Treatment with Ascorbyl Glucoside Arginine Complex ameliorates solar lentigos

Treatment with Ascorbyl Glucoside Arginine Complex ameliorates solar lentigos | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Treatment with Ascorbyl Glucoside Arginine Complex ameliorates solar lentigos Mariko Takada, Kayoko Numano, Masahiko Nakano, Akio Yamamoto, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5332003/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Little is known about anti-pigmenting effects of whitening agents on solar lentigo (SLs). To characterize the anti-pigmenting effects of newly designed ascorbyl glucoside arginine complex (AGAC) on SLs, lotions with or without 28% AGAC were applied twice daily for 24 weeks in a double-blind half-face study of 27 Japanese females with SLs. Pigmentation scores were evaluated using a photo-scale and skin colors were assessed using a color difference meter and a Mexameter. Treatment with the test lotion elicited a significant decrease in pigmentation scores at 24 weeks com-pared to week 0, with a significant decrease in pigmentation scores at 24 weeks compared to the placebo lotion-treated SLs. In the test lotion-treated SLs, the lightness (L) values and melanin index (MI) reflecting pigmentation level significantly (p<0.0001) increased and decreased, respectively, at 12 and 24 weeks of treatment compared to week 0. Comparisons of increased L values or decreased MI values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly higher increased L or decreased MI values than the placebo lotion-treated SLs both at 12 and 24 weeks of treatment. The sum of our results strongly indicates that AGAC is distinctly effective in ameliorating the hyperpigmentation levels of SLs at a visibly recognizable level by the subjects without any hypo-pigmenting effects or skin irritation. Ascorbyl Glucoside Arginine Complex solar lentigo double-blind half-face study pigmentation L value melanin index Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Solar lentigos (SLs) are hyperpigmented lesions that frequently occur on sun-exposed skin, especially on the face and the dorsum of the hands of Asian subjects [ 20 ]. Based on the frequency of the final diagnosis of patients with various pigmentary disorders in Japan, SLs have the highest incidence, occurring in approximately 60% of all patients with hyperpigmentary disorders, while melasma and post-inflammatory hyperpigmentation (including ultraviolet B (UVB) melanosis) occur in as few as 5.2% and 3.3% of patients, respectively [ 20 ]. In general, hyperpigmentary disorders, including UVB-melanosis, SLs and melasma, are targeted by anti-pigmenting agents. However, an effective anti-pigmenting treatment for SLs is difficult especially for dark-skinned individuals because the treatment is required to reduce the hyperpigmentation without causing any undesirable hypopigmentation or contact irritation in the surrounding normally appearing pigmented skin. It is thought that hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders including SLs [ 1 , 12 , 16 , 27 , 38 ], but treatment with HQ often causes skin irritation [ 5 , 15 , 26 , 33 , 41 , 50 , 52 ]. Although many whitening agents are available [2, 3, 4, 7, 8, 14, 17, 18, 25, 30, 34, 37, 39, 44, 46, 47. 48, 50], some of which are targeted and approved especially to treat UVB-induced hyperpigmentation in Japan, little is known about the potential anti-pigmenting effects of those whitening agents on SLs because clinical evaluations of SLs are not required for approval as a whitening agent in Japan. Excluding kojic acid and rhododendrol, which have been reported to elicit hypopigmentation on the faces of dark-skinned individuals [ 21 , 31 , 32 , 40 ], other whitening agents approved in Japan are suitable candidates for investigating their potential anti-pigmenting effects on SLs because they have been proven to be substantially safe in terms of hypopigmentation and skin irritation since they have been commercially available for a long period of time. Among those whitening agents, ascorbic acid derivatives are thought to be invaluable agents especially from the skin safety point of view. L-ascorbate-2-phosphate Mg (AMP), a whitening agent first approved in Japan, was reported to have a significant ameliorating effect on chloasma by acting as a tyrosinase inhibitor after it is enzymatically degraded by intrinsic epidermal phosphatases to release free ascorbic acid [9. 23, 28, 35, 42, 43, 49, 51]. L-ascorbate-2-phosphate trisodium salt (APS) is another approved whitening agent in Japan [ 9 , 13 , 36 ]. APS is a modified derivative of AMP that improves its stability, namely its aggregation due to the Mg salt [ 24 ]. Ascorbyl glucoside (AG) is also a whitening agent approved in Japan that has been documented to have a depigmenting effect on UVB-hyperpigmentation by acting as a tyrosinase inhibitor itself or after it is converted by intrinsic epidermal glucosidase to free ascorbic acid [ 34 ]. Based on the requirement for approval of whitening agents in Japan, it is well established that topical application of AMP, APS or AG for 21 days on UVB (2MED)-exposed human skin significantly inhibits the UVB-increased pigmentation measured as L values at 21 days post-UVB irradiation. However, there had been no published data on the anti-pigmenting effect of topical treatment with APS, AMP or AG on SLs in a double-blind half-face study, although a whole-face study using AMP on SLs for 3 months was reported to have some efficacy [ 23 ], the study of which was flawed due to the lack of a placebo control. We have recently reported for the first time that in a double-blind half-face study of 27 Japanese female subjects with SLs using lotions with or without 6% APS (test lotion and placebo lotion, respectively) applied twice a day for 24 weeks, APS has a weak but significant anti-pigmenting effect on SLs and also a significant whitening effect even on normally pigmented non-lesional surrounding skin (NLS) [ 22 ]. However, that clinical study was not satisfactory for clinical evaluation because there was no significant difference in the pigmentation scores of SLs judged by a dermatologist between the test and placebo lotions. This prompted us to characterize the anti-pigmenting effects of ascorbyl glucoside arginine complex (AGAC) on SLs via the modification of AG by making a complex with arginine to reduce its acidity and to increase the concentration used to 28%. In this study, we conducted a double-blind half-face study of 27 Japanese female subjects with SLs using lotions with or without 28% AGAC (test lotion and placebo lotion, respectively) applied twice a day for 24 weeks. Here we show that repeated topical treatment with AGAC has a significant anti-pigmenting effect on SLs with a significant difference in pigmentation scores of SLs judged by a dermatologist between the test and the placebo lotions. Those results were corroborated by mechanical evaluations using a color difference meter and a Mexameter, and show that there was a significant whitening effect even on normally pigmented NLS without any hypo-pigmenting effects at a mechanical skin color level. Materials and Methods Test materials The chemical structure of AGAC is shown in Fig. 1 . The test and placebo lotions were provided by Doctor’s Choice Co., Ltd. The test lotion was comprised of 28% AGAC and other ingredients as listed in Table 1 . The placebo lotion was comprised of the same components as the test lotion except it did not include AGAC. Ascorbyl glucoside and arginine were purchased from Technoble Co., Ltd. (Osaka, Japan) and Technoscience Co., Ltd. (Kashiwa, Japan), respectively. Table 1 Full ingredients list of the test lotion (containing 28% AGAC) and the placebo lotion (without AGAC). Ingredients With or without 28% Ascorbyl Glucoside Arginine Complex (AGAC), Water, Propanediol, Butylene Glycol (BG), Betaine, Pentylene Glycol, 1,2-Hexanediol, PEG‐60 Hydrogenated Castor Oil, PEG‐9M, Citric Acid, Sodium Citrate, Glycerin, Sphingolipids, Hydrolyzed Elastin, Hydrolyzed Collagen Study design This study was performed from September 2023 to March 2024, in the Ebisu Skin Research Center, Inforward Co. LTD, Tokyo, Japan. Twenty seven Japanese female volunteers with SLs were instructed to apply lotions with or without 28% AGAC (test lotion and placebo lotion, respectively) twice a day for 24 weeks on the entire right and left sides of their faces. They were also instructed in a double-blind manner to treat with the test lotion on one-half of their face and with the placebo lotion on the opposite side. Compliance with ethical standards Ethical approval All procedures performed in this study involving human subjects were in accordance with the ethical standards of the Institutional and/or National Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The present study adhered to the tenets of the Declaration of Helsinki and was reviewed and approved by the Japanese Ethics Committee, Clinical Research Review Center, General Incorporated Association (Approval No: CrrC23-020). A formal informed consent was obtained from each subject before the study. The UMIN study ID was assigned as UMIN000052115. Following screening by a dermatologist, 27 Japanese female volunteers with SLs on the entire right and left sides of their faces were enrolled in this study. Evaluation of pigmentation level Pigmentation levels in previously assigned SLs were evaluated using a photo-scale ranging from 1.0 to 5.0 of SLs on the face by a trained dermatologist (KN). Mechanical evaluation of pigmentation levels was performed according to the methods described previously [22]. In brief, the pigmentation levels in the same previously assigned SLs and non-lesional surrounding skin (NLS) were measured using a color difference meter CM-700 d (Konica Minolta Japan, Inc) to determine L values [22] and using a Mexameter MX18 (Courage+Khazaka Electronic GmbH) [6, 10, 11, 29] to measure Melanin Index (MI) values at 0, 12 and 24 weeks of treatment. The average values of 5 times measurements at the same SLs and NLS were used as data