Abstract
Background: Although an aggressive use of tretinoin along with hydroquinone enables an efficient treatment of hyperpigmented skin lesions, irritant dermatitis remains to be solved.
Objective: To determine the effectiveness of 10 % all-trans retinol (ROL) gel for improvement of skin hyperpigmentation.
Methods: 10 % ROL gel was used instead of 0.1 % tretinoin gel in our two-phased bleaching protocol (bleaching and healing phases). 5% hydroquinone and 7% lactic acid ointment was used along with ROL gel in the bleaching phase (2-6 weeks). 5% hydroquinone and 7% ascorbic acid ointment was used alone during the healing phase (4-6 weeks). Twenty-one Japanese patients with hyperpigmented lesions on the face were enrolled in this study, and 18 patients followed up for more than 10 weeks were analyzed.
Results: Improvement of pigmentation was seen in 16 of 18 patients after an average treatment period of 11.3 weeks, and in 6 patients pigmentation was almost eliminated after treatment. Erythema and scaling were seen, however, during the bleaching phase as well as the bleaching treatment with tretinoin gel.
Conclusion: ROL can improve skin hyperpigmentation to a similar extent to tretinoin when used at high concentration, whereas it induces irritant dermatitis as well.

Introduction
Tretinoin and hydroquinone have been used successfully for treatment of skin hyperpigmentation since Kligman and Willis [1] introduced their bleaching formula. Several modified protocols have been reported [2], and the authors also proposed an aggressive bleaching therapy, in which tretinoin and hydroquinone were used separately and corticosteroids were not used [3-5]. In our facility, more than 8,500 cases with various kinds of hyperpigmented lesions have been successfully treated with our protocol and its modifications since 1995. Virtually the only adverse side effect of the strong bleaching treatment is irritant dermatitis. Further investigations and clinical trials would be worthwhile, in order to reduce the displeasing side effects without losing bleaching ability.
All-trans retinol (ROL) and all-trans retinal (RAL) have been considered to be less irritating than tretinoin if ROL or RAL was applied at relatively low (0.1-1.6 %) concentration [6, 7]. However, since binding affinity to specific nuclear receptors (retinoic acid receptors; RARs) of ROL or RAL is far less than that of tretinoin, beneficial effects are also expected to be very moderate compared to tretinoin. Our previous study revealed that HB-EGF mRNA, which play a critical role in epidermal growth after retinoids treatment [8, 9], was upregulated by ROL or RAL to a similar extent as tretinoin, but only when ROL or RAL was used at 40-100 times higher concentration than tretinoin [10]. We assumed that HB-EGF mRNA is one of the important indexes of retinoids at least when they are used for bleaching.
Based on the data mentioned above, we prepared 10% ROL aqueous gel, because ROL gel of 100 times higher concentration than tretinoin can be expected to show similar clinical effects to tretinoin gel (0.1%). The ROL gel was clinically used for depigmenting in order to estimate its bleaching potential and the extent of adverse side effects.

Patients and Methods
Preparation of Ointments: ROL aqueous gel (10%) was originally prepared and packed into small stainless tubes (5 g each) at the Department of Pharmacy, University of Tokyo Graduate School of Medicine. The precise regimen of ROL aqueous gel for 100 g was as follows: all-trans retinol (Sigma Chemical, St. Louis, MO) 10 g, Carbopol 940 (Goodrich Chemical, Hounlow, UK) 1 g, polyoxyethylene oleyl ether (Kao, Tokyo, Japan) 2.5 g, methyl p-hydroxybenzoate (Wako Pure Chemical Industries, Osaka, Japan) 0.026 g, propyl p-hydroxybenzoate (Wako Pure Chemical Industries, Osaka, Japan) 0.014 g, 10% sodium hydroxide aqueous solution 0.6 ml, purified water 86 g. 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. Plastibase (petrolatum polyethylene ointment base, Taisho Pharmacology, Osaka, Japan) was used as the ointment base of the HQ-LA ointment, while the hydrophilic ointment was used for the HQ-AA ointments. Because ROL gel, HQ-LA, and HQ-AA ointments (especially ROL gel) are pharmacologically unstable, fresh ointments were prepared at least once a month and stored in a dark and cool (4oC) place.
Evaluations of results:
Photographs were taken for every patient at baseline and after treatment with a high-resolution digital camera (Canon EOS-D30). The percentage of pigmentary clearance was evaluated via the photographs by two experienced plastic surgeons that did not perform this treatment. The mean data of the pigmentary clearance of each patient were classified into 4 categories: "excellent" (90% or more clearance), "good" (60% to less than 90% clearance), "fair" (30% to less than 60% clearance), and "poor" (less than 30% clearance).
Patients: Each ointment was topically applied under signed informed consent in 21 Japanese women with hyperpigmented skin lesions on the face, and 18 of them followed up for more than 10 weeks were analyzed in this study. The other 3 patients stopped treatment because of irritant dermatitis induced by ROL gel and/or hydroquinone. The age of patients varied from 20 to 67 years old (age=43.2±7.0; mean S.D.). Thirteen patients had solar lentigines, 5 had melasma, and 4 had ephelides; 4 patients had two kinds of pigmented lesions.
Treatment protocol: Our bleaching protocol is composed of two phases, a bleaching phase and a healing phase. In the bleaching phase, the pigmentation is aggressively treated, and transient adverse skin effects such as erythema and irritation are usually observed. Once satisfactory improvement is obtained, the healing phase is started in order to reduce the erythema and inflammation, taking care not to induce new postinflammatory hyperpigmentation.
1) bleaching phase: 10 % ROL gel and HQ-LA ointment were applied twice a day. ROL gel was carefully applied only on pigmented areas using a small cotton-tip applicator, and subsequently HQ-LA ointment was widely applied with the fingers beyond the pigmented area (e.g. all over the face). Patients were requested to visit our hospital at 1, 2, 4, 6 and 8 weeks after starting this treatment, and every 2 weeks afterwards. In most cases, it took 2 to 6 weeks to finish this phase.
2) healing phase: After sufficient improvement of pigmentation was obtained, the application of ROL 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, and it took 4-6 weeks to complete this phase. Topical corticosteroids were not employed either in the bleaching or healing phase.

