PIGMENT CELL RES 16: 101–110.

2003 Copyright
Blackwell Munksgaard 2003

Printed in UK—all rights reserved ISSN 0893-5785

Review: Innovative Technology

Chemical and Instrumental Approaches to Treat Hyperpigmentation

STEFANIA BRIGANTI, EMANUELA CAMERA and MAURO PICARDO

San Gallicano Dermatological Institute, Via Elio Chianesi, Rome, Italy

*Address reprint requests to Mauro Picardo, San Gallicano Dermatological Institute, Via Elio Chianesi 52, 00144 Rome, Italy.
E-mail: picardo@ifo.it
Received 23 December 2002; in final form 16 January 2003

Many modalities of treatment for acquired skin hyperpigmen-
tation are available including chemical agents or physical
therapies, but none are completely satisfactory. Depigmenting
compounds should act selectively on hyperactivated melano-
cytes, without short- or long-term side-effects, and induce a
permanent removal of undesired pigment. Since 1961 hydro-
quinone, a tyrosinase inhibitor, has been introduced and its
therapeutic efficacy demonstrated, and other whitening agents
specifically acting on tyrosinase by different mechanisms have
been proposed. Compounds with depigmenting activity are now
numerous and the classification of molecules, based on their
mechanism of action, has become difficult. Systematic studies
to assess both the efficacy and the safety of such molecules are
necessary. Moreover, the evidence that bleaching compounds
are fairly ineffective on dermal accumulation of melanin has
prompted investigations on the effectiveness of physical ther-
apies, such as lasers. This review which describes the different
approaches to obtain depigmentation, suggests a classification
of whitening molecules on the basis of the mechanism by which
they interfere with melanogenesis, and confirms the necessity to
apply standardized protocols to evaluate depigmenting treat-
ments.
Key words: Melanin, Melanocyte, Melanogenesis, Hyperpig-
mentation, Depigmenting agents, Classification, Tyrosinase,
TRP-1, ROS, Peroxidase, Melanosome transfer, Laser

INTRODUCTION
Increased production and accumulation of melanins char-
acterize a large number of skin diseases, which include
acquired hyperpigmentation, such as melasma, postinflam-
matory melanoderma, solar lentigo, etc. (1, 2). Epidermal
and dermal hyperpigmentation can be dependent on either
increased numbers of melanocytes or activity of melano-
genic enzymes (3). Ultraviolet light, chronic inflammation,
and rubbing of the skin as well as abnormal a-melanocyte
stimulating hormone (a-MSH) release, are triggering
factors for these disorders (4, 5). As a result of their
prevalent localization in photo-exposed areas, acquired
hyperpigmentation have psychosocial and cosmetic rele-
vance, and many efforts have been devoted to screening
recognized and putative depigmenting agents. Moreover,
as bleaching compounds are fairly ineffective on dermal
accumulation of melanin, physical therapies, such as
lasers, have been proposed and are currently under
investigation.
This review reports different approaches to achieve depig-
mentation, classifies the active molecules on the basis of their
mechanisms of interference with melanogenesis and focuses
on innovative drugs.
DEPIGMENTING AGENTS
The ideal depigmenting compound should have a potent,
rapid and selective bleaching effect on hyperactivated mel-
anocytes, carry no short- or long-term side-effects and lead to
a permanent removal of undesired pigment, acting at one or
more steps of the pigmentation process.

Abbreviations – a-MSH, a-melanocyte stimulating hormone; TRP-1, tyrosinase related protein-1; TRP-2, tyrosinase related protein-2; MITF,
microphthalmia transcription factor; ERK, extracellular signal-regulated kinase; AKT, serine-threonine kinase; PKB, protein kinase-B; PKC, protein
kinase C; ATRA, tretinoin, all-trans retinoic acid; l-DOPA, l-3,4-dihydroxyphenylalanine; HQ, hydroquinone, dihydroxybenzene; ROS, reactive
oxygen species; 4-SCAP, 4-S-cystaminylphenol; DHI, 5,6-dihydroxyindole; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; NF-kB, nuclear factor
kappa B; MG, methyl ester of gentisic acid, 2,5-dihidroxybenzoic acid; MBEH, hydroquinone monobenzyl ether; EA, ellagic acid; AZA, azelaic acid;
AsA , ascorbic acid; VC-PMG , magnesium-l-ascorbyl-2-phosphate; a-Toc, a-tocopherol; a-Toc-F, a-tocopherol ferulate; PAR-2, protease-activated
receptor 2; STI, soybean trypsin inhibitor; BBI, Bowman-Birk protease inhibitor; ET-1, endothelin)1; ECE, endothelin-1 converting enzyme.

