Pharmacology and Treatment
Received: October 16, 2001
Dermatology 2002;204:281–286
Assessment of Topical Hypopigmenting
Agents on Solar Lentigines of Asian
Women
J.F. Hermanns L. Petit C. Piérard-Franchimont P. Paquet G.E. Piérard
Department of Dermatopathology, University Medical Center Sart Tilman and Sauvenière, Liège, Belgium
Key Words
Melanin W Tyrosinase W Corneomelametry W Reflectance
spectrophotometry W Ultraviolet light reflection
Abstract
Background: So-called darkened age spots encompass
distinct pathological processes. The efficacy of topical
depigmenting agents is difficult to objectivate. Objective:
To assess the hypopigmenting effect of three cosmetic
formulations using objective biometrological methods.
Methods: 50 women of South-East Asian ancestry were
enrolled in this pilot study. They had solar lentigines
according to dermoscopic criteria. The lesions were
treated by topical hypopigmenting formulations. Prod-
ucts were applied twice daily for 2 or 3 months. Assess-
ments at 1-month intervals were made using narrow-
band reflectance spectrophotometry, image analysis of
video-recorded ultraviolet light reflection and photoden-
sitometry- and image-analysis-assisted corneomelame-
try. Results: A 20% azelaic acid formulation and another
one containing 5% ascorbyl glucosamine, 1% kojic acid
and ·-hydroxyacid esters appeared inefficacious on so-
lar lentigines. A stabilized soy extract showed a better
although modest lightening effect when assessed by cor-
© 2002 S. Karger AG, Basel
ABC 1018–8665/02/2044–0281$18.50/0
Fax + 41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
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Accepted: November 15, 2001
neomelametry. The subclinical or faint mottled skin re-
vealed by ultraviolet light examination better responded
(p ! 0.05) to treatments. Conclusion: Focal epidermal
hyperpigmentation is better controlled by topical whiten-
ing agents when the increase in melanin content reflects
a modest functional hyperactivity of melanocytes.
Copyright © 2002 S. Karger AG, Basel
Introduction
The innate melanin pigmentation of skin is modulated
during lifetime by a series of factors including specific
neuromediators, hormones and cytokines. Ultraviolet
light, chronic inflammation and rubbing of the skin are
the most important triggering factors. In youth, the var-
ious physiological stimulations of melanogenesis increase
evenly the eumelanin pigmentation or induce phaeomel-
anin-enriched freckles without any prominent change in
the melanocyte density. By contrast, intense and chronic
ultraviolet light exposures result in a mottled appearance
typical of photo-ageing and PUVA-induced changes [1–
6]. Indeed, the number, distribution and synthetic activi-
ty of melanocytes become uneven in such conditions, as
well as the capacity to transfer melanosomes to keratino-
Prof. G.E. Piérard, MD, PhD
Department of Dermatopathology
CHU Sart-Tilman
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B–4000 Liège (Belgium)
Tel. +32 4 366 2408, Fax +32 4 366 2976, E-Mail Gerald.Pierard@ulg.ac.be
Univ. of California San Diego
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cytes. As a result, focal excess in the eumelanin load can
lead to darkened age spots. Control of these hyperpigmen-
tations is becoming ever more important as the aged pop-
ulation continues to grow. In particular, Asian-type skin
is reputed to be particularly prone to such skin changes
[6, 7].
The objective assessment of pigmentary changes bene-
fits from a series of distinct sensitive methods [8, 9].
Among them, the narrow-band remittance spectropho-
tometry yields the melanin and erythema indices, assess-
ing the skin contents in melanin and haemoglobin, respec-
tively [8–13]. Any discrete mottled hyperpigmentation is
better revealed under long-wave ultraviolet light illumina-
tion [4, 5, 14–19]. This method increases the contrast
between the faint or almost invisible hyperpigmentation
and the surrounding skin. This is the result of the greater
reflection of ultraviolet wavelengths than visible light by
collagen, and their absorption by melanin [17]. Ultravio-
let light photography is normally used as a diagnostic tool.
It is less suited for measuring pigmentation [8] because
casual equipments generate shadows superimposed on
the melanin pigmentation. Video recording of small skin
areas is more satisfactory for computerized quantifica-
tions [16, 17].
Corneomelametry (after corneocyte, melanin, metry)
is another objective method designed to quantify the
