Improvement in Wrinkles by a Fairy Chemical, 2-Aza-8-oxohypoxanthine

Abstract

2-aza-8-oxohypoxanthine (AOH) has been shown to improve skin barrier function according to DNA microarray studies of normal human epidermal keratinocytes. This study aimed to evaluate the cosmetic efficacy of AOH in wrinkle improvement. A 12-week clinical trial involved 23 women (mean age 44 ± 8 years), who applied 0.01% or 0.05% AOH lotion to each half of their face twice daily. Skin assessments were conducted before and after application at 4, 8, and 12 weeks by measuring stratum corneum water content using a Corneometer and evaluating wrinkles through image analysis of replicas. Application of the lotion enhanced stratum corneum water content. Use of 0.01% AOH reduced the wrinkle area percentage and maximum wrinkle depth at 8 and 12 weeks post-application, respectively. The 0.05% lotion showed a significant reduction in the wrinkle area percentage and mean maximum wrinkle depth at 12 weeks and a significant decrease in maximum wrinkle depth at 4 weeks. A between-group comparison revealed that the 0.05% AOH lotion significantly reduced mean maximum wrinkle depth compared to the 0.01% AOH lotion at 8 and 12 