for figure description. General clinical evaluation A general clinical evaluation was performed for dryness, scaling, erythema, papules, stinging sensations or itchiness with 1 to 5 scoring grades which correspond to none, weak, mild, moderate and severe by a trained dermatologist (KN) in the entire half faces at 0, 12 and 24 weeks of treatment. Evaluation of skin redness level For mechanical evaluation of skin irritation, skin redness levels in the same previously assigned SLs and NLS were evaluated using a Mexameter MX18 as erythema index values, which reflect skin redness levels based on hemoglobin levels related to skin blood flow [10]. The average values of 5 times measurements at the same SLs and NLS were used as data for figure description. Statistics All data are expressed as means ± standard deviation (SD) unless noted otherwise. Prism 10 from Graph Pad was used as a software for statistics analysis. For pairwise comparisons, t test was used. ANOVA and Tukey’s comparison test were used to evaluate pigmentation scores and changes in L values, MI values and erythema index values. Friedman’s test and Dunn’s multiple comparisons test were used for skin scoring. p values < 0.05 are considered statistically significant. Results Visible pigmentation level of SLs Topical applications of lotions with or without 28% AGAC (test lotion and placebo lotion, respectively) were carried out twice daily for 24 weeks on the entire right and left sides of the subjects’ faces with SLs. The pigmentation levels of the same previously assigned SLs on the subjects’ faces were assessed at 0, 12 and 24 weeks using a photo-scale ranging from 1.0 to 5.0 by a dermatologist (KN) and these results are shown in Figure 2 . The results indicated that although the placebo lotion-treated SLs had significant (p<0.05 and p<0.01) decreases in pigmentation scores at 12 and 24 weeks compared to 0 week, the test lotion-treated SLs had more marked significant (p<0.001 and p<0.0001) decreases than the placebo’s ones in pigmentation scores at 12 and 24 weeks compared to week 0, together with a significant (p<0.01) decrease in pigmentation scores at 24 weeks compared to the placebo’s ones ( Figure 2 ). These results suggest that there was a distinct anti-pigmenting effect of the test lotion on SLs at a visible clinical level. Clinical photographs of SLs and NLS Representative photographs of the faces of subjects #7 ( Figure 3A ) and #12 ( Figure 3B ) before and after treatment for 24 weeks showed that the pigmentation level of the test lotion-treated SLs (red arrows represent measurement sites) slightly decreased whereas the pigmentation level of the test lotion-treated NLS (blue arrows represent measurement sites) did not have any distinct changes ( Figure 3 ). In contrast, the pigmentation level of the placebo lotion-treated SLs (red arrows represent measurement sites) and the NLS (blue arrows represent measurement sites) at 24 weeks remained unchanged compared to week 0 ( Figure 3 ) L and MI values of SLs To evaluate anti-pigmenting effects on SLs, lightness (L) values and melanin index (MI) values reflecting pigmentation levels (see ref 6, 10, 11, 22, 29) were measured in the test lotion or the placebo lotion-treated (previously assigned) SLs using a color difference meter and a Mexameter MX18, respectively, at 0, 12 and 24 weeks of treatment. In these mechanical evaluations, an increase in L values and a decrease in MI values could reflect a decrease in the pigmentation levels (see ref 6, 10, 11, 22, 29). In the test lotion-treated SLs, the L values significantly (p<0.0001) increased at 12 and 24 weeks of treatment compared to week 0 with a significant (p<0.0001) increase between 12 and 24 weeks ( Figure 4A ). In the placebo lotion-treated SLs, the L values also significantly (p<0.01) increased at 12 and 24 weeks of treatment compared to week 0 without any significant increase between 12 and 24 weeks ( Figure 4A ). Comparisons of increased (△) L values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly (p<0.05, p<0.0001 and p<0.001) higher △L values than the placebo lotion-treated SLs at 0~12, 0~24 and 12~24 weeks, respectively, of treatment ( Figure 4B ). While over a 2.0 △L and a 1.6 △L are distinctly or slightly, respectively, recognizable levels by the subjects [22], the ratio of subjects with over a 2.0 △L or 1.6 △L of the SLs was 6 or 15 of 27 for the test lotion and 0 or 1, respectively, of 27 for the placebo lotion at 0~24 weeks ( Figure 4B ). The MI values were significantly (p<0.0001) decreased at 12 and 24 weeks of treatment compared to week 0 in the test lotion-treated SLs with a significant (p<0.0001) decrease between 12 and 24 weeks ( Figure 4C ), while in the placebo lotion-treated SLs, the MI values also significantly (p<0.0001) decreased at 12 and 24 weeks of treatment compared to week 0 with a significant (p<0.01) decrease between 12 and 24 weeks ( Figure 4C ). Comparisons of decreased (△) MI values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly (p<0.001, p<0.0001 and p<0.001) lower △MI values than the placebo lotion-treated SLs at 0~12, 0~24 and 12~24 weeks, respectively, of treatment ( Figure 4D ). While over a 50 △MI is a distinctly recognizable level [22], the ratio of subjects with over a 50 △MI of the SLs was 13 of 27 for the test lotion and 2 of 27 for the placebo lotion at 0 ~24 weeks ( Figure 4D ). These findings suggest that the test lotion but not the placebo lotion has a distinct anti-pigmenting effect on SLs at both the color difference and MI levels. L and MI values of NLS To evaluate the whitening effects on NLS, L values and MI values were measured in the test lotion or the placebo lotion-treated (previously assigned) NLS using a color difference meter and a Mexameter MX18, respectively, at 0, 12 and 24 weeks of treatment. In the test lotion-treated NLS, L values significantly (p<0.05 and p<0.001) increased at 12 and 24 weeks, respectively, of treatment compared to week 0 ( Figure 5A), whereas in the placebo lotion-treated NLS, L values did not increase at 12 and 24 weeks of treatment compared to week 0. Comparisons of △L values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS had a significantly (p<0.0001 and p<0.01) higher △L value than the placebo lotion-treated NLS at 0~24 and 12~24 weeks of treatment ( Figure 5B ). While over a 1.6 △L is a slightly recognizable level [22], the ratio of subjects with over a 1.6△L of NLS was 3 of 27 for the test lotion and 0 of 27 for the placebo lotion. In the test lotion-treated NLS, MI values significantly (p<0.0001) decreased at both 12 and 24 weeks of treatment compared to week 0 with a significant (p<0.0001) decrease between 12 and 24 weeks ( Figure 5C) , while in the placebo lotion-treated NLS, MI values also significantly (p<0.0001) decreased at both 12 and 24 weeks of treatment compared to week 0, but not accompanied with any significant decrease between 12 and 24 weeks. Comparisons of △ MI values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS had a significantly (p<0.001, p<0.0001 and p<0.01) lower △MI value than the placebo lotion-treated NLS at 0~12, 0~24 and 12~24 weeks, respectively, of treatment ( Figure 5D ). While over a 30 △MI is a slightly recognizable level [22], the ratio of subjects with over a 30 △MI of NLS was 10 of 27 for the test lotion and 1 of 27 for the placebo lotion. These findings suggest that the test lotion but not the placebo lotion has a distinct whitening effect on NLS at the color difference and MI levels. Correlations between L and MI values in SLs and NLS Correlation plots between L and MI values are shown in Figure 6 for the SLs, the NLS and the SLs+ the NLS which indicated that L and MI values have a good correlation in the SLs, the NLS and the SLs+ the NLS. Figure 7 shows correlation plots between L and MI values in the test or the placebo lotion-treated SLs and NLS. Those correlation plots demonstrated that in the test lotion-treated SLs ( Figure 7A ), while the slopes of the correlation plots occur at a similar level at 0, 12 and 24 weeks of treatment, their intercepts distinctly decrease with a parallel shift through 0, 12 and 24 weeks of treatment. Those results indicate that the test lotion has a distinct potential to increase L values and decrease MI values, reflecting its distinct anti-pigmenting effects. In contrast, treatment with the placebo lotion did not have a tendency similar to the test lotion-treated SLs ( Figure 7B ). In the test lotion-treated NLS ( Figure 7C ), while the slopes of the correlation plots occur at a similar level at 0, 12 and 24 weeks of treatment, their intercepts distinctly decrease with a parallel shift through 0, 12 and 24 weeks of treatment. That indicates that the test lotion has a distinct potential capable of increasing L values and decreasing MI values, reflecting the whitening effects on NLS. In contrast, in the placebo lotion-treated NLS, the correlation plots did not have a such tendency ( Figure 7D ). General clinical evaluation During the test period of 24 weeks, there were no skin problems including skin irritation or hypopigmentation in the test lotion or the placebo lotion-treated skin. Therefore, the test lotion was considered by a trained dermatologist to be safe for long term use. A general clinical evaluation was also performed by a trained dermatologist (KN) at 0, 12 and 24 weeks of treatment. That evaluation demonstrated that treatments with the test lotion or the placebo lotion induced a significant decrease in the scores of scaling and stinging sensations at 24 weeks ( Figure 8B, F ), whereas the same treatments did not cause any changes in dryness, itchiness, erythema, or papules at 12 and 24 weeks ( Figure 8A, C, D, E ). Stinging sensations were significantly (p<0.001) ameliorated after treatment with the test lotion or the placebo lotion for 12 and 24 weeks compared with week 0 ( Figure 8F ). These findings indicate that both the test and the placebo lotions have a distinct ameliorating effect on scaling and stinging sensation without any skin irritations. Clinical evaluation of skin redness based on