melanin content present in the stratum corneum. For that
purpose, cyanoacrylate skin surface strippings (CSSS) are
collected, stained using argentaffin histochemistry and
submitted to quantitative photodensitometry and com-
puterized image analysis under the microscope.
Upregulation and downregulation of melanin synthe-
sis depend on multi-step enzymatic processes [20, 21].
The current strategies for developing new compounds
controlling the excess in eumelanin pigmentation target
several of these biological mechanisms. The main targets
include: (a) the levels of expression of tyrosinase and
of the 2 tyrosinase-related proteins TRP-1 and TRP-2;
(b) thiol conjugation (e.g. with glutathione or cysteine)
leading to the formation of phaeomelanins; (c) the ·-mela-
nocyte-stimulating hormone and its receptor which is
coupled to the adenylate cyclase/protein kinase A path-
way; (d) the product of the agouti locus; (e) the regulation
of melanosome formation and clumping; (f) the melano-
cyte dendritogenesis, and (g) the mechanisms regulating
the uptake and distribution of melanosomes in recipient
keratinocytes [18, 22, 23]. Any interference at these levels
decreases the amount of melanin in the epidermis and/or
contributes to the formation of various forms of melanins
with different light absorption characteristics.
282 Dermatology 2002;204:281–286
The aim of the present study was to assess the hypopig-
menting effect of three cosmetic formulations upon dark-
ened age spots present on the dorsal forearms and back of
the hands in Asian women.
Volunteers and Methods
Study 1
A total of 30 healthy women of South-East Asian ancestry were
enrolled in this study conducted during the winter season in Liège
after approval by the University Ethics Committee. They were aged
between 42 and 57 years and presented hyperpigmented macules on
the dorsal forearms and back of the hands. At least one of these
lesions on each limb had an area over 50 mm2. Dermoscopy [24, 25]
was used to select solar lentigines and to rule out any lesion corre-
sponding to seborrhoeic keratosis or malignant melanoma.
This open randomized trial was conducted for 2 months. Twice
daily, the volunteers applied a stabilized soy extract on the target
lesions and the surrounding skin on one limb, the symmetrical site on
the other limb serving as reference. Objective assessments of the skin
melanization were performed at entry in the study, and after 1 and 2
months of treatment. At each site, three complementary assessments
were performed using the Mexameter® (C + K Electronic, Cologne,
Germany), the Visioscan® (C + K Electronic) and corneomelametry,
respectively.
The Mexameter was used as previously described [8, 9] to derive
the melanin and erythema indices of the target solar lentigines on
each forearm. Three measures were taken on each site, and the
median value was recorded. Corneomelametry was assessed on the
same sites. For this purpose, CSSS were harvested from the target
solar lentigines and the surrounding normal-looking skin. They were
stained by Masson’s argentaffin method. The samples were placed
under a photomicroscope equipped with an internal photodensitom-
eter. The recorded values which increased with the staining intensity
were an estimate of the melanin load inside corneocytes. Microscopic
photographs of these CSSS were also taken at a !220 magnification
and digitalized in a 300 ! 300 pixel frame. Grey level discrimination
on a 0–10 scale was used to assess the pigment densities.
Visioscan recordings were made on 3 contiguous fields outside
the solar lentigines. Each of them measuring 1 ! 0.8 cm was digital-
ized in a 512 ! 400 pixel frame. Image analysis of the subclinical
melanin spots was performed as previously described [16, 17].
Study 2
A randomized double-blind study was performed in 20 other
Asian menopausal women, aged from 51 to 56 years and exhibiting
solar lentigines on the dorsum of the hands. Two commercial topical
products were tested comparatively. One formulation (Melanex duo,
Paraphar) contained 5% ascorbyl glucosamine, 1% kojic acid, vita-
min C and E, and ·-hydroxyacid esters. The other formulation (Ski-
noren, Schering) contained 20% azelaic acid. The volunteers applied
twice daily for 3 months one of the two formulations on a given hand,
the other product being applied on the other hand. The biometrologi-
cal assessments of skin pigmentation were identical to those used in
the first study.
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 Table 1. Biometrological assessments of the hypopigmenting effect of a stabilized soy extract

