A Clinical Trial of Topical Bleaching Treatment with Nanoscale
Tretinoin Particles and Hydroquinone for Hyperpigmented
Skin Lesions
KATSUJIRO SATO, MD, DAISUKE MATSUMOTO, MD, FUMIKO IIZUKA, MD, EMIKO AIBA-KOJIMA, MD,CHIAKI MACHINO, MD, HIROTAKA SUGA, MD, ASAMI WATANABE-ONO, MD,y KEITA INOUE, MD,KOICHI GONDA, MD, AND KOTARO YOSHIMURA, MDBACKGROUND Although combined use of tretinoin (all-trans-retinoic acid; atRA) and hydroquinone
improves various hyperpigmented lesions, the pharmacologic instability of atRA and atRA-induced irritant dermatitis are difficult unsolved problems.
OBJECTIVE The objective was to evaluate the efficacy and adverse effects of a newly formulated gel
containing inorganic-coated atRA nanoscale particles (nano-atRA gel).
METHODS Nano-atRA gel was used in our two-phased bleaching protocol: 5% hydroquinone and 7%
lactic acid ointment were used along with nano-atRA gel in the bleaching phase (2–8 weeks), and 5%hydroquinone and 7% ascorbic acid ointment were used alone during the healing phase (4–8 weeks).
Eighty-four patients with facial hyperpigmented lesions were enrolled in this study, and 77 of them (88lesions) followed up for more than 10 weeks were analyzed.
RESULTS Hyperpigmentation was improved in 84 of 88 lesions (95.5%) after a mean treatment period of
14.3 weeks and was almost eliminated in 52 lesions (59.1%). Nano-atRA gel caused exfoliation andscaling similar to that seen with conventional atRA gel, whereas the erythema seen in the bleachingphase appeared to be weaker.
CONCLUSION Nano-atRA gel can improve hyperpigmentation to a similar extent as conventional atRA
gel. It also induces irritant dermatitis, but with less erythema.
The authors have indicated no significant interest with commercial supporters.

T

he combination of topical tretinoin (all-transretinoic acid; atRA) and hydroquinone iseffective on various hyperpigmented skin disorders.
Kligman and Willis1 introduced this treatment, andseveral modified protocols2 have been reported. In
these therapeutic regimens, topical corticosteroids
are used in combination with atRA and hydroquinone to reduce adverse effects such as irritation anderythema. We have previously proposed aggressive
bleaching protocols,3–5 in which atRA andhydroquinone were used separately. Corticosteroids
were not used, because corticosteroids reduce themelanin-discharging effect of atRA.6–9 Corticosteroids can lead to postinflammatory hyperpigmentation (PIH) in bleaching therapy, especially in coloredskin, probably by suppressing epidermal turnoverand melanin discharge. Since 1995, we have successfully treated more than 15,000 cases of various
skin lesions with epidermal hyperpigmentation using
our bleaching protocols.
In our combined bleaching treatment with atRA andhydroquinone, atRA discharges melanin granules
from the epidermis by accelerating epidermal turnover,8–10 whereas hydroquinone strongly suppresses
new melanin production.11 The hyperpigmented
epidermis is replaced by less-pigmented epidermis
in a few weeks. atRA promotes the discharge ofmelanin granules in epidermis by (1) accelerating
epidermal turnover (differentiation of keratinocytes)