Pigment Cell Res. 16, 2003 101

 Fig. 1. Schematic illustration of the possible
approaches to interfere with melanogenesis
pathway. Tyr, tyrosinase; M, melanosomes;
ROS, reactive oxygen species.

Depigmentation can be achieved by regulating (i) the
transcription and activity of tyrosinase, tyrosinase related
protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2),
and/or peroxidase; (ii) the uptake and distribution of
melanosomes in recipient keratinocytes and (iii) melanin
and melanosome degradation and turnover of ÔpigmentedÕ
keratinocytes (Fig. 1, Table 1).
melanocytes (10) whereas it enhances the pigmentation of low
melanized (S91 murine) melanoma cells, and decreases that of
highly pigmented normal human melanocytes after UV
Table 1. Classification of depigmenting agents. Compounds have been
divided in categories on the basis of the reported mechanisms of
interference with melanin synthesis and deposition

Before melanin synthesis

REGULATION OF MELANOGENIC ENZYMES Tyrosinase transcription
C2-ceramide Tretinoin
As a result of the key role played by tyrosinase in melanin Tyrosinase glycosylation
PaSSO3Ca
biosynthesis, most whitening agents act specifically to reduce
the function of this enzyme by means of several mechanisms: During melanin synthesis
(i) interference with its transcription and/or glycosylation, Tyrosinase inhibition
Hydroquinone Kojic acid
(ii) inhibition by different modalities, (iii) reduction of 4-hydroxy-anisole Methyl Gentisate
by-products and (iv) post-transcriptional control. 4-S-CAP & derivatives Ellagic Acid
Arbutin Resveratrol
Aloesin Oxyresveratrol
Transcriptional Control of Tyrosinase Expression Azelaic acid
Peroxidase inhibition
Transcription of genes encoding tyrosinase and TRP-1 is Methimazole Phenols/catechols
under transcriptional control of the microphthalmia Product reduction and ROS scavengers
Ascorbic acid a-Toc
transcription factor (MITF) (6–8). Substances able to inhibit Ascorbic Acid Palmitate D, L-a TF
MITF expression and activity, as well as the extracellular VC-PMG Hydrocumarins
signal-regulated kinase (ERK) and serine-threonine kinase Thioctic acid
(AKT)/protein kinase B (PKB) pathways, could represent After melanin synthesis
depigmenting agents. Tyrosinase degradation
Linoleic acid a-Linolenic acid
Among these, tretinoin (all-trans retinoic acid; ATRA), Inhibition of melanosome transfer
acting on retinoid-activated transcription factors, interferes Serine protease inhibitors Niacinamide
with melanocyte development and melanogenesis. ATRA has Lecthins and Neoglycoproteins RW-50353
bimodal effects on melanocytes: it stimulates the differenti- Soybean/milk extracts
Skin turnover acceleration
ation of melanocyte precursors, inducing transcription of Lactic acid Retinoic acid
tyrosinase by protein kinase C (PKC) activation and MITF Glycolic acid Linoleic acid
expression, and removes differentiated melanocytes by pro- Liquiritin
moting apoptosis via caspase-3 pathway and bcl-2 down-
PaSSO3Ca, Calcium D-pantetheine-S-sulphonate; 4-SCAP, 4-S-cyst-
modulation (9). ATRA does not inhibit melanogenesis in skin aminylphenol; VC-PMG, magnesium-L-ascorbyl-2-phosphate; a-Toc,
equivalents or monolayer cultures of murine and human a-tocopherol; a-Toc-F, a-Tocopherol ferulate.