erythema index values measure d using a Mexameter MX18 Since erythema index values obtained using a Mexameter MX18 can serve as a measure of skin redness [10], erythema index values of the test lotion-treated (previously assigned) SLs were measured at 0, 12 and 24 weeks of treatment and were compared with those of the placebo lotion-treated (previously assigned) SLs. In the test lotion-treated SLs, erythema index values did not change at 12 and 24 weeks of treatment compared to week 0 ( Figure 9A) . In contrast, in the placebo lotion-treated SLs, erythema index values significantly (p<0.05) increased at 24 weeks of treatment compared to week 0 ( Figure 9A) . Comparisons of increased (△) erythema index values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had similar △erythema index values compared to the placebo lotion-treated SLs at both 12 and 24 weeks of treatment ( Figure 9C ). In the test lotion-treated (previously assigned) NLS, erythema index values did not change at 12 and 24 weeks of treatment compared to week 0 ( Figure 9B) . However, in the placebo lotion-treated NLS, erythema index values significantly (p<0.0001) increased at 12 and 24 weeks of treatment compared to week 0 ( Figure 9B) . Comparisons of increased erythema index values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS had significantly (p<0.05) lower △L erythema index values than the placebo lotion-treated NSL at both 12 and 24 weeks of treatment ( Figure 9D ). These results indicate that the test lotion but not the placebo lotion has a distinct potential to ameliorate skin redness, which may reflect a weak anti-inflammatory effect of the test lotion. Discussion AGAC is thought to serve as a tyrosinase inhibitor following its conversion to ascorbic acid via its deglucosylation by epidermal α-glucosidases after it penetrates into the epidermis. α‑glucosidases are known to exist in the epidermis as a protein glycosylation processing enzyme that can break the glucose of asparagine-linked carbohydrate moieties bound to proteins to release glucose in the Golgi area of keratinocytes [19]. In order for the action of tyrosinase inhibitors to be effective, it was essential to know whether the hyperpigmentation of SLs is accompanied by an accentuated expression of the key melanogenic enzyme tyrosinase in the lesional melanocytes. In this connection, it had already been reported that the hyperpigmentation in SLs occurs in concert with the up-regulated mRNA levels of tyrosinase in the increased numbers of tyrosinase-positive melanocytes in the SLs lesional epidermis [20]. The present double-blind half-face study of subjects with SLs demonstrated that, although the placebo lotion-treated SLs had significant (p<0.05 and p<0.01) decreases in pigmentation scores at 12 and 24 weeks, respectively, compared to 0 week, the test lotion-treated SLs had more marked significant (p<0.001 and p<0.0001) decreases than the placebo’s ones in pigmentation scores at 12 and 24 weeks, respectively, compared to week 0, accompanied with a significant (p<0.01) decrease in pigmentation scores at 24 weeks compared to the placebo’s ones (see Figure 2). These results suggest that AGAC has a weak but distinct potential to ameliorate the clinical hyperpigmentation level of SLs. This clinical anti-pigmenting effect was corroborated by mechanical observations using a color difference meter and a Mexameter. In those mechanical evaluations, although both the test and the placebo lotions significantly increased L values or decreased MI values at 12 and 24 weeks of treatment, comparisons of the increased L (△L) values or the decreased (△) MI values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly higher △L and △MI values than the placebo lotion-treated SLs at 0~12, 12~24 and 0~24 weeks of treatment (see Figure 4). Since the significant anti-pigmenting effects of the placebo lotion might reflect seasonal changes in skin color from September to March during this clinical study, the significant differences observed in both the △L and △MI values at 12 and 24 weeks compared to week 0 between the test and the placebo lotions suggest that AGAC has a distinct anti-pigmenting effect on SLs at the color difference and MI levels. Further, our findings that the ratio of subjects with distinctly recognizable levels of over a 2.0 △L value or a 50 △MI value of SLs was 6 or 13 of 27 for the test lotion, and 0 or 2, respectively, of 27 for the placebo lotion at 0~24 weeks (see Figure 4) strongly suggest that AGAC is distinctly effective in diminishing the hyperpigmentation levels of SLs at a visibly recognizable level by the subjects themselves. In the time course of the anti-pigmenting effects measured as L values especially by a color difference meter, the significant effects of the test lotion on SLs occurred in a step-by-step manner at 12 and 24 weeks of treatment with significantly increased changes even at 12~24 weeks. In contrast, those significant effects of the placebo lotion also occurred at 12 and 24 weeks of treatment, but not accompanied with any significant changes at 12~24 weeks (see Figure 4A). These time course trends of the anti-pigmenting effects by the test lotion could provide an insight into predicting more distinct anti-pigmenting effects by possibly further prolonged treatments with the test lotion. Of considerable interest is that in the test lotion-treated NLS, both the L and MI values significantly increased or decreased at 12 and 24 weeks of treatment compared to week 0. In contrast, in the placebo lotion-treated NLS, the L values did not increase at 12 and 24 weeks of treatment compared to week 0, while the MI values significantly decreased at 12 and 24 weeks of treatment (see Figure 5C). Although the significant decrease of the MI values in the placebo lotion-treated NLS seems to occur due to the seasonal variation from September to March during this clinical study, comparisons of increased L (△L) values or decreased MI (△MI) values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS have significantly higher △L and lower △MI values than the placebo lotion-treated NLS at 0~24 and 12~24 weeks of treatment (see Figure 5). Although the increased or decreased levels of L or MI values occur at lower levels than 2.0 △L or 50 △MI (distinctly recognizable levels by the subjects), respectively, at 0~24 week, the ratio of subjects with over a 1.6△L or a 30 △MI (slightly recognizable levels by the subjects) of NLS at 0~24 weeks was 3 and 10 of 27 for the test lotion, and 0 and 1, respectively, of 27 for the placebo lotion (see Figure 5). The sum of these findings indicates that the test lotion has a significantly higher whitening effect on NLS than the placebo lotion and suggests that AGAC has a weak but significant whitening effect on NLS at visibly recognizable levels. It is of considerable interest to compare the anti-pigmenting effects on SLs and the whitening effects on NLS between AGAC and ASP, because the latter has been reported to have both anti-pigmenting and whitening effects in subjects with SLs [22]. Although both compounds have similar anti-pigmenting and whitening effects on SLs and NLS, respectively, as revealed by evaluation of L and MI values, a major difference occurs at the clinical scoring levels of pigmentation in SLs in which AGAC but not ASP exhibited a significant (p<0.0001) decrease in pigmentation scores at 24 weeks compared to week 0, accompanied with a significant (p<0.01) decrease in pigmentation scores at 24 weeks compared to the placebo lotion-treated SLs (see Figure 2). This indicates a slight superiority of AGAC to ASP from a clinical point of view although the production cost is much higher for AGAC than ASP. It is likely that the slight superiority of AGAC to ASP can be ascribed to the higher concentration used, i.e. 28% AGAC compared to 6% ASP, despite the fact that the rate of penetration into the epidermis is thought to be much higher for ASP than AGAC. A major skin problem that occurs during the long-term topical application of anti-pigmenting agents is skin irritation as is frequently observed for HQ [5.15,52]. Since such a long time of topical applications is required to attain a distinct anti-pigmenting effect in dark-skinned individuals with SLs, skin irritation that happens during the treatment is a major causative factor for not being able to continue the topical applications. Therefore, general clinical evaluations of skin symptoms, including skin irritation, are important and were carried out in this study by a trained dermatologist (KN). These clinical evaluations demonstrated that, while the test lotion rather significantly ameliorated scaling and stinging sensations at 24 weeks, there was no appearance of erythema, papules or itchiness during the 24 weeks of treatment. Further, based on the evidence that erythema index values measured using a Mexameter can serve as a reflection of skin redness due to hemoglobin levels in the blood flow [10], our evaluations revealed that the test lotion but not the placebo lotion has a distinct potential capable of diminishing skin redness. These findings strongly suggest that AGAC could act as an anti-pigmenting agent with a weak anti-inflammatory effect. In conclusion, the sum of the above findings indicates that AGAC has a weak but significant anti-pigmenting effect on SLs and a significant whitening effect even on normally pigmented skin without the risk of eliciting hypopigmentation or skin irritation. This provides skin safety at a sufficient level to use for a long period time of topical applications, which is an essential requirement to achieve distinct anti-pigmenting effects on SLs. Abbreviations SLs, solar lentigos; AGAC, Ascorbyl Glucoside Arginine Complex; EDN, endothelin; SCF, stem cell factor; NLS, non-lesional surrounding skin; AG, Ascorbyl Glucoside; AMP, L-ascorbate-2-phosphate Mg; APS, L-ascorbate-2-phosphate trisodium salt; HQ, hydroquinone; MI, melanin index; TNF, tumor necrosis factor; UVB, ultraviolet B Declarations Author Contributions: A.Y. and G.I. designed this study. M.T., K.N. and M.N. analyzed the data and M.T. and G.I. wrote the manuscript. M.N. and A.Y. financially supported this study. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national re-search committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Data Availability Statement: All relevant data are included in the manuscript. Acknowledgments: Dr. Vincent Hearing has critically read this manuscript and made some kind and useful suggestions. Conflicts of Interest: The authors state no conflict of interest. Authors, Masahiko Nakano and Akio Yamamoto were employed by the company, Doctor’s Choice Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ORCID Mariko Takada https://orcid.org/0009-0005-2469-0047 Kayoko Numano https://orcid.org/0009-0000-2836-7574 Masahiko Nakano https://orcid.org/0000-0002-0133-6396 Akio Yamamoto https://orcid.org/0000-0002-4919-8535 Genji Imokawa https://orcid.org/0000-0001-7312-4475 References Arndt, K. A., & Fitzpatrick, T. B. (1965). Topical use of hydroquinone as a depigmenting agent. Jama, 194 (9), 965-967. Breathnach, A. C., Nazzaro-Porro, M., Passi, S., & Zina, G. (1989). Azelaic acid therapy in disorders of pigmentation. Clin Dermatol, 7 (2), 106-119. doi:10.1016/0738-081x(89)90061-8 Cabanes, J., Chazarra, S., & Garcia-Carmona, F. (1994). Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. J Pharm Pharmacol, 46 (12), 982-985. doi:10.1111/j.2042-7158.1994.tb03253.x Chen, Y. S., Lee, S. M., Lin, C. C., Liu, C. Y., Wu, M. C., & Shi, W. L. (2013). Kinetic study on the tyrosinase and melanin formation inhibitory activities of carthamus yellow isolated from Carthamus tinctorius L. J Biosci Bioeng, 115 (3), 242-245. doi:10.1016/j.jbiosc.2012.09.013 Cheong, K. A., Kim, H. J., Kim, J. Y., Kim, C. H., Lim, W. S., Noh, M., & Lee, A. Y. (2014). Retinoic acid and hydroquinone induce inverse expression patterns on cornified envelope-associated proteins: implication in skin irritation. 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Comparative Studies on the Photoreactivity, Efficacy, and Safety of Depigmenting Agents. Pharmaceuticals (Basel), 17 (1). doi:10.3390/ph17010055 Murtaza, F., Bangash, A. R., Khushdil, A., & Noor, S. M. (2016). Efficacy of Trichloro-Acetic Acid Peel Alone Versus Combined Topical Magnesium Ascorbyl Phosphate for Epidermal Melasma. J Coll Physicians Surg Pak, 26 (7), 557-561. Nayama, S., Takehana, M., Kanke, M., Itoh, S., Ogata, E., & Kobayashi, S. (1999). Protective effects of sodium-L-ascorbyl-2 phosphate on the development of UVB-induced damage in cultured mouse skin. Biol Pharm Bull, 22 (12), 1301-1305. doi:10.1248/bpb.22.1301 Pillaiyar, T., Manickam, M., & Namasivayam, V. (2017). Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. J Enzyme Inhib Med Chem, 32 (1), 403-425. doi:10.1080/14756366.2016.1256882 Rajaratnam, R., Halpern, J., Salim, A., & Emmett, C. (2010). Interventions for melasma. 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Histopathological, morphometric and stereological studies of ascorbic acid and magnesium ascorbyl phosphate in a skin care formulation. Int J Cosmet Sci, 22 (3), 169-179. doi:10.1046/j.1467-2494.2000.00017.x Son, K. H., & Heo, M. Y. (2013). The evaluation of depigmenting efficacy in the skin for the development of new whitening agents in Korea. Int J Cosmet Sci, 35 (1), 9-18. doi:10.1111/ics.12012 Tai, Y., Wang, C., Wang, Z., Liang, Y., Du, J., He, D., . . . Liu, B. (2017). Involvement of Transient Receptor Potential Cation Channel Member A1 activation in the irritation and pain response elicited by skin-lightening reagent hydroquinone. Sci Rep, 7 (1), 7532. doi:10.1038/s41598-017-07651-5 Thawabteh, A. M., Jibreen, A., Karaman, D., Thawabteh, A., & Karaman, R. (2023). Skin Pigmentation Types, Causes and Treatment-A Review. Molecules, 28 (12). doi:10.3390/molecules28124839 Tse, T. W., & Hui, E. (2013). Tranexamic acid: an important adjuvant in the treatment of melasma. J Cosmet Dermatol, 12 (1), 57-66. doi:10.1111/jocd.12026 Verallo-Rowell, V. M., Verallo, V., Graupe, K., Lopez-Villafuerte, L., & Garcia-Lopez, M. (1989). Double-blind comparison of azelaic acid and hydroquinone in the treatment of melasma. Acta Derm Venereol Suppl (Stockh), 143 , 58-61. doi:10.2340/000155551435861 Wang, P. C., Huang, Y. L., Hou, S. S., Chou, C. H., & Tsai, J. C. (2013). Lauroyl/palmitoyl glycol chitosan gels enhance skin delivery of magnesium ascorbyl phosphate. J Cosmet Sci, 64 (4), 273-286. Won, Y. K., Loy, C. J., Randhawa, M., & Southall, M. D. (2014). Clinical efficacy and safety of 4-hexyl-1,3-phenylenediol for improving skin hyperpigmentation. Arch Dermatol Res, 306 (5), 455-465. doi:10.1007/s00403-014-1439-9 Yamamoto, K., Shichiri, H., Ishida, T., Kaku, K., Nishioka, T., Kume, M., . . . Hirai, M. (2017). Effects of Ascorbyl-2-phosphate Magnesium on Human Keratinocyte Toxicity and Pathological Changes by Sorafenib. Biol Pharm Bull, 40 (9), 1530-1536. doi:10.1248/bpb.b17-00386 Zheng, Y., Du, X., Zhang, L., Jia, T., Zhang, H., Peng, B., . . . Geng, S. (2023). Hydroquinone-induced skin irritant reaction could be achieved by activating mast cells via mas-related G protein-coupled receptor X2. Exp Dermatol, 32 (4), 436-446. doi:10.1111/exd.14723 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5332003","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370516730,"identity":"c3fc58cb-9479-40e2-a4a2-4a1093933b41","order_by":0,"name":"Mariko Takada","email":"","orcid":"","institution":"Utsunomiya University","correspondingAuthor":false,"prefix":"","firstName":"Mariko","middleName":"","lastName":"Takada","suffix":""},{"id":370516731,"identity":"61e9b596-89d0-45f1-8be3-92f86f49500d","order_by":1,"name":"Kayoko Numano","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kayoko","middleName":"","lastName":"Numano","suffix":""},{"id":370516732,"identity":"8a151555-695d-46fb-9d86-6ceeb73de390","order_by":2,"name":"Masahiko Nakano","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Masahiko","middleName":"","lastName":"Nakano","suffix":""},{"id":370516733,"identity":"1e737392-6d37-4610-8551-d84baf4100bf","order_by":3,"name":"Akio Yamamoto","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Akio","middleName":"","lastName":"Yamamoto","suffix":""},{"id":370516734,"identity":"4cbd0989-9fea-4a1b-baca-f03fc31ff222","order_by":4,"name":"Genji Imokawa","email":"data:image/png;base64,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","orcid":"","institution":"Utsunomiya University","correspondingAuthor":true,"prefix":"","firstName":"Genji","middleName":"","lastName":"Imokawa","suffix":""}],"badges":[],"createdAt":"2024-10-25 11:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5332003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5332003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67680110,"identity":"f95a19e5-b81e-4f9c-88e1-b8db6faee7ac","added_by":"auto","created_at":"2024-10-28 15:40:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical structure of AGAC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/d4dc0dcc7fba14d8eaf2e5ee.png"},{"id":67680109,"identity":"b453236d-fdc5-468b-b1c9-6ae977dd7a5e","added_by":"auto","created_at":"2024-10-28 15:40:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":153277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of pigmentation scores of previously assigned SLs at 0, 12 and 24 weeks of treatment. \u003c/strong\u003eBar represents mean ± SD. n=27, ****: p\u0026lt;0.0001, ***: p\u0026lt;0.001, **: p\u0026lt;0.01, *: p\u0026lt;0.05 by Tukey’s comparison test, followed by ANOVA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/e9aafef0fc0470750659bde3.png"},{"id":67680112,"identity":"f5d6bfc6-a71f-4256-a7d5-1c0cb60c5953","added_by":"auto","created_at":"2024-10-28 15:40:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":693633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical photographs of SLs and NLS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eSubject #7) Test Lotion/SLs (red arrows) at 0 and 24 weeks: ΔL value: 2.23, ΔMI value: 44.33. Test Lotion/NLS (blue arrows) at 0 and 24 weeks: ΔL value: 0.49, ΔMI value: 20.00. Placebo Lotion/SLs (red arrows) at 0 and 24 weeks: ΔL value: -0.24, ΔMI value: 40.00. Placebo Lotion/NLS (blue arrows) at 0 and 24 weeks: ΔL value: 0.21, ΔMI value: 3.67. \u003cstrong\u003eB: \u003c/strong\u003eSubject #12) Test Lotion/SLs (red arrows) at 0 and 24 weeks: ΔL value: 2.57, ΔMI value: 76.00. Test Lotion/NLS (blue arrows) at 0 and 24 weeks: ΔL value: 0.22, ΔMI value: 49.00, Placebo Lotion/SLs (red arrows) at 0 and 24 weeks: ΔL value: 1.27, ΔMI value: 44.00. Placebo Lotion/NLS (blue arrows) at 0 and 24 weeks: ΔL value: 1.20, ΔMI value: 21.0.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/bf3b032c326281dcebc5e1b3.png"},{"id":67681415,"identity":"1c4c761e-8731-4d66-bc0a-5aed4d082cc8","added_by":"auto","created_at":"2024-10-28 15:56:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in L values and MI values of SLs after treatment for 24 weeks.\u003c/strong\u003e \u003cstrong\u003eA/C:\u003c/strong\u003e Time course study, Bar represents mean ± SD. n=27, ****: p\u0026lt;0.0001, **: p\u0026lt;0.01 by Tukey’s comparison test, followed by ANOVA, \u003cstrong\u003eB/D:\u003c/strong\u003e Increased (△) L values and decreased (△) MI values between 0 and 12, 0 and 24, and 12 and 24 weeks. Bar represents mean ± SD. n=27, ****: p\u0026lt;0.0001, ***: p\u0026lt;0.001, *: p\u0026lt;0.05 by paired t test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/f69d64be98ce2a1ad7e725e1.png"},{"id":67680114,"identity":"5f29d3a4-6490-4dd6-8e56-cb21c8f1b73a","added_by":"auto","created_at":"2024-10-28 15:40:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in L values and MI values of NLS after treatment for 24 weeks., A/C: \u003c/strong\u003eTime course study, Bar represents mean ± SD. n=27, ****: p\u0026lt;0.0001; ***: p\u0026lt;0.001, **: p\u0026lt;0.01 by Tukey’s comparison test, followed by ANOVA, \u003cstrong\u003eB/D:\u003c/strong\u003e Increased (△) L values and decreased (△) MI values between 0 and 12. 