Department of Plastic Surgery; University of Tokyo School of Medicine, Tokyo; yRitz Medical Clinic, Tokyo, Japan& 2007 by the American Society for Dermatologic Surgery, Inc.  Published by Blackwell Publishing 
ISSN: 1076-0512  Dermatol Surg 2007;33:937–944  DOI: 10.1111/j.1524-4725.2007.33195.x
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in a direct manner and (2) promoting epidermal
growth (proliferation of keratinocytes) in an indirectmanner; the latter effect was found to be mediatedby heparin-binding epidermal growth factor–like
growth factor (HB-EGF) secreted by suprabasal
keratinocytes.10,12 Thus, atRA has a specific effect as
a discharger of epidermal melanin that cannot be
performed by any other reagents or any peelingprocedures such as a-hydroxyl acid peeling or
microdermabrasion. These exfoliating procedures
simply induce subsequent normal wound healing andonly minimal acceleration of epidermal turnover.
It is well known, however, that atRA frequently
induces irritant dermatitis, especially when usedaggressively. Because all-trans-retinol (ROL) andall-trans-retinal had been considered to be lessirritating than atRA,13,14 we tried using 10% ROL
aqueous gel instead of 0.1% atRA gel and achieved
similar effectiveness, but failed to reduce the adverse
effects.7 As yet, there is no way to reduce the irritantdermatitis without losing the bleaching efficacy ofretinoids.
Nanoscale atRA (nano-atRA) particles were developed as a novel drug delivery system through the useof a boundary-organized nanoscale reaction.15
The poor stability of atRA to heat and light is aserious pharmacologic problem: our handmade,aqueous atRA gel requires monthly preparation.3–9
Nano-atRA showed improved stability comparedwith atRA15 and can be stocked for 6 months. In
addition, nano-atRA particles showed improved
permeation of the stratum corneum, and atRAshould be gradually released in the epidermis as
nanoscale micells degrade. In murine skin, nano-atRA
gel contributed to accelerated epidermal turnover, epidermal hyperplasia, decreasing melanin content, increased expression of hyaluronic acid, and increased
HB-EGF mRNA levels in epidermal keratinocytes.15
Enhanced production of HB-EGF by suprabasal keratinocytes is known to be one of the importantphenomena seen after topical retinoid treatment12
and is the most likely mechanism by which retinoids
accelerate the discharge of epidermal melanins.10

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Improved permeation and slow release of atRA
from nanoscale particles may enhance its clinicallybeneficial effects and/or reduce its adverse effects,
such as retinoid dermatitis. In this trial study, we
prepared 0.1, 0.2, and 0.4% nano-atRA gels forclinical use to estimate the bleaching potentialof nano-atRA gel and the extent of its adverse
side effects.

Methods
Preparation of Ointments
atRA was commercially obtained from Wako Pure
Chemical Industries Ltd. (Osaka, Japan), and 0.1,
0.2, and 0.4% nano-atRA gels were prepared using
boundary-organized nanoscale reaction droplets aspreviously reported15 (Figure 1). The size andstructure of the nano-atRA particles were confirmedby freeze fracture transmittance electron microscopy(ff-TEM, Hitachi Co. Ltd, Tokyo, Japan). The nanoatRA gels were prepared at Institute of Medical
Science, St. Marianna University School of Medicine.
An ointment including 5% hydroquinone and 7%
lactic acid (HQ-LA ointment) and an ointment including 5% hydroquinone and 7% ascorbic acid
(HQ-AA ointment) were also prepared. Petrolatum
polyethylene ointment base (Plastibase, Taisho
Pharmacology, Osaka, Japan) was used as the ointment base of the HQ-LA ointment, whereas ahydrophilic ointment (Taisho Pharmacology) wasused for the HQ-AA ointment.
Patients
Each ointment was applied topically by 83 Japanesewomen and 1 man with facial hyperpigmented skinlesions, and the 77 patients (76 women and 1 man)
who were followed up for more than 10 weeks wereanalyzed in this study. Because 11 patients had twolesions, there were 88 hyperpigmented skin lesions
in all, including solar lentigines (n = 10), melasma(n = 36), ephelides (n = 9), and PIH (n = 33). Dermalmelanosis and melanocytosis were not targeted. The
other 7 patients stopped the treatment because of
their private reasons or adverse side effects such as

S AT O E T A L

Figure 1. Schematic of the preparation of nano-atRA particles with their core/shell (CaCO3) structure.

irritation and erythema. The age of the patients
ranged from 16 to 84 years (mean 7 SD,
45.2 7 15.4 years).
Informed consent was obtained from all subjects.
The study protocol conformed to the guidelines of
the 1975 Declaration of Helsinki and was approvedby our institutional review board.