102 Pigment Cell Res. 16, 2003

exposure (11). Topical ATRA stimulates tyrosinase activity
only in individuals with low skin type and (12) improves the
irregular hyperpigmentation associated with photo-aging or
inflammation (13, 14). Agents able to decrease MITF expres-
sion include C2-ceramide, which inhibits cell proliferation and
melanogenesis in a mouse melanocyte cell line, probably
through an initial inhibition and a late activation of AKT/
PKB and ERK pathways (15, 16).
Glycosylation and Maturation of Melanogenic Enzymes
The major post-translational modification of the melano-
genic enzymes consists of glycosylation at asparagine resi-
dues. The inhibition of N-glycosylation by a variety of agents
results in the reduction of melanosome maturation, and of
melanosomal enzyme activities (17, 18). The modification of
the glycosylation pattern makes the protein inactive or less
active affecting the intracellular transport or deposition
within melanosomes. Glucosamine or tunicamycin, specific
inhibitors of lipid carrier-dependent glycosylation of protein,
have been found to induce, in melanoma cells, a marked loss
of pigmentation associated with ultrastructural alterations
of melanogenic compartments (19). Calcium d-pantetheine-
S-sulphonate (PaSSO3Ca), probably through the alteration
of tyrosinase and TRP-1 glycosylation, causes an inhibition
of melanogenic enzymes, without modifying their expression,
and produces a reversible loss of pigmentation in normal
human melanocytes and in melanoma cell lines (20).
Control of Tyrosinase Activity
The classification of tyrosinase inhibitors (Table 2) is difficult
because of the capability of several compounds to work in
different ways, interacting with both catalytic and regulatory

Table 2. Chemical structures of modulators of melanogenic enzyme activity. Most of the compounds show chemical analogy with l-tyrosinase the
natural substrate of tyrosinase. In the table adjunctive effects, which could contribute to the depigmenting action have been reported

a
Kinetics study on mushroom tyrosinase, bKinetics study on melanocyte or melanoma cell cultures.

