property affects
the lipid peroxidation of membranes and increases the
intracellular glutathione content42. 6-Hydroxy-3,4-dihydro-
cumarins, another novel type of antioxidant, have an anti-
melanogenic activity in cultured normal human melano-
cytes at non-cytotoxic concentrations without interfering
with tyrosinase activity43. The acceleration of glutathione
synthesis and the inhibition of tyrosinase transfer may be
the mechanism of action44. α-lipoic acid, a disulfide
derivative of octanoic acid, has been reported to prevent
UV-induced oxidative damage, mainly through the down-
modulation of NF-κB activation. In addition, this agent is
known to inhibit tyrosinase activity by possibly chelating
the copper ions45. Peroxidase is involved in the polymeri-
zation of melanogenic intermediates46. Based on this, the
inhibition of peroxidase can decrease melanogenesis by
reducing the polymerization of eumelanin47. Methima-
zole, an antithyroid agent belonging to the thionamide
group, shows inhibitory action towards tyrosinase and
peroxidase48. Mild to moderate inhibition of melanization
can be expected with morphological changes of melano-
cytes in animal models.
3) Combination of multifunction hypopigmenting agents
Recently, we reported that terrein, a bioactive fungal
metabolite isolated from a Penicillium species, reduces
melanin synthesis by reducing tyrosinase production via
ERK activation, and that this is followed by MITF downre-
gulation49. Interestingly, we also found that terrein decrea-
ses melanogenesis through ubiquitin-dependent proteaso-
mal degradation as well as decreased expression of its
mRNA50. Thus, terrein can be an example of a hypopig-
menting agent that inhibits melanogenesis by dual action
including the down-regulation of transcription and up-
regulation of degradation.
In contrast to terrein with multifunction, the combined use
of two agents with different action mechanisms can be
additive in terms of total effects. As already described,
4-n-butylresorcinol did not induce ERK or Akt activation,

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Topical Hypopigmenting Agents for Pigmentary Disorders and Their Mechanisms of Action
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5
or MITF degradation, and also had no effect on cAMP
response element binding protein phosphorylation, which
stimulates MITF expression26. However, 4-n-butylresor-
cinol showed an additive effect in combination with hino-
kitiol, which reduces MITF expression. Thus, the combina-
tion of these two agents with different action mechanisms
can be another strategy to increase the efficacy of these
agents.
CONCLUSION
Recently, melanocyte biology has made remarkable pro-
gress. However, the pathogenic mechanisms underlying
acquired hyperpigmentation have not been completely
understood. Even though these mechanisms need to be
explored further, our present understanding has improved
greatly, which has contributed to the enhancement of
diagnosis and treatment of pigmentary conditions. In
particular, the finding that the dermal microenvironment
can affect epidermal pigmentation through dermal dege-
neration or vascular dilatation has had a significant in-
fluence. These findings suggest that environmental effects
on melanogenesis are very important. In addition, the
combined use of multiple agents with different actions can
show additive effects. As already described, 4-n-butylre-
sorcinol is a strong tyrosinase inhibitor, whereas a signal
regulator such as terrain affects pigmentation through
ERK-induced MITF degradation. Thus, the combination of
multiple agents with different mechanisms of action can
be another strategy to increase the efficacy of these agents.
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