Results
The average treatment period of 18 patients was 11.3 weeks, ranged from 8 to 16 weeks. Erythema and scaling started to be seen during the first week in most cases. Comparing with thousands of our cases treated with 0.1 % tretinoin gel (our preparation regimen is available in ref. #4), we thought that 10 % ROL gel induced slightly less degrees of erythema and higher degrees of scaling in the first two weeks, although statistical analysis was not performed. In addition, tolerance to the active reagent seemed to be acquired earlier in the ROL treatment than with tretinoin. In some patients who achieved tolerance as early as 2 weeks, further improvement was unlikely, so the results were categorized as "poor" and "fair".
The clinical results were summarized in Table 1. Six patients (33.3 %) were evaluated as "excellent", 7 cases (38.9 %) as "good", 3 cases (16.7 %) as "fair", and 2 cases (11.1 %) as "poor". Some improvement was seen in 16 of 18 patients (88.9 %). Most of "fair" and "poor" cases had very mild or no skin reactions such as scaling and erythema during the bleaching phase. The representative 4 cases are shown in Figs. 1-4.


Discussion
In bleaching treatment with tretinoin and hydroquinone, we believe that the role of tretinoin is to wash out of melanin granules out of epidermis, while that of hydroquinone is to strongly suppress new melanin production [10, 11]. It is suggested that tretinoin shows a depigmenting effect by: 1) accelerating epidermal turnover (differentiation of keratinocytes), and also 2) promoting epidermal growth (proliferation of keratinocytes); the latter of the two effects was found to be mediated by heparin-binding EGF-like growth factor (HB-EGF) secreted by suprabasal keratinocytes [8, 9, 12]. Thus, the mechanism of this bleaching therapy is very simple; accelerate the output, and suppress the input, but appears to be only effective for epidermal pigmentation. Indeed, we confirmed histologically that accumulated melanin granules around the basal layer were cleared up after treating with tretinoin, but the melanin deposits in the dermis appeared not to change in acquired bilateral nevus of Ota-like macules, which has both epidermal and dermal pigmentation (in preparation).
The authors recently reported that ROL and RAL can induce markedly HB-EGF mRNA upregulation at pharmacological concentration (0.01-1 mM) in cultured human keratinocytes [10]. However, in cases of ROL and RAL, 40-100 times higher concentration was required in order to upregulate HB-EGF mRNA to a similar extent to tretinoin [307]. In the present study, we prepared and used 10 % ROL gel, which is 100 times higher concentration that 0.1% tretinoin gel, and our subjective opinion is that 10% ROL gel is as effective as 0.1% tretinoin gel in improving skin hyperpigmentation based on our experience with thousands of patients treated with 0.1% tretinoin gel.
ROL and RAL are considered to work after conversion to tretinoin. The binding affinities of Rol and Ral to RARs are quite low [13], so that their biological activity should result from their oxidative transformation into tretinoin by epidermal keratinocytes. This conversion to tretinoin after topical application of ROL and RAL was revealed to occur in a study using skin organ culture [14]. However, there have been some reports suggesting the existence of other pathways than that mediated by nuclear receptors [15]. ROL is known to show much less side effects such as irritation than tretinoin, at least when used at similar concentration, thus has been thought to be more tolerable, and may be of great value in clinical use. However, the present study revealed that even ROL can induce irritant contact dermatitis to a similar extent as tretinoin when used at 100 times higher concentration, although it also showed beneficial effects similar to tretinoin.
It remains unknown what percentages of ROL in the prepared ROL gel was absorbed into the skin. Unlike the 0.1 % tretinoin gel we use in which more than 96 % of the gel is composed of water, the 10 % ROL gel contains 10 % ROL, while water formed only 86 % of it. The permeability of ROL in the 10 % ROL gel may be much less than that of tretinoin in the 0.1% tretinoin gel. Therefore, 5 % ROL gel which should contain more water may show similar depigmenting effects to the 10 % ROL gel.
In this study, we showed that ROL can play a depigmenting role as well as tretinoin. However, the cost performance of the ROL gel is very poor, compared to the tretinoin gel, as far as our preparations are concerned. Purified ROL is commercially available, but 1 gram of it costs almost the same as 1 gram of tretinoin. Since 100 times amount of the working ingredient was required to prepare the ROL gel, it costs 100 times more. In light of this, it seems to be impractical to clinically use the 10 % ROL gel we prepared.
Conclusions
The present clinical trial revealed that 10 % ROL gel clinically show a considerable bleaching effect, which seems to be as good as that of 0.1 % tretinoin gel, but the ROL gel also induce adverse side effects, such as irritant dermatitis, to a similar degrees as tretinoin gel.