Treatment Protocol
Our bleaching protocol consists of two phases, ableaching phase and a healing phase.6–9 In thebleaching phase, discharge of epidermal melaninis accelerated by nano-atRA gel and melanin
production is suppressed by HQ-LA ointment.
In the healing phase, the discharge of epidermalmelanin is discontinued and great care is taken not to
induce new PIH, by using hydroquinone alone.
In the bleaching phase, 0.1% nano-atRA gel and
HQ-LA ointment were applied twice a day. All patients were started with 0.1% atRA, and higher
percentages (0.2 or 0.4%) of tretinoin were used
only when patients did not see skin reactions such as
scaling. Nano-atRA gel was carefully applied only topigmented areas, using a small cotton-tip applicator,and subsequently HQ-LA ointment was widely applied with the fingers beyond the pigmented area
(i.e., all over the face). Patients were asked to visit
our hospital at 1, 2, 4, 8, and 12 weeks after starting
this treatment. In most cases, it took 2 to 8 weeks tofinish this phase. When scaling was not observed at
1 week, the concentration of tretinoin was increased
to 0.2 or 0.4%. The concentration of tretinoin andfrequency of its application were appropriately
modified according to the skin condition and thedegree of skin reaction.
The healing phase was begun after pigmentation had
improved sufficiently or 8 weeks had passed. The
application of nano-atRA gel and HQ-LA ointment
was discontinued, and application of HQ-AA ointment all over the face was started. HQ-AA ointment
was used until the erythema was almost eliminated; it
took 4 to 8 weeks to complete this phase. Topicalcorticosteroids were not employed in the bleachingor healing phase. In melasma patients, a second
treatment, which is a complete treatment cycle
including bleaching and healing phases, was performed after a 1-month interval if the patient
requested it.

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TABLE 1. Clinical Results of Nano-atRA Treatment
Number of lesions
Skin pigmentation
disorderMean treatment
period (weeks)

Excellent

Good

Fair

Poor

Total

Solar lentigines
MelasmaEphelides
PIH
Total

13.6
14.1
16.5
14.2
14.3

1 (10.0%)
17 (47.2%)
2 (22.2%)
27 (81.8%)
47 (53.4%)

3 (30.0%)
11 (30.5%)
3 (33.3%)
5 (15.2%)
22 (25.0%)

4 (40.0%)
6 (16.7%)
4 (44.4%)
1 (3.3%)15 (17.0%)

2 (20.0%)
2 (5.6%)0 (0%)
0 (0%)
4 (4.5%)10 (100%)
36 (100%)
9 (100%)33 (100%)
88 (100%)

PIH, postinflammatory hyperpigmentation.

Evaluation of Results
Photographs of each patient were taken at baselineand during and after treatment with a high-resolution digital camera (D70, Nikon, Tokyo, Japan). The
percentage of pigmentary clearance was evaluated
via the photographs by two experienced plastic surgeons who did not perform this treatment. The meandata of the pigmentary clearance of each patientwere classified into four categories: excellent (80%clearance or better), good (50% to less than 80%
clearance), fair (0% to less than 50% clearance), andpoor (no change or worse).

long-term effects. Continuous use of atRA always
induced less response of skin (such as scaling) in
our conventional atRA treatment, and thus wefrequently had to change the concentration of atRAby the fourth week. The resistance to atRA was also
seen with the nano-atRA treatment.
The results of this study are summarized in Tables 1
and 2. Forty-seven lesions (53.5%) were evaluated as‘‘excellent,’’ 22 (25.0%) as ‘‘good,’’ 15 (17.0%) as‘‘fair,’’ and 4 (4.5%) as ‘‘poor.’’ Most of the fair andpoor cases had minimal skin reactions, such as
scaling, during the bleaching phase. Representative
cases are shown in Figures 2 through 4.

Results
The 88 hyperpigmented lesions were treated for amean period of 14.3 weeks. Almost all patients had
sufficient improvement without serious adverse
effects. Mild erythema appeared in the first week
in most cases and scaling was seen at the same time.
Clinical evaluations of patients at 1 and 2 weeks
suggested that nano-atRA gel appeared to induce alesser degree of erythema and a similar degree of
scaling in the first 2 weeks compared to our thousands of cases treated with conventional atRA gel,3–9
although statistical analysis of erythema was not
performed. Scaling is a clinical sign of accelerating
epidermal turnover in this treatment, and if scaling
was not seen during the first or second week oftreatment, we changed the concentration of atRA
from 0.1% to 0.2 or 0.4%. Scaling was observed
even in the initial stage of the healing phase,suggesting that the slow-release nano-atRA gel had