Evaluation Melanin index Corneomelametry Corneomelametry

time photodensitometry value melanin area
untreated treated untreated treated untreated treated

Baseline 491B34 504B28 101B16 109B17 2,310B823 2,524B747

1 month 492B33 497B26 102B17 103B17 2,298B856 2,435B796
2 months 491B35 481B27 103B16 91B18 2,336B858 2,230B732

Data are expressed in arbitrary units as means B SD.

Statistical Analysis
Intent-to-treat evaluations were performed. Absolute values were
recorded. The mean and SD were calculated for each parameter at
each evaluation time. The two-tailed paired t test was used to com-
pare the data both at entry and at completion of the studies on the
two comparative test sites. Variance analysis was performed to assess
the changes occurring in time. A p value lower than 0.05 was consid-
ered significant. Due to large interindividual differences at inclusion,
percentage variations from baseline values were also computed to
describe the relative changes in time.

Results

Study 1
At entry in the study, the treated and control solar len-
tigines showed almost similar mean values for each of the
biometrological parameters. No significant change oc-
curred in time for any of the parameters at the control
lesional site and for the erythema index at the treated site.
The perilesional normal-looking skin remained unaf-
fected by the treatment.
A modest non-significant decrease in the melanin
index of the treated solar lentigines took place in time (ta-
ble 1), reaching –1.4% after 1 month and –4.4% at com-
pletion of the study. The melanin density in CSSS (fig. 1)
as assessed by photodensitometer-assisted corneomelam-
etry was 4- to 6-fold higher on the solar lentigines than on
the surrounding clinically normal-looking skin. The value
on lesional skin was reduced during treatment, reaching
–4.8% after 1 month and –15.1% (p ! 0.05) at comple-
tion of the 2-month study (table 1). Computerized image
analysis of these samples showed that the melanin load
was obviously reduced in 40% (12/30) of the subjects
(fig. 2a, b).
The absolute values of area of the ultraviolet-revealed
melanin spots showed a modest trend to decrease under
treatment (table 1). However, the reduction in percentage
Fig. 1. Microscopic aspect of a CSSS performed on a solar lentigo.
The melanin load inside corneocytes is prominent.
which was only –4.4% after 1 month reached significance
with –12.3% (p ! 0.01) at completion of the study.
Study 2
No significant difference was yielded between the
effect of the two test formulations upon the pigmentation
of solar lentigines. Indeed, the changes in the melanin
index and corneomelametry from baseline remained in-
conspicuous after the 3-month treatment. The erythema
index was unaffected by both treatments.
The subclinical and faint melanin spots revealed under
ultraviolet light showed a similar trend in lightening for
both formulations (table 2). Each improvement reached
significance (p ! 0.01) at completion of the 3-month study
(Melanex duo: –8.7%; Skinoren: –10.4%).

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Fig. 2. Image-analysis-assisted corneomela-
metry showing the decreased area (pixels)
and darkness (arbitrary units, AU) of melan-
in pigmentation of the stratum corneum.
Comparison of values before (a) and after
(b) a 2-month treatment using the stabilized
soy extract.