References

1. Kligman AM, Willis I. A new formula for depigmenting human skin. Arch Dermatol 1975; 111: 40-8.
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5. Yoshimura K, Harii K, Masuda Y, Takahashi M, Aoyama T, Iga T. Usefulness of a narrow-band reflectance spectrophotometer in evaluating effects of depigmenting treatment. Aesthetic Plast Surg 2001; 25: 129-33.
6. Kang S, Duell EA, Fisher GJ, et al. Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation. J Invest Dermatol 1995 ;105: 549-56.
7. Fluhr JW, Vienne MP, Lauze C, Dupuy P, Gehring W, Gloor M. Tolerance profile of retinol, retinaldehyde and retinoic acid under maximized and long-term clinical conditions. Dermatology 1999; 199(S1): 57-60.
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10. Yoshimura K, Uchida G, Okazaki M, Kitano Y, Harii K. Differential expression of heparin-binding EGF-like growth factor (HB-EGF) mRNA in normal human keratinocytes induced by a variety of natural and synthetic retinoids. Exp Dermatol, in press.
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Figure Legends

Fig.1. Case 1. A 46-year-old woman with a solar lentigine and melasma on her cheek underwent the treatment (A: before treatment); 10 % ROL gel was used for 4 weeks together with HQ-LA ointment (B: at 4 weeks; after bleaching treatment), followed by application of HQ-AA ointment alone for 4 weeks. Both the solar lentigine and melasma almost disappeared after 8 weeks of treatment (C: at 8 weeks).

Fig.2. Case 2. A 54-year-old woman with several solar lentigines on her right cheek underwent the treatment (A: before treatment); 10 % ROL gel was used for 2 weeks together with HQ-LA ointment, followed by application of HQ-AA ointment alone for 6 weeks. Significant erythema and irritation on the treated area were observed 1-3 weeks after starting the treatment (B: at 2 weeks; after bleaching phase). The erythema almost disappeared after 6 weeks' healing phase, although slight postinflammatory hyperpigmentation was left on the treated areas (C: at 8 weeks).

Fig.3. Case 3. A 32-year-old woman with melasma and some solar lentigines underwent the treatment (A: before treatment). 10 % ROL gel was used for 4 week combined with HQ-LA ointment, followed by single application of HQ-AA ointment for 4 weeks. Irritant dermatitis was seen on the treated areas throughout the bleaching phase, but pigmentation was improved significantly by day 28 (B: at 4 weeks; after bleaching phase). After 4 weeks of healing phase, erythema disappeared without leaving postinflammatory hyperpigmentation (C: at 8 weeks).

Fig.4. Case 4. A 34-year-old woman with ephelides underwent the treatment (A: before treatment). Bleaching was performed with 10 % ROL gel and HQ-LA ointment for 4 weeks, thereafter HQ-AA ointment alone was used for 6 weeks. The pigmented spots were almost eliminated and mild rosy glow was still seen at 10 weeks (B: at 10 weeks).