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Nano-atRA improved all four kinds of skin pigmentation disorders. Of 36 cases of melasma,
28 (79.3%) ranked excellent or good. The mean
treatment period of melasma patients was 14.1
weeks. Thirty-two of 33 PIH cases ranked excellent
or good. There were no poor results in the PIH
group. There were only 9 cases of ephelides, morethan half of which achieved excellent or good results.
TABLE 2. Clinical Results of Cases with Melasma
Number of cases
Number of
Excellenttreatments Excellent Good Fair Poor Total cases (%)
One
12treatmentTwo5
treatments
Total
17

10

5

1

28

42.8

1

1

1

8

62.5

11

6

2

36

47.2

S AT O E T A L

Figure 2. Case 1: A 40-year-old woman with a solar lentigine and melasma on her right cheek, first shown before treatment(A). The solar lentigine was first treated with a Q-switched ruby laser, but 4 weeks later PIH appeared at the original position(B). Bleaching treatment with 0.1% nano-atRA gel and HQ-LA ointment was performed for 4 weeks; the patient is shown at
2 weeks (C) and at 4 weeks (D). This was followed by healing treatment with HQ-AA ointment alone for 4 weeks. Only
minimal erythema appeared during the bleaching phase. PIH was completely eliminated and the melasma was also
apparently improved at 8 weeks (E).

Only 4 of 88 lesions (2 of melasma and 2 senile
lentigines) showed poor treatment results.

Discussion
In our recent report, we used histology to confirm
that accumulated melanin granules around the basal

layer were cleared after treatment with atRA andhydroquinone. In acquired dermal melanocytosis,
melanin deposits in the dermis appeared not tochange.8 Thus, the bleaching therapy is only effective
for epidermal pigmentation, and we therefore focused on various types of hyperpigmentation of theepidermis in this trial.

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Figure 3. Case 2: A 38-year-old woman with melasma on
both cheeks is shown before the treatment (A). Bleachingwith 0.1% nano-atRA gel and HQ-LA ointment was performed for 12 weeks, followed by application of HQ-AA
ointment alone for 4 weeks. Mild erythema appeared during
the bleaching phase, shown at 4 weeks (B). The melasma
was almost cleared at 16 weeks (C).

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Figure 4. Case 3: A 53-year-old woman with a solar lentigine
and melasma on her left cheek before the treatment (A).
0.1% nano-atRA gel was used together with HQ-LA ointment
in bleaching phase for 12 weeks, and HQ-AA ointment wasapplied alone in the healing phase for 9 weeks. Mild erythema was seen during the bleaching phase, shown at 4
weeks (B). The melasma almost disappeared, whereas thesolar lentigine became lighter, as seen at 21 weeks (C).

Using nano-atRA gel based on the newly developed
drug delivery system technology, we obtained results
as good as those of previous studies that used ourS AT O E T A L

former atRA gel. The present treatment resulted insatisfactory improvement of melasma, with a mean
treatment period of 14.1 weeks. Roughly two-thirdsof the melasma patients required only one session of
the bleaching treatment (Table 2), whereas treatmentof melasma with our former atRA gel was sometimes
performed in two or three sessions.8 Of the various
hyperpigmented lesions treated, PIH was most improved. This has always been true in our treatmentof lesions with conventional atRA. The number ofpatients with ephelides enrolled in this study was
small, but more than half of the patients showed
excellent or good results. Among the four types ofhyperpigmented lesions studied, the percentage of
excellent and good results was lowest in the senile
lentigines. Senile lentigines, especially those that are
resident for a long period, frequently show excessive
development of horny layers (hyperkeratosis) that
may obstruct the penetration of atRA into the epidermis. This may also be the case with nano-atRA
gel, although high permeation of nano-atRA wasreported in murine skin.15 We recommend that irradiation by Q-switched ruby laser for removal of
hyperkeratinization of senile lentigines (CO2 laser
for debulking extreme hyperkeratinization of seborrheic keratoses) is first performed as done in Case 1(Figure 2). In colored skin, PIH after laser treatments
can frequently be a disfiguring problem. PIH afterlaser irradiation, however, is always easily treated by
our aggressive bleaching treatment with atRA andhydroquinone3,4,8,9 especially when the laser irradiation is only once and the bleaching treatment
started early (e.g., 4 weeks after Q-switched laser
irradiation).
In our conventional bleaching treatment, there are
two major problems: one is dermatitis induced byaggressive use of atRA, and the other is the biochemical instability of atRA in our ointments. We
applied atRA only on the pigmented area but used it
aggressively, which almost always led to irritantdermatitis on the area where it was applied. The
dermatitis is the most serious side effect and remains
to be resolved. In animal experiments, nano-atRAgel showed slower diffusion into the epidermis andhigher degrees of epidermal alterations, as assessedby retinoid signals such as elevated HB-EGF mRNA
expression, enhanced hyaluronic acid deposition,and epidermal hyperplasia, with a lesser degree oferythema.15 These advantages of nano-atRA appeared to be partly confirmed in this clinical trial, in
which similar degrees of bleaching effectiveness andscaling were seen, with a lesser degree of erythema.
In addition, the improved biochemical stability ofnano-atRA to heat and light is another advantage,15
because we had to prepare the conventional atRA gelevery month because of its pharmacological instability. The fact that nano-atRA gel can be stored forat least 6 months without significantly losing atRA
chemical activity may increase its commercial profitability. atRA has long been used for treating acne,
photoaging-associated symptoms such as fine wrinkles, and hyperpigmentation. When used aggressively, atRA can be an extremely powerful tool fordischarging epidermal melanin. Reducing retinoidinduced dermatitis remains a major challenge. New
generations of synthetic retinoids or new drug delivery systems, such as the nano-atRA presented
here, may resolve the problem.