0 and 24, and 12 and 24 weeks. Bar represents mean ± SD, n=27, ****: p\u0026lt;0.0001, ***: p\u0026lt;0.001 by paired t test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/b5d9f8bf564965cd6c70a994.png"},{"id":67680425,"identity":"de150bc0-88c5-4644-8195-f691c42e6dae","added_by":"auto","created_at":"2024-10-28 15:48:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":125682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between L and MI values in the SLs, the NLS and the SLs + the NLS during this clinical study. A: \u003c/strong\u003eCorrelation between L and MI values in SLs,\u003cstrong\u003e B: \u003c/strong\u003eCorrelation between L and MI values in the NSL, \u003cstrong\u003eC: \u003c/strong\u003eCorrelation between L and MI values in the SLs + the NSL.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/33b169ab7e13dfd564d0966c.png"},{"id":67680117,"identity":"7127dab6-4216-4b41-a5ea-5fa4712486e2","added_by":"auto","created_at":"2024-10-28 15:40:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":156055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between L and MI values in the SLs and the NLS at 0, 12 and 24 weeks of treatment with the test or the placebo lotion.\u003c/strong\u003e \u003cstrong\u003eA:\u003c/strong\u003eCorrelation between L and MI values in the test lotion-treated SLs, \u003cstrong\u003eB:\u003c/strong\u003eCorrelation between L and MI values in the placebo lotion-treated SLs, \u003cstrong\u003eC: \u003c/strong\u003eCorrelation between L and MI values in the test lotion-treated NSL, D: Correlation between L and MI values in the placebo lotion-treated NSL.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/367a6e32fbf836ed0247c33e.png"},{"id":67680426,"identity":"272ed2ef-b477-4f56-9780-c6c1bbe50cac","added_by":"auto","created_at":"2024-10-28 15:48:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":77084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical effects of the test and placebo lotions on facial skin symptoms of subjects with SLs \u003c/strong\u003eA: Dryness, B: Scaling, C: Itchy Sensation, D: Erythema, E: Papules, F: Stinging Sensation. Clinical scoring was performed at 0, 12 and 24 weeks according to the criteria described in the Materials and Methods section. n=27, *: p\u0026lt;0.05, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001, compared to week 0, All data were analyzed by Friedman test and Dunn’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/10924e5691624a53350533a7.png"},{"id":67680115,"identity":"0e3a22a8-e79e-4d40-b99e-36f431957d74","added_by":"auto","created_at":"2024-10-28 15:40:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":391249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical effects of the test and the placebo lotions on the intensity of skin redness measured as erythema index with a Mexameter MX18.\u003c/strong\u003e \u003cstrong\u003eA: \u003c/strong\u003eTime course study in the lotions- treated SLs, Bar represents mean ± SD. n=27, *: p\u0026lt;0.05, by Tukey’s multiple comparison test, followed by ANOVA, \u003cstrong\u003eB:\u003c/strong\u003eTime course study in the lotions-treated NLS, Bar represents mean ± SD. n=27, ****: p\u0026lt;0.0001, by Tukey’s multiple comparison test, followed by ANOVA, \u003cstrong\u003eC: \u003c/strong\u003eIncreased (△) erythema index values between 0 and 12, 0 and 24, and 12 and 24 weeks in the lotions-treated SLs. Bar represents mean ± SD. n=27, \u003cstrong\u003eD:\u003c/strong\u003e Increased (△) erythema index values between 0 and 12, 0 and 24, and 12 and 24 weeks in the lotions-treated NSL, Bar represents mean ± SD. n=27, *: p\u0026lt;0.05 by paired t test.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/db675a0f0938e4b3047a4a5e.png"},{"id":67886616,"identity":"8f16d500-a7a1-4a04-9af2-35a3ce1eed29","added_by":"auto","created_at":"2024-10-30 18:31:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2983682,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5332003/v1/45d77960-5804-47c4-8b0d-a80ecc6d00e4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Treatment with Ascorbyl Glucoside Arginine Complex ameliorates solar lentigos","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSolar lentigos (SLs) are hyperpigmented lesions that frequently occur on sun-exposed skin, especially on the face and the dorsum of the hands of Asian subjects [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on the frequency of the final diagnosis of patients with various pigmentary disorders in Japan, SLs have the highest incidence, occurring in approximately 60% of all patients with hyperpigmentary disorders, while melasma and post-inflammatory hyperpigmentation (including ultraviolet B (UVB) melanosis) occur in as few as 5.2% and 3.3% of patients, respectively [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn general, hyperpigmentary disorders, including UVB-melanosis, SLs and melasma, are targeted by anti-pigmenting agents. However, an effective anti-pigmenting treatment for SLs is difficult especially for dark-skinned individuals because the treatment is required to reduce the hyperpigmentation without causing any undesirable hypopigmentation or contact irritation in the surrounding normally appearing pigmented skin. It is thought that hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders including SLs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], but treatment with HQ often causes skin irritation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Although many whitening agents are available [2, 3, 4, 7, 8, 14, 17, 18, 25, 30, 34, 37, 39, 44, 46, 47. 48, 50], some of which are targeted and approved especially to treat UVB-induced hyperpigmentation in Japan, little is known about the potential anti-pigmenting effects of those whitening agents on SLs because clinical evaluations of SLs are not required for approval as a whitening agent in Japan. Excluding kojic acid and rhododendrol, which have been reported to elicit hypopigmentation on the faces of dark-skinned individuals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], other whitening agents approved in Japan are suitable candidates for investigating their potential anti-pigmenting effects on SLs because they have been proven to be substantially safe in terms of hypopigmentation and skin irritation since they have been commercially available for a long period of time. Among those whitening agents, ascorbic acid derivatives are thought to be invaluable agents especially from the skin safety point of view.\u003c/p\u003e \u003cp\u003eL-ascorbate-2-phosphate Mg (AMP), a whitening agent first approved in Japan, was reported to have a significant ameliorating effect on chloasma by acting as a tyrosinase inhibitor after it is enzymatically degraded by intrinsic epidermal phosphatases to release free ascorbic acid [9. 23, 28, 35, 42, 43, 49, 51]. L-ascorbate-2-phosphate trisodium salt (APS) is another approved whitening agent in Japan [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. APS is a modified derivative of AMP that improves its stability, namely its aggregation due to the Mg salt [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Ascorbyl glucoside (AG) is also a whitening agent approved in Japan that has been documented to have a depigmenting effect on UVB-hyperpigmentation by acting as a tyrosinase inhibitor itself or after it is converted by intrinsic epidermal glucosidase to free ascorbic acid [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the requirement for approval of whitening agents in Japan, it is well established that topical application of AMP, APS or AG for 21 days on UVB (2MED)-exposed human skin significantly inhibits the UVB-increased pigmentation measured as L values at 21 days post-UVB irradiation. However, there had been no published data on the anti-pigmenting effect of topical treatment with APS, AMP or AG on SLs in a double-blind half-face study, although a whole-face study using AMP on SLs for 3 months was reported to have some efficacy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the study of which was flawed due to the lack of a placebo control. We have recently reported for the first time that in a double-blind half-face study of 27 Japanese female subjects with SLs using lotions with or without 6% APS (test lotion and placebo lotion, respectively) applied twice a day for 24 weeks, APS has a weak but significant anti-pigmenting effect on SLs and also a significant whitening effect even on normally pigmented non-lesional surrounding skin (NLS) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, that clinical study was not satisfactory for clinical evaluation because there was no significant difference in the pigmentation scores of SLs judged by a dermatologist between the test and placebo lotions. This prompted us to characterize the anti-pigmenting effects of ascorbyl glucoside arginine complex (AGAC) on SLs via the modification of AG by making a complex with arginine to reduce its acidity and to increase the concentration used to 28%. In this study, we conducted a double-blind half-face study of 27 Japanese female subjects with SLs using lotions with or without 28% AGAC (test lotion and placebo lotion, respectively) applied twice a day for 24 weeks. Here we show that repeated topical treatment with AGAC has a significant anti-pigmenting effect on SLs with a significant difference in pigmentation scores of SLs judged by a dermatologist between the test and the placebo lotions. Those results were corroborated by mechanical evaluations using a color difference meter and a Mexameter, and show that there was a significant whitening effect even on normally pigmented NLS without any hypo-pigmenting effects at a mechanical skin color level.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTest materials\u003c/h2\u003e \u003cp\u003eThe chemical structure of AGAC is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The test and placebo lotions were provided by Doctor\u0026rsquo;s Choice Co., Ltd. The test lotion was comprised of 28% AGAC and other ingredients as listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The placebo lotion was comprised of the same components as the test lotion except it did not include AGAC. Ascorbyl glucoside and arginine were purchased from Technoble Co., Ltd. (Osaka, Japan) and Technoscience Co., Ltd. (Kashiwa, Japan), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFull ingredients list of the test lotion (containing 28% AGAC) and the placebo lotion (without AGAC).