Pigment Cell Res. 16, 2003 103

sites of the enzyme or being metabolized to a product that, in
turn, can act as either a non-competitive or a competitive
inhibitor. Tyrosine hydroxylation is the rate limiting step for
melanogenesis, but requires l-3,4-dihydroxyphenylalanine
(l-DOPA), the substrate of tyrosinase’s second activity, as
a cofactor. Thus, tyrosinase activity cannot be easily exam-
ined through kinetic analyses. Moreover controversial results
were obtained when purified mushroom tyrosinase or cell
lysates of melanoma cells were employed. Most of the
inhibitors are phenol/catechol derivatives, structurally sim-
ilar to tyrosine or DOPA, which act as alternative substrates
of tyrosinase without producing pigment (21, 22).
Alternative Phenolic Substrates
The most popular depigmenting agent is hydroquinone
(dihydroxybenzene; HQ) introduced for clinical use since
1961. Several trials have demonstrated its therapeutic efficacy
alone or in association with other compounds (23–28). HQ
seems to exert its effects mainly in melanocytes with active
tyrosinase activity, such as epidermal hyperactivated mel-
anocytes (29) and has been considered as a reference
standard in evaluating depigmenting agents (30, 31). The
oxidation products are quinones and reactive oxygen species
(ROS), which lead to an oxidative damage of membrane
lipids and proteins, including tyrosinase, and depletion of
glutathione contributes to the lightening action (32). HQ may
interfere with pigmentation even through: (i) the covalent
binding to histidine or interaction with coppers at the active
site of tyrosinase (33), (ii) the inhibition of DNA and RNA
synthesis and (iii) the alteration of melanosome formation
and melanization extent (34). However, because of the
hazard of long-term treatments (35), the use of hydroquinone
in cosmetics has been banned by the European Committee
(24th Dir. 2000/6/EC) and formulations are available only by
prescription of physicians and dermatologists. HQ mono-
benzyl ether (MBEH) is metabolized to reactive free radicals
inside the cells, causing permanent depigmentation even at
sites distant from those of application. The clinical use is
limited to obtain a generalized depigmentation in patients
with diffuse vitiligo (36). Several other phenolic compounds
have been screened as depigmenting agents and studies have
been performed on the relationship between chemical struc-
ture and tyrosinase inhibitory activity. The presence of
hydroxylic group and of an electron donator group in
position para has been suggested as a fundamental require-
ment for an effective action as alternative substrate of
tyrosinase. In fact, in para-hydroxylated monophenols,
methylation of one hydroxyl favours nucleophilic attack at
the active site enhancing the catalytic efficiency (37). The fact
that 4-hydroxyanisole (para-hydroxy-methoxybenzene),
oxidized by tyrosinase (Km ¼ 62–80 lM), exhibits strong
melanocytotoxicity and clinical effects have been demonstra-
ted in the treatment of solar lentiges and related hyperpig-
mented lesions, in combination with tretinoin (38–40).
4-Tertiary butylphenol has been reported to induce in human
melanocytes a competitive inhibition of both tyrosine
hydroxylase and DOPA oxidase properties of tyrosinase
without cytotoxicity (41). The presence of thio-ether side
chains in para position on the phenolic ring enhances
104
tyrosinase oxidation rate and cytotoxic effects (42). 4-S-
cystaminylphenol (4-SCAP) and its N-acetylated derivatives
have both depigmenting and anti-melanoma effects. Topical
treatment in Yucatan pigs induces a stable but reversible
depigmentation with slight irritant effects when compared
with HQ (43, 44). The activity seems to be related to: (i) a
marked decrease of the functioning melanocytes and melan-
ized melanosome number, (ii) a reduced transfer of melano-
somes to keratinocytes, and (iii) a selective degeneration of
melanocytes and deposition of melanin–like material in the
Golgi apparatus, where tyrosinase is located (45). Catechols
(o-diphenols) bearing electron donor groups in position para
were found to be more effective in inhibiting tyrosinase and
inducing melanocytotoxic effects than mono-phenolic deriv-
atives (21, 46). However, when a derivative with methyl
groups in position ortho and meta, such as 2,5,6-trimethyl-p-
hydroxy-methoxybenzene, is used as substrate of mushroom