Acknowledgments Nano-atRA gels used in thisstudy were gifts from Dr Yoko Yamaguchi and
Dr Rie Igarashi of Institute of Medical Science,
St. Marianna University School of Medicine.

References
1. Kligman AM, Willis I. A new formula for depigmenting humanskin. Arch Dermatol 1975;111:40–8.
2. Gano SE, Garcia RL. Topical tretinoin, hydroquinone, and betamethasone valerate in the therapy of melasma. Cutis
1979;23:239–41.
3. Yoshimura K, Harii K, Aoyama T, et al. A new bleaching protocol
for hyperpigmented skin lesions with a high concentration of
all-trans retinoic acid aqueous gel. Aesthetic Plast Surg 1999;
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4. Yoshimura K, Harii K, Aoyama T, Iga T. Experience with a strong
bleaching treatment for skin hyperpigmentation in Orientals. Plast
Reconstr Surg 2000;105:1097–108.
5. Yoshimura K, Harii K, Masuda Y, et al. Usefulness of a narrowband reflectance spectrophotometer in evaluating effects ofdepigmenting treatment. Aesthetic Plast Surg 2001;25:129–33.

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6. Yoshimura K, Momosawa A, Watanabe A, et al. Cosmetic color
improvement of the nipple-areola complex by optimal use oftretinoin and hydroquinone. Dermatol Surg 2002;28:1153–8.
7. Yoshimura K, Momosawa A, Aiba E, et al. Clinical trial of
bleaching treatment with 10% all-trans retinol gel. Dermatol Surg
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8. Momosawa A, Yoshimura K, Uchida G, et al. Combined therapy
using Q-switched ruby laser and bleaching treatment withtretinoin and hydroquinone for acquired dermal melanocytosis.
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9. Yoshimura K, Sato K, Aiba-Kojima E, et al. Repeated treatment
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10. Yoshimura K, Uchida G, Okazaki M, et al. Differential expressionof heparin-binding EGF-like growth factor (HB-EGF) mRNA in
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11. Yoshimura K, Tsukamoto K, Okazaki M, et al. Effects of all-transretinoic acid on melanogenesis in pigmented skin equivalents andmonolayer culture of melanocytes. J Dermatol Sci 2001;27(Suppl 1):S68–75.
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epidermal basal cell growth by suprabasal retinoic acid receptors.
EMBO J 1999;18:1539–48.
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14. Fluhr JW, Vienne MP, Lauze C, et al. Tolerance profile ofretinol, retinaldehyde and retinoic acid under maximized andlong-term clinical conditions. Dermatology 1999;199(Suppl 1):
57–60.
15. Yamaguchi Y, Nagasawa T, Nakamura N, et al. Successfultreatment of photo-damaged skin of nano-scale atRA particles
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Address correspondence and reprint requests to:
Kotaro Yoshimura, MD, Department of Plastic Surgery,
University of Tokyo School of Medicine, 7-3-1 Hongo;
Bunkyo-ku, Tokyo 113-8655, Japan, ore-mail: yoshimura-pla@h.u-tokyo.ac.jp