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWith or without 28% Ascorbyl Glucoside Arginine Complex (AGAC), Water, Propanediol, Butylene Glycol (BG), Betaine, Pentylene Glycol, 1,2-Hexanediol, PEG‐60 Hydrogenated Castor Oil, PEG‐9M, Citric Acid, Sodium Citrate, Glycerin, Sphingolipids, Hydrolyzed Elastin, Hydrolyzed Collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eThis study was performed from September 2023 to March 2024, in the Ebisu Skin Research Center, Inforward Co. LTD, Tokyo, Japan. Twenty seven Japanese female volunteers with SLs were instructed to apply lotions with or without 28% AGAC (test lotion and placebo lotion, respectively) twice a day for 24 weeks on the entire right and left sides of their faces. They were also instructed in a double-blind manner to treat with the test lotion on one-half of their face and with the placebo lotion on the opposite side.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this study involving human subjects were in accordance with the ethical standards of the Institutional and/or National Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The present study adhered to the tenets of the Declaration of Helsinki and was reviewed and approved by the Japanese Ethics Committee, Clinical Research Review Center, General Incorporated Association (Approval No: CrrC23-020). A formal informed consent was obtained from each subject before the study. The UMIN study ID was assigned as UMIN000052115. Following screening by a dermatologist, 27 Japanese female volunteers with SLs on the entire right and left sides of their faces were enrolled in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of pigmentation level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePigmentation levels in previously assigned SLs were evaluated using a photo-scale ranging from 1.0 to 5.0 of SLs on the face by a trained dermatologist (KN). Mechanical evaluation of pigmentation levels was performed according to the methods described previously [22]. In brief, the pigmentation levels in the same previously assigned SLs and non-lesional surrounding skin (NLS) were measured using a color difference meter CM-700 d (Konica Minolta Japan, Inc) to determine L values [22] and using a Mexameter MX18 (Courage+Khazaka Electronic GmbH) [6, 10, 11, 29] to measure Melanin Index (MI) values\u0026nbsp;at 0, 12 and 24 weeks of treatment. The average values of 5 times measurements at the same SLs and NLS were used as data for figure description.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral clinical evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA general clinical evaluation was performed for dryness, scaling, erythema, papules, stinging sensations or itchiness with 1 to 5 scoring grades which correspond to none, weak, mild, moderate and severe by a trained dermatologist (KN) in the entire half faces at 0, 12 and 24 weeks of treatment.\u0026nbsp; \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of skin redness level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor mechanical evaluation of skin irritation, skin redness levels in the same previously assigned SLs and NLS were evaluated using a Mexameter MX18 as erythema index values, which reflect skin redness levels based on hemoglobin levels related to skin blood flow\u0026nbsp;[10].\u0026nbsp;The average values of 5 times measurements at the same SLs and NLS were used as data for figure description.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are expressed as means ± standard deviation (SD) unless noted otherwise. Prism 10 from Graph Pad was used as a software for statistics analysis. For pairwise comparisons, t test was used. ANOVA and Tukey’s comparison test were used to evaluate pigmentation scores and changes in L values, MI values and erythema index values. Friedman’s test and Dunn’s multiple comparisons test were used for skin scoring. p values \u0026lt; 0.05 are considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eVisible pigmentation level of SLs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTopical applications of lotions with or without 28% AGAC (test lotion and placebo lotion, respectively) were carried out twice daily for 24 weeks on the entire right and left sides of the subjects\u0026rsquo; faces with SLs. The pigmentation levels of the same previously assigned SLs on the subjects\u0026rsquo; faces were assessed at 0, 12 and 24 weeks using a photo-scale ranging from 1.0 to 5.0 by a dermatologist (KN) and these results are shown in \u003cstrong\u003eFigure 2\u003c/strong\u003e. The results indicated that although the placebo lotion-treated SLs had significant (p\u0026lt;0.05 and p\u0026lt;0.01) decreases in pigmentation scores at 12 and 24 weeks compared to 0 week, the test lotion-treated SLs had more marked significant (p\u0026lt;0.001 and p\u0026lt;0.0001) decreases than the placebo\u0026rsquo;s ones in pigmentation scores at 12 and 24 weeks compared to week 0, together with a significant (p\u0026lt;0.01) decrease in pigmentation scores at 24 weeks compared to the placebo\u0026rsquo;s ones (\u003cstrong\u003eFigure 2\u003c/strong\u003e). These results suggest that there was a distinct anti-pigmenting effect of the test lotion on SLs at a visible clinical level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical photographs of SLs and NLS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative photographs of the faces of subjects #7 (\u003cstrong\u003eFigure 3A\u003c/strong\u003e) and #12 (\u003cstrong\u003eFigure 3B\u003c/strong\u003e) before and after treatment for 24 weeks showed that the pigmentation level of the test lotion-treated SLs (red arrows represent measurement sites) slightly decreased whereas the pigmentation level of the test lotion-treated NLS (blue arrows represent measurement sites) did not have any distinct changes (\u003cstrong\u003eFigure 3\u003c/strong\u003e). In contrast, the pigmentation level of the placebo lotion-treated SLs (red arrows represent measurement sites) and the NLS (blue arrows represent measurement sites) at 24 weeks remained unchanged compared to week 0 (\u003cstrong\u003eFigure 3\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL and MI values of SLs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate anti-pigmenting effects on SLs, lightness (L) values and melanin index (MI) values reflecting pigmentation levels (see ref 6, 10, 11, 22, 29) were measured in the test lotion or the placebo lotion-treated (previously assigned) SLs using a color difference meter and a Mexameter MX18, respectively, at 0, 12 and 24 weeks of treatment. In these mechanical evaluations, an increase in L values and a decrease in MI values could reflect a decrease in the pigmentation levels (see ref 6, 10, 11, 22, 29). In the test lotion-treated SLs, the L values significantly (p\u0026lt;0.0001) increased at 12 and 24 weeks of treatment compared to week 0 with a significant (p\u0026lt;0.0001) increase between 12 and 24 weeks (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). In the placebo lotion-treated SLs, the L values also significantly (p\u0026lt;0.01) increased at 12 and 24 weeks of treatment compared to week 0 without any significant increase between 12 and 24 weeks (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). Comparisons of increased (△) L values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly (p\u0026lt;0.05, p\u0026lt;0.0001 and p\u0026lt;0.001) higher\u0026nbsp;△L values than the placebo lotion-treated SLs at 0~12, 0~24 and 12~24 weeks, respectively, of treatment (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). While over a 2.0\u0026nbsp;△L and a 1.6\u0026nbsp;△L are distinctly or slightly, respectively, recognizable levels by the subjects [22], the ratio of subjects with over a 2.0\u0026nbsp;△L or 1.6\u0026nbsp;△L of the SLs was 6 or 15 of 27 for the test lotion and 0 or 1, respectively, of 27 for the placebo lotion at 0~24 weeks (\u003cstrong\u003eFigure 4B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe MI values were significantly (p\u0026lt;0.0001) decreased at 12 and 24 weeks of treatment compared to week 0 in the test lotion-treated SLs with a significant (p\u0026lt;0.0001) decrease between 12 and 24 weeks (\u003cstrong\u003eFigure 4C\u003c/strong\u003e), while in the placebo lotion-treated SLs, the MI values also significantly (p\u0026lt;0.0001) decreased at 12 and 24 weeks of treatment compared to week 0 with a significant (p\u0026lt;0.01) decrease between 12 and 24 weeks (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). Comparisons of decreased (△) MI values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly (p\u0026lt;0.001, p\u0026lt;0.0001 and p\u0026lt;0.001) lower\u0026nbsp;△MI values than the placebo lotion-treated SLs at 0~12, 0~24 and 12~24 weeks, respectively, of treatment (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). While over a 50\u0026nbsp;△MI is a distinctly recognizable level [22], the ratio of subjects with over a 50\u0026nbsp;△MI of the SLs was 13 of 27 for the test lotion and 2 of 27 for the placebo lotion at 0 ~24 weeks (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). These findings suggest that the test lotion but not the placebo lotion has a distinct anti-pigmenting effect on SLs at both the color difference and MI levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL and MI values of NLS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the whitening effects on NLS, L values and MI values were measured in the test lotion or the placebo lotion-treated (previously assigned) NLS using a color difference meter and a Mexameter MX18, respectively, at 0, 12 and 24 weeks of treatment. In the test lotion-treated NLS, L values significantly (p\u0026lt;0.05 and p\u0026lt;0.001) increased