tyrosinase, the oxidation rate is low and the initial acceler-
ation (lag period) is not observed (47). Arbutin, a naturally
occurring HQ b-d-gluconopyranoside, which is depigmented
at non-cytotoxic concentrations (48), acts similar to HQ
(IC50 ¼ 17 mM) (49). In both normal human melanocytes
and melanoma cells, arbutin induces a decrease of tyrosinase
activity without affecting messenger RNA (mRNA) expres-
sion, inhibits the 5,6-dihydroxyindole-2-carboxylic acid
(DHICA) polymerase activity (pmel17/silver protein) and
exerts an inhibitory effect on melanosome maturation (50).
An interference with the uptake of tyrosine into melanocytes
has also been postulated (51). However, despite encouraging
initial results (52) other authors have not confirmed the
activity on intact melanocytes and in clinical trial (30). Kojic
acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) is an
antibiotic produced by many species of Asperigillus and
Penicillum. The depigmenting action is attributed to the
chelating ability (53), even if an interference with different
steps of melanin synthesis (54) and inhibition of nuclear
factor-kappa B (NF-jB) activation in keratinocytes, con-
trasting with the hyperpigmentation associated with inflam-
matory response, have been described (55). Clinical trials
have reported a skin lightening effect of kojic acid (56). The
methyl ester of gentisic acid (2,5-dihidroxybenzoic acid;
MG), a natural product of Gentiana’s root, inhibits melanin
synthesis in murine melanocytes at concentrations unable to
induce cytotoxicity (57). MG can act as pro-drug, inhibiting
tyrosinase following the intracellular release of HQ, even if
kinetic studies have demonstrated a direct effect on the
enzyme, possibly through the binding of the copper ions
(30). Clinical trials have demonstrated the efficacy of the
compound (58). Aloesin, a natural hydroxychromone deriv-
ative isolated from Aloe Vera, inhibits mushroom (59) and
melanoma tyrosinase at non-cytotoxic concentration (60, 61)
probably acting as a competitive inhibitor on DOPA
oxidation and as an non-competitive on tyrosine hydroxy-
lase activity. In vivo, aloesin and arbutin co-treatment
inhibits UV-induced melanogenesis in a synergistic manner
(62).
Most hydroxystilbene compounds like resveratrol, natur-
ally contained in herbal medicines, have potent inhibitory
effects on mushroom tyrosinase. Resveratrol, one of the
ingredients of red wine, possesses several biological properties,
Pigment Cell Res. 16, 2003
such as free radical scavenging, anti-inflammatory and
anticancer activities (63, 64). A dose-dependent inhibition
of l-tyrosine oxidation by tyrosinase, without affecting the
protein expression and synthesis, has been described in B-16
murine melanoma. Resveratrol is probably metabolized by
tyrosinase leading to the formation of an o-hydroxy deriv-
ative, which is a competitive inhibitor of the enzyme (65).
Oxyresveratrol, extracted from Morus alba, is a stronger
inhibitor (IC50 ¼ 1 lM) on mushroom DOPA oxidase
activity than resveratrol (IC50 ¼ 54.6 lM) (66), suggesting
that the number and the position of hydroxyl substituents
have an important role in determining the activity. Ellagic
acid (EA), a polyphenol widely distributed in plants, is
capable of preventing pigmentation caused by sunburn (67).
EA inhibits tyrosinase non-competitively in a dose-depend-
ent manner, through its capacity to chelate copper, even if
other mechanisms, such as a scavenger effect have been
suggested. Interestingly, in brownish guinea-pigs, EA
induced a reversible inhibition of melanin synthesis only in
UV-activated melanocytes.
Azelaic Acid
Compounds able to bind either amino or carboxyl groups
may block the access of tyrosine to the active site, behaving
as competitive inhibitors. Among these, azelaic acid (AZA),
a naturally occurring 9-carbon dicarboxylic acid compound
isolated from cultures of Pityrosporum Ovale, is included.
AZA inhibits tyrosinase activity in vitro (Ki ¼ 2.73 ·
10)3 M) (68) and may also interfere with DNA synthesis
and mitochondria activity in hyperactive and abnormal
melanocytes (69). AZA has been used to treat melasma and
postinflammatory hyperpigmentation (23) and to arrest the
progression of lentigo maligna to melanoma (70). Topical
azelaic acid (15–20%) has been reported to be efficacious in
improving melasma (23, 71), even if not all the authors are
in agreement with these therapeutic effects.