Table 2. Image-analysis-assisted corneomelametry determining the Discussion

area in arbitrary units (means B SD) of pigmented spots on skin
treated by two commercial formulations
In Asian typology, the balance in skin hue is genetically
Evaluation time Melanex duo Skinoren regulated by the amount in eumelanin. The diffuse and
spotty skin hyperpigmentations are usually perceived as a
Baseline 2,174B694 2,215B861 disturbing disfigurement. These conditions may be tem-
1 month 2,258B766 2,188B613 porary, but some prove to be chronic and may require
2 months 2,050B709 2,005B755
treatment.
3 months 1,985B679 1,985B782

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 Most commercial whitening cosmetics, over-the-coun- solar lentigines. By contrast, no significant effect was
ter products and dermatological formulations incorporate yielded by the two other formulations.
tyrosinase inhibitors to reduce melanin production. Hy- Image analysis of subclinical or faint melanin spots as
droquinone used to be very popular because of its effica- revealed by ultraviolet light appeared to be very sensitive
cy. Unfortunately, this compound has significant disad- to disclose the hypopigmenting effect of the three test for-
vantages due to its toxic and mutagenic effects on mam- mulations. The sensitivity of the method is indisputable
malian cells [22]. Other products target the same or other [18], also improving the objectivity in comparative assess-
distinct steps of melanin biosynthesis and transfer to kera- ments. In our experience, this approach is less suitable for
tinocytes [18, 22, 23, 26–28]. The effectiveness of many of darkened age spots due to the very weak amount of light
these compounds appears quite limited on darkened age reaching the skin surface.
spots. The low susceptibility to various topical hypopig- Data gathered by the three presently used biometrolog-
menting agents can, however, be improved by combina- ical methods cannot distinguish hypopigmenting agents
tion treatments [27]. Despite improved emerging strate- according to their mechanisms of action. Indeed, the
gies in this field, the topical management of darkened age reduction in the melanin load in the stratum corneum can
spots remains difficult. Indeed, these lesions are heteroge- be the result of a reduction in the melanocyte density, an
neous in nature. They may correspond to seborrhoeic ker- inhibition in melanin synthesis, a decreased transfer of
atoses, melano-acanthomas or solar lentigines. An incipi- melanosomes into keratinocytes or an increased degrada-
ent malignant melanoma must be ruled out. In this study, tion of melanosomes during the transit time of cells
we used dermoscopic criteria to select only lesions looking throughout the epidermis.
like solar lentigines [24, 25]. In conclusion, the present study explored the biological
The discovery and development of novel topical hypo- activity of hypopigmenting formulations in vivo. Al-
pigmenting products benefit from in vitro technologies though an effect was proven on lightly pigmented skin,
including enzymatic and cell-based assays [26]. Unfortu- clinically relevant improvement was barely reached on
nately, the extrapolation to the in vivo efficacy is unsatis- solar lentigines. It is inferred that functional disturbances
factory. In addition, most animal models for hypopig- in melanin load such as epidermal melasma might benefit
menting efficacy are inadequate [22]. Hence, clinical from topical treatments. In contrast, melanocytic hyper-
trials performed in human volunteers are critical to deter- plasia with melanin overproduction would better benefit
mine the efficacy profile of these topical products. The from selective destruction therapies such as those pro-
objective assessment of the extent and severity of skin pig- vided by lasers.
mentation disorders benefits from a series of methods,
but only a few of them are well suited for spotty changes
[6, 9, 14–18]. In this study, we used a narrow-band reflec-
tance spectrophotometer equipped with a small-diameter
probe. The melanin content was derived from the mela-
nin index representing the changes in wavelengths of the
light emitted by the diodes of the device and those re-
emitted by the skin [9]. This method did not reveal any
significant decrease in pigmentation of solar lentigines
during treatment with the three test formulations. The
lack of significant variation in the erythema index values
indicated absence of erythema. It was inferred that the
tolerance of the test products was satisfactory.
Corneomelametry assisted by photodensitometry and
computerized image analysis is a novel quantitative
method to assess the load in argentaffin compounds
present inside the superficial layers of the stratum cor-
neum. Compared to the normal-looking skin, solar lentig-
ines exhibited a 4- to 6-fold increase in the photodensi-
tometric values. The stabilized soy extract induced a sig-
nificant lightening effect after a 2-month treatment of the

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