at 12 and 24 weeks, respectively, of treatment compared to week 0 (\u003cstrong\u003eFigure 5A),\u0026nbsp;\u003c/strong\u003ewhereas in the placebo lotion-treated NLS, L values did not increase at 12 and 24 weeks of treatment compared to week 0. Comparisons of\u0026nbsp;△L values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS had a significantly (p\u0026lt;0.0001 and p\u0026lt;0.01) higher\u0026nbsp;△L value than the placebo lotion-treated NLS at 0~24 and 12~24 weeks of treatment (\u003cstrong\u003eFigure 5B\u003c/strong\u003e). While over a 1.6\u0026nbsp;△L is a slightly recognizable level [22], the ratio of subjects with over a 1.6△L of NLS was 3 of 27 for the test lotion and 0 of 27 for the placebo lotion. In the test lotion-treated NLS, MI values significantly (p\u0026lt;0.0001) decreased at both 12 and 24 weeks of treatment compared to week 0 with a significant (p\u0026lt;0.0001) decrease between 12 and 24 weeks (\u003cstrong\u003eFigure 5C)\u003c/strong\u003e, while in the placebo lotion-treated NLS, MI values also significantly (p\u0026lt;0.0001) decreased at both 12 and 24 weeks of treatment compared to week 0, but not accompanied with any significant decrease between 12 and 24 weeks. Comparisons of\u0026nbsp;△\u0026nbsp;MI values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS had a significantly (p\u0026lt;0.001, p\u0026lt;0.0001 and p\u0026lt;0.01) lower\u0026nbsp;△MI value than the placebo lotion-treated NLS at 0~12, 0~24 and 12~24 weeks, respectively, of treatment (\u003cstrong\u003eFigure 5D\u003c/strong\u003e). While over a 30 △MI is a slightly recognizable level [22], the ratio of subjects with over a 30 △MI of NLS was 10 of 27 for the test lotion and 1 of 27 for the placebo lotion. These findings suggest that the test lotion but not the placebo lotion has a distinct whitening effect on NLS at the color difference and MI levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelations between L and MI values in SLs and NLS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrelation plots between L and MI values are shown in \u003cstrong\u003eFigure 6\u003c/strong\u003e for the SLs, the NLS and the SLs+ the NLS which indicated that L and MI values have a good correlation in the SLs, the NLS and the SLs+ the NLS. \u003cstrong\u003eFigure 7\u0026nbsp;\u003c/strong\u003eshows correlation plots between L and MI values in the test or the placebo lotion-treated SLs and NLS. Those correlation plots demonstrated that in the test lotion-treated SLs (\u003cstrong\u003eFigure 7A\u003c/strong\u003e), while the slopes of the correlation plots occur at a similar level at 0, 12 and 24 weeks of treatment, their intercepts distinctly decrease with a parallel shift through 0, 12 and 24 weeks of treatment. Those results indicate that the test lotion has a distinct potential to increase L values and decrease MI values, reflecting its distinct anti-pigmenting effects. In contrast, treatment with the placebo lotion did not have a tendency similar to the test lotion-treated SLs (\u003cstrong\u003eFigure 7B\u003c/strong\u003e). In the test lotion-treated NLS (\u003cstrong\u003eFigure 7C\u003c/strong\u003e), while the slopes of the correlation plots occur at a similar level at 0, 12 and 24 weeks of treatment, their intercepts distinctly decrease with a parallel shift through 0, 12 and 24 weeks of treatment. That indicates that the test lotion has a distinct potential capable of increasing L values and decreasing MI values, reflecting the whitening effects on NLS. In contrast, in the placebo lotion-treated NLS, the correlation plots did not have a such tendency (\u003cstrong\u003eFigure 7D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral clinical evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the test period of 24 weeks, there were no skin problems including skin irritation or hypopigmentation in the test lotion or the placebo lotion-treated skin. Therefore, the test lotion was considered by a trained dermatologist to be safe for long term use. A general clinical evaluation was also performed by a trained dermatologist (KN) at 0, 12 and 24 weeks of treatment. That evaluation demonstrated that treatments with the test lotion or the placebo lotion induced a significant decrease in the scores of scaling and stinging sensations at 24 weeks (\u003cstrong\u003eFigure 8B, F\u003c/strong\u003e), whereas the same treatments did not cause any changes in dryness, itchiness, erythema, or papules at 12 and 24 weeks (\u003cstrong\u003eFigure 8A, C, D, E\u003c/strong\u003e). Stinging sensations were significantly (p\u0026lt;0.001) \u0026nbsp;ameliorated after treatment with the test lotion or the placebo lotion for 12 and 24 weeks compared with week 0 (\u003cstrong\u003eFigure 8F\u003c/strong\u003e). These findings indicate that both the test and the placebo lotions have a distinct ameliorating effect on scaling and stinging sensation without any skin irritations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical evaluation of skin redness based on erythema index values measure\u003c/strong\u003ed\u003cstrong\u003e\u0026nbsp;using a Mexameter MX18\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince erythema index values obtained using\u0026nbsp;a Mexameter MX18\u0026nbsp;can serve as a measure of skin redness [10], erythema index values\u0026nbsp;of the test lotion-treated (previously assigned) SLs were measured at 0, 12 and 24 weeks of treatment and were compared with those of the placebo lotion-treated (previously assigned) SLs. In the test lotion-treated SLs, erythema index values did not change at 12 and 24 weeks of treatment compared to week 0 (\u003cstrong\u003eFigure 9A)\u003c/strong\u003e. In contrast, in the placebo lotion-treated SLs, erythema index values significantly (p\u0026lt;0.05) increased at 24 weeks of treatment compared to week 0 (\u003cstrong\u003eFigure 9A)\u003c/strong\u003e. Comparisons of increased (△) erythema index values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had similar\u0026nbsp;△erythema index values compared to the placebo lotion-treated SLs at both 12 and 24 weeks of treatment (\u003cstrong\u003eFigure 9C\u003c/strong\u003e). In the test lotion-treated (previously assigned) NLS, erythema index values did not change at 12 and 24 weeks of treatment compared to week 0 (\u003cstrong\u003eFigure 9B)\u003c/strong\u003e. However, in the placebo lotion-treated NLS, erythema index values significantly (p\u0026lt;0.0001) increased at 12 and 24 weeks of treatment compared to week 0 (\u003cstrong\u003eFigure 9B)\u003c/strong\u003e. Comparisons of increased erythema index values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS had significantly (p\u0026lt;0.05) lower\u0026nbsp;△L erythema index values than the placebo lotion-treated NSL at both 12 and 24 weeks of treatment (\u003cstrong\u003eFigure 9D\u003c/strong\u003e). These results indicate that the test lotion but not the placebo lotion has a distinct potential to ameliorate skin redness, which may reflect a weak anti-inflammatory effect of the test lotion.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAGAC is thought to serve as a tyrosinase inhibitor following its conversion to ascorbic acid via its deglucosylation by epidermal α-glucosidases after it penetrates into the epidermis. α‑glucosidases are known to exist in the epidermis as a protein glycosylation processing enzyme that can break the glucose of asparagine-linked carbohydrate moieties bound to proteins to release glucose in the Golgi area of keratinocytes [19]. In order for the action of tyrosinase inhibitors to be effective, it was essential to know whether the hyperpigmentation of SLs is accompanied by an accentuated expression of the key melanogenic enzyme tyrosinase in the lesional melanocytes. In this connection, it had already been reported that the hyperpigmentation in SLs occurs in concert with the up-regulated mRNA levels of tyrosinase in the increased numbers of tyrosinase-positive melanocytes in the SLs lesional epidermis [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present double-blind half-face study of subjects with SLs demonstrated that, although the placebo lotion-treated SLs had significant (p\u0026lt;0.05 and p\u0026lt;0.01) decreases in pigmentation scores at 12 and 24 weeks, respectively, compared to 0 week, the test lotion-treated SLs had more marked significant (p\u0026lt;0.001 and p\u0026lt;0.0001) decreases than the placebo’s ones in pigmentation scores at 12 and 24 weeks, respectively, compared to week 0, accompanied with a significant (p\u0026lt;0.01) decrease in pigmentation scores at 24 weeks compared to the placebo’s ones (see Figure 2). These results suggest that AGAC has a weak but distinct potential to ameliorate the clinical hyperpigmentation level of SLs. This clinical anti-pigmenting effect was corroborated by mechanical observations using a color difference meter and a Mexameter. In those mechanical evaluations, although both the test and the placebo lotions significantly increased L values or decreased MI values at 12 and 24 weeks of treatment, comparisons of the increased L (△L) values or the decreased (△) MI values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly higher\u0026nbsp;△L and\u0026nbsp;△MI values than the placebo lotion-treated SLs at 0~12, 12~24 and 0~24 weeks of treatment (see Figure 4). Since the significant anti-pigmenting effects of the placebo lotion might reflect seasonal changes in skin color from September to March during this clinical study, the significant differences observed in both the\u0026nbsp;△L and\u0026nbsp;△MI values at 12 and 24 weeks compared to week 0 between the test and the placebo lotions suggest that AGAC has a distinct anti-pigmenting effect on SLs at the color difference and MI levels. Further, our findings that the ratio of subjects with distinctly recognizable levels of over a 2.0\u0026nbsp;△L value or a 50\u0026nbsp;△MI value of SLs was 6 or 13 of 27 for the test lotion, and 0 or 2, respectively, of 27 for the placebo lotion at 0~24 weeks (see Figure 4) strongly suggest that AGAC is