Peroxidase Inhibitors
The involvement of peroxidase in the polymerization of
melanogenic intermediates has been suggested by the high
efficiency of peroxidase in the oxidation of 5,6-dihydroxyin-
dole (DHI) with the generation of hydrogen peroxide (H2O2)
as a by-product (72, 73). Intracellular H2O2, generated after
UV irradiation or in response to cytokines, such as tumor
necrosis factor-a (TNF-a) or transforming growth factor-b
(TGF-b) (74–76), can induce a transient reduction of
tyrosinase and other melanogenic protein activities, through
the down-regulation of the MITF transcription factor (77).
The inhibition of peroxidase, however, results in depigmen-
tation, reducing the polymerization rate of eumelanin in
different experimental models (78). Moreover, several mel-
anocytotoxic phenol/catechol derivatives, are good sub-
strates of either tyrosinase or peroxidase (21), and the
inhibition of peroxidase could play a role in their mechanism
of action as well as of depigmenting agents, such as
glucocorticoids or ascorbic acid, that do not exhibit any
effects against the melanogenic enzymes (79, 80). Methimaz-
ole, an antithyroid agent belonging to the thionamide group,
Pigment Cell Res. 16, 2003
exerts inhibitory action towards both mushroom tyrosinase
and peroxidase (81) and, in brown guinea-pigs, induces mild
to moderate inhibition of melanization, with morphologic
changes of melanocytes.
Redox Agents and ROS Scavengers
Compounds with redox properties can have depigmenting
effects by interacting with o-quinones, thus avoiding the
oxidative polymerization of melanin intermediates, or with
the copper at the active site. Moreover, redox agents, by
scavenging ROS generated in the skin following UV expo-
sure, can inhibit possible second messengers which is able to
stimulate epidermal melanogenesis either directly or indi-
rectly (82).
Ascorbic acid (AsA) interferes with the different steps of
melanization, by interacting with copper ions at the
tyrosinase active site (83) and reducing dopaquinone and
by blocking DHICA oxidation (84). However, AsA is
highly instable, being quickly oxidized and decomposed in
aqueous solution and, because of its prevalent hydrophilic
nature, has a low degree of penetration into the skin.
Different esters, such as magnesium-l-ascorbyl-2-phosphate
(VG-PMG), stable in water and in alkaline medium and
absorbed percutaneously, have been used. These com-
pounds are able to induce a lightening effect in normal
and in hyperactive melanocytes (85). a-Tocopherol (a-Toc)
derivatives inhibit tyrosinase in vitro (86) and melanogenesis
in epidermal melanocytes (87). The antioxidant properties
of a-Toc, which interferes with lipid peroxidation of
melanocyte membranes and increases the intracellular
glutathione content, could explain its depigmenting effect
(88, 89). Co-treatment with AsA, which regenerates a-Toc
from its radical form, results in more effective and
longlasting antioxidant response. Topical application of
a-Toc and AsA, in vivo, decreases the tanning response
inhibiting the UV-induced melanogenesis and proliferation
of melanocytes (90). An alternative compound is a-toco-
pherol ferulate (a-Toc-F), a derivative of a-Toc linked by
an ester bond to ferulic acid, an antioxidant, which provides
stabilization to a-Toc, similar to AsA. The a-Toc-F inhibits
melanin synthesis in normal human melanocytes without
interfering with the tyrosinase pathway (87).
6-Hydroxy-3,4-dihydrocumarins, novel antioxidants with
a-Toc like chemical structures (91) have been recently
reported to have an anti-melanogenic activity in cultured
normal human melanocytes at non-cytotoxic concentration
without interfering with tyrosinase activity. The acceleration
of the biosynthesis of glutathione within melanocytes,
leading to the inhibition of tyrosinase transfer to premelan-
osomes, could be the mechanism of action (92). Thioctic acid
(a-lipoic acid), a disulfide derivative of octanoic acid, exhibits
several biological effects, which include the quenching of
ROS, metal chelation, interaction and regeneration of other
antioxidants, redox regulation of protein thiol groups, and
effects on gene expression and apoptosis (93). Thioctic acid
has been reported to prevent UV-induced photo-oxidative
damage, mainly through the down-modulation of NF-jB
activation and to inhibit tyrosinase activity probably by
chelating the copper ions (94).
105

Post-Transcriptional Control of Tyrosinase
Substances which are able to regulate melanin synthesis
without affecting the expression of melanogenic proteins are
likely to exert a post-transcriptional control of melanogenic
enzymes. Unsaturated fatty acids, such as oleic acid (C18:1),
linoleic acid (C18:2), or a-linolenic acid (C18:3), suppress
pigmentation, in vitro, correlated with the degree of unsat-
uration, whereas saturated fatty acids, such as palmitic acid,
increase the rate of melanogenesis (95). Linoleic acid in vivo
showed the greatest lightening effect in UVB induced
pigmentation, without toxic effects on melanocytes (95).
The evidence that no changes in TRP-1 or TRP-2 proteins
have been found, suggests that tyrosinase is selectively
targeted by fatty acids, which seem to act on the degradation
of the enzyme during the physiologic proteasome-dependent
mechanism (96), leading to the alteration of tyrosinase
protein content in hyperactive melanocytes. Linoleic acid
accelerates the process whereas palmitic acid works in an
antagonistic manner mimicking protease inhibitors (97).
Inhibition Melanosome Transfer
Several studies have focused on the identification of
regulatory factors of the melanosome movement in dend-
rites and of the interaction between keratinocytes and
melanocytes during the transfer process (98, 99). The
activation of protease-activated receptor 2 (PAR-2), a seven
trans-membrane G-protein coupled receptor, which is
expressed in keratinocytes and not in melanocytes, was
found to activate keratinocyte phagocytosis, enhancing the
pigment transfer (100). Inhibition of PAR-2 cleavage by
serine protease inhibitors, such as RWJ-50353, completely
avoids the UVB-induced pigmentation of epidermis analogs
(101, 102). In dark skinned Yucatan microswine, topical
treatment with serine protease inhibitors, such as the
soybean trypsin inhibitor (STI) or Bowman–Birk protease
inhibitor (BBI) in a liposomal delivery system, exerts a
significant skin lightening effect without toxic effects on
melanocytes. Soybean milk extracts reduced the melanin
deposition within the swine epidermis and prevented UVB
induced pigmentation in vivo, similar to STI, without the
addition of any delivery vehicle (103). Even niacinamide,
the biologically active form of vitamin B3, inhibits melan-
ogenesis, both in vitro and in vivo, probably through the
suppression of melanosome transfer. Purified tyrosinase, in
fact, is not inhibited and pigmentation is reduced in co-
culture system or in reconstructed epidermis and not in
melanocyte monocultures (104).
Melanin Dispersion and Acceleration of Skin Turnover
The capacity of several compounds to disperse melanin
pigment and/or accelerate epidermal turnover can result in
skin lightening. Chemical substances used as exfoliantes,
such a-hydroxy acids, free fatty acids and retinoic acid,
stimulates cell renewal facilitating the removal of melanized
keratinocyte, leading to melanin pigment loss (10, 95).
Topical application has been shown to reduce the visibility
of age spots, without reducing their size or number (105), and
106
can be useful in the treatment of melasma (106). Liquiritin, a
flavonoid glycoside of liquorice, has been found to induce a
significant improvement of hyperpigmentation in patients
with bilateral and symmetrical idiopathic epidermal melasma
(107). The mechanisms proposed involve melanin dispersion
by means of the pyran ring of its flavonoidal nucleus, and
acceleration of epidermal renewal.
Inhibitors of Inflammation-Induced Melanogenic Response
Some mediators produced by keratinocytes after exposure to
pro-inflammatory stimuli or UV exposure, such as interleu-
kin-1a (IL-1a) and endothelin 1 (ET-1), are able to promote
melanogenesis. Therefore, anti-inflammatory compounds
could be useful for the prevention or treatment of postin-
flammation hyperpigmentation. ET-1 shows an unique
behavior in exerting stimulatory effects both on DNA
synthesis and melanization by human melanocytes (108).
Activation of epidermal ETs is determined by the enzymatic
cleavage of inactive prepolypeptides by an endopeptidase
termed ET converting enzyme (ECE), regulated by the pro-
inflammatory cytokine IL-1a (109). Topical application of
M. chamomilla extract inhibits UVB-induced pigmentation,
by avoiding ET-1-induced DNA synthesis, but not the IL-a-
induced ET-1 production and tyrosinase activation (108).
Among anti-inflammatory drugs, topical corticosteroids
have been included in several clinical trials for the treatment
of melasma even to decrease the irritation caused by
depigmenting agents (110–112). The possible mechanism is
the suppression of cytokines through the inhibition of NF-kB
activation, which may explain their short-lived effect with a
transient loss of color. Glabridin, the main component of
hydrophobic fraction of liquorice extracts decreases tyrosin-
ase activity in B16 melanoma cells, without affecting DNA
synthesis, and, in guinea-pigs, inhibits UVB-induced skin
pigmentation, as well as erythema (113). The capability to
inhibit cyclooxygenase activity and superoxide anion pro-
duction suggests that the anti-inflammatory effect involves
an interference with the arachidonic acid cascade and that
protection against oxidative stress has a key role in modu-
lating melanogenesis. Variations in the chemical structure
modulate the effect, because melanogenesis inhibition is lost
by replacing both hydroxyl groups and reduced by substitu-
tion at the 4¢ position.
Laser Treatment of Pigmented Lesions
Therapeutic options, other than chemical agents, have been
proposed for hyperpigmentation, such as cryotherapy with
liquid nitrogen, laser surgery, chemical peeling and superfi-
cial dermoabrasion (116, 117). Since the first demonstration
(118), treatments of pigmented lesions with argon laser, CO2