distinctly effective in diminishing the hyperpigmentation levels of SLs at a visibly recognizable level by the subjects themselves. In the time course of the anti-pigmenting effects measured as L values especially by a color difference meter, the significant effects of the test lotion on SLs occurred in a step-by-step manner at 12 and 24 weeks of treatment with significantly increased changes even at 12~24 weeks. In contrast, those significant effects of the placebo lotion also occurred at 12 and 24 weeks of treatment, but not accompanied with any significant changes at 12~24 weeks (see Figure 4A). These time course trends of the anti-pigmenting effects by the test lotion could provide an insight into predicting more distinct anti-pigmenting effects by possibly further prolonged treatments with the test lotion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOf considerable interest is that in the test lotion-treated NLS, both the L and MI values significantly increased or decreased at 12 and 24 weeks of treatment compared to week 0. In contrast, in the placebo lotion-treated NLS, the L values did not increase at 12 and 24 weeks of treatment compared to week 0, while the MI values significantly decreased at 12 and 24 weeks of treatment (see Figure 5C). Although the significant decrease of the MI values in the placebo lotion-treated NLS seems to occur due to the seasonal variation from September to March during this clinical study, comparisons of increased L (△L) values or decreased MI (△MI)\u0026nbsp;values between the test and placebo lotion-treated NLS demonstrated that the test lotion-treated NLS have significantly higher\u0026nbsp;△L and lower\u0026nbsp;△MI values than the placebo lotion-treated NLS at 0~24 and 12~24 weeks of treatment (see Figure 5). Although the increased or decreased levels of L or MI values occur at lower levels than 2.0\u0026nbsp;△L or 50\u0026nbsp;△MI (distinctly recognizable levels by the subjects), respectively, at 0~24 week, the ratio of subjects with over a 1.6△L or a 30\u0026nbsp;△MI (slightly recognizable levels by the subjects) of NLS at 0~24 weeks was 3 and 10 of 27 for the test lotion, and 0 and 1, respectively, of 27 for the placebo lotion (see Figure 5). The sum of these findings indicates that the test lotion has a significantly higher whitening effect on NLS than the placebo lotion and suggests that AGAC has a weak but significant whitening effect on NLS at visibly recognizable levels.\u003c/p\u003e\n\u003cp\u003eIt is of considerable interest to compare the anti-pigmenting effects on SLs and the whitening effects on NLS between AGAC and ASP, because the latter has been reported to have both anti-pigmenting and whitening effects in subjects with SLs [22]. Although both compounds have similar anti-pigmenting and whitening effects on SLs and NLS, respectively, as revealed by evaluation of L and MI values, a major difference occurs at the clinical scoring levels of pigmentation in SLs in which AGAC but not ASP exhibited a significant (p\u0026lt;0.0001) decrease in pigmentation scores at 24 weeks compared to week 0, accompanied with a significant (p\u0026lt;0.01) decrease in pigmentation scores at 24 weeks compared to the placebo lotion-treated SLs (see Figure 2). This indicates a slight superiority of AGAC to ASP from a clinical point of view although the production cost is much higher for AGAC than ASP. It is likely that the slight superiority of AGAC to ASP can be ascribed to the higher concentration used, i.e. 28% AGAC compared to 6% ASP, despite the fact that the rate of penetration into the epidermis is thought to be much higher for ASP than AGAC.\u003c/p\u003e\n\u003cp\u003eA major skin problem that occurs during the long-term topical application of anti-pigmenting agents is skin irritation as is frequently observed for HQ [5.15,52]. Since such a long time of topical applications is required to attain a distinct anti-pigmenting effect in dark-skinned individuals with SLs, skin irritation that happens during the treatment is a major causative factor for not being able to continue the topical applications. Therefore, general clinical evaluations of skin symptoms, including skin irritation, are important and were carried out in this study by a trained dermatologist (KN). These clinical evaluations demonstrated that, while the test lotion rather significantly ameliorated scaling and stinging sensations at 24 weeks, there was no appearance of erythema, papules or itchiness during the 24 weeks of treatment. Further,\u0026nbsp;based on the evidence that\u0026nbsp;erythema index values measured using a Mexameter\u0026nbsp;can serve as a reflection of skin redness\u0026nbsp;due to hemoglobin levels in the blood flow\u0026nbsp;[10],\u0026nbsp;our evaluations revealed that the test lotion but not the placebo lotion has a distinct potential capable of diminishing skin redness. These findings strongly suggest that AGAC could act as an anti-pigmenting agent with a weak anti-inflammatory effect.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the sum of the above findings indicates that AGAC has a weak but significant anti-pigmenting effect on SLs and a significant whitening effect even on normally pigmented skin without the risk of eliciting hypopigmentation or skin irritation. This provides skin safety at a sufficient level to use for a long period time of topical applications, which is an essential requirement to achieve distinct anti-pigmenting effects on SLs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSLs, solar lentigos; AGAC, Ascorbyl Glucoside Arginine Complex; EDN, endothelin; SCF, stem cell factor; NLS, non-lesional surrounding skin; AG, Ascorbyl Glucoside; AMP, L-ascorbate-2-phosphate Mg; APS, L-ascorbate-2-phosphate trisodium salt; HQ, hydroquinone; MI, melanin index; TNF, tumor necrosis factor; UVB, ultraviolet B\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eA.Y. and G.I. designed this study. M.T., K.N. and M.N. analyzed the data and M.T. and G.I. wrote the manuscript. M.N. and A.Y. financially supported this study. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national re-search committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eAll relevant data are included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eDr. Vincent Hearing has critically read this manuscript and made some kind and useful suggestions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors state no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors, Masahiko Nakano and Akio Yamamoto were employed by the company, Doctor\u0026rsquo;s Choice Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eORCID\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMariko Takada\u0026nbsp;\u003c/strong\u003e\u003cu\u003ehttps://orcid.org/0009-0005-2469-0047\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKayoko Numano\u0026nbsp;\u003c/strong\u003ehttps://orcid.org/0009-0000-2836-7574\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMasahiko Nakano\u003c/strong\u003ehttps://orcid.org/0000-0002-0133-6396\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAkio Yamamoto\u003c/strong\u003ehttps://orcid.org/0000-0002-4919-8535\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenji Imokawa\u0026nbsp;\u003c/strong\u003ehttps://orcid.org/0000-0001-7312-4475\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArndt, K. A., \u0026amp; Fitzpatrick, T. B. (1965). 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Hydroquinone-induced skin irritant reaction could be achieved by activating mast cells via mas-related G protein-coupled receptor X2. \u003cem\u003eExp Dermatol, 32\u003c/em\u003e(4), 436-446. doi:10.1111/exd.14723\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ascorbyl Glucoside Arginine Complex, solar lentigo, double-blind half-face study, pigmentation, L value, melanin index","lastPublishedDoi":"10.21203/rs.3.rs-5332003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5332003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLittle is known about anti-pigmenting effects of whitening agents on solar lentigo (SLs). To characterize the anti-pigmenting effects of newly designed ascorbyl glucoside arginine complex (AGAC) on SLs, lotions with or without 28% AGAC were applied twice daily for 24 weeks in a double-blind half-face study of 27 Japanese females with SLs. Pigmentation scores were evaluated using a photo-scale and skin colors were assessed using a color difference meter and a Mexameter. Treatment with the test lotion elicited a significant decrease in pigmentation scores at 24 weeks com-pared to week 0, with a significant decrease in pigmentation scores at 24 weeks compared to the placebo lotion-treated SLs. In the test lotion-treated SLs, the lightness (L) values and melanin index (MI) reflecting pigmentation level significantly (p\u0026lt;0.0001) increased and decreased, respectively, at 12 and 24 weeks of treatment compared to week 0. Comparisons of increased L values or decreased MI values between the test and placebo lotion-treated SLs demonstrated that the test lotion-treated SLs had significantly higher increased L or decreased MI values than the placebo lotion-treated SLs both at 12 and 24 weeks of treatment. The sum of our results strongly indicates that AGAC is distinctly effective in ameliorating the hyperpigmentation levels of SLs at a visibly recognizable level by the subjects without any hypo-pigmenting effects or skin irritation.\u003c/p\u003e","manuscriptTitle":"Treatment with Ascorbyl Glucoside Arginine Complex ameliorates solar lentigos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-28 15:40:07","doi":"10.21203/rs.3.rs-5332003/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"376ba159-f43d-4743-a5ca-33bd8f2dae6c","owner":[],"postedDate":"October 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-30T18:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-28 15:40:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5332003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5332003","identity":"rs-5332003","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}