laser, Nd:YAG laser or Q-switched ruby laser have been
reported (114). The theory of a selective photothermolysis
suggests that laser therapy would allow discriminating
destruction of pigment without injuring the surrounding
tissue (119). Selective melanin photothermolysis can be
obtained with any laser light having a wavelength in the
absorption spectrum of melanin and sufficient energy levels
to target melanosomes (120). Laser induces extreme heating
Pigment Cell Res. 16, 2003
of melanosomes with subsequent thermal expansion, local
vaporization and generation of acoustic waves that damage
the nucleus and eventually destroy the pigment-laden cells.
The released melanin is then removed through trans-epider-
mal elimination or phagocytosis by dermal macrophages. To
be effective and specific, wavelengths that avoid absorption
by other skin chromophores and penetrate to the desired
depth have to be used. Different laser wavelengths induce
similar melanosome alterations, but the threshold doses and
penetration are substantially different. Shorter wavelengths
(<600 nm) damage pigmented cells with lower energy
fluences, while longer wavelengths (>600 nm) penetrate
deeper into the skin but need more energy to induce
melanosome disruption. Thus, short wavelengths damage
only superficial pigmented lesions, leaving deeper structures
intact, while longer wavelengths can target pigmented lesions
in the dermis. Equally important is the pulse duration, which
should be shorter than the thermal relaxation time of
melanin. Clinically, the immediate effect of sub-microsecond
pulses in pigmented skin is tissue-whitening, which corres-
ponds to melanosome disruption and is an end-point during
treatment. There are four main short-pulsed pigment-select-
ive lasers in clinical use nowadays: the pigmented lesion
dye laser (PLDL) (510 nm, 300 ns), the Q-switched ruby
laser (694 nm, 24–40 ns), the Q-switched alexandrite laser
(755 nm, 50–100 ns), and the Q-switched Nd:YAG laser
(1064 nm, 5–10 ns), which can be frequency-doubled to emit
a green light at 532 nm of the same pulse duration.
Epidermal melasma responds to laser treatment similar to
the use of depigmenting agents or chemical peels, but the
dermal and mixed types are resistant to laser therapy.
However, several months after treatment with a Q-switched
Fig. 2. Possible multi-step protocol for the
evaluation of putative depigmenting com-
pounds.
Pigment Cell Res. 16, 2003
ruby laser, both epidermal repigmentation and an increased
number of dermal macrophages have been observed (121).
The poor response may be referred to the hyperactivation of
melanocytes and to the laser-mediated inflammatory
response, that leads to rapid reoccurrence or even darkening
of the treated areas. To improve the efficacy, the develop-
ment of a combination laser treatment has been proposed. A
pilot study has described the effectiveness of a combination
laser therapy (pulsed CO2 laser followed by Q-switched
alexandrite laser) in treatment of dermal-type melasma (122).
Given the epidermal trauma and worsening in pigmentation
induced by laser impact, it is not surprising that laser
treatment of postinflammatory hyperpigmentation has been
discouraging (123).
CONCLUSIONS
The knowledge of melanocyte biology and processes under-
lying melanin synthesis has made remarkable progresses over
the last years opening new paths in the pharmacologic
approach to the treatment of hyperpigmentation. At the
same time, the topic has become more complex and
the classification of the molecules more difficult. Moreover,
the pathogenetic mechanisms underlying acquired hyperpig-
mentation have not been completely clarified, and the
therapeutic approaches are focused on the outcome of the
process. Numerous are the candidate depigmenting agents
and, deeper studies and clinical trials are needed to assess
their safety. HQ is one of the most potent whitening agents
first discovered, but since its introduction some adverse
effects have been recognized. Recent molecules, even if very
promising, need extended follow-up and could disclose
107
undesired effects, not yet identified, or secondary effects,
which may accompany the main function. The diverse sites of
action lead to a difficult comparison of their efficacy. To
render the scenario more complex, different models and
methods have been used to assay the depigmenting activity.
From a methodological point of view, according to Virador
et al. (124) and Lei et al. (125) a multi-step process could be
covered (Fig. 2). Initial evaluation of depigmenting proper-
ties should be performed on purified tyrosinase and other
melanogenic enzymes including peroxidase, thereafter
employing melanocyte cultures, cytotoxicity and effects on
melanin synthesis, should be assayed. For the biological
evaluation, co-culture systems and reconstructed skin seem
to be reliable methods to screen the capability of new agents
to interfere with the pigmentary process, in particular
following triggering stimuli such as UV irradiation, or
exposure to a-MSH or proinflammatory mediators. Finally,
the in vivo activity of hypopigmenting formulation should be
assessed by using non-invasive techniques, such as remittance
spectrophotometry and UV light photography to obtain
comparable values (126).
Based on the review of the literature and on our clinical
experience, we can conclude that the identification of the ideal
depigmenting agent is difficult and, for a clinical use, a
formulation which include compounds acting at different steps
of pigmentation should be advantageous. In mixtures, the
possible synergistic effect of different compounds could allow
application of lower concentrations of each agent, possibly
reducing adverse effects and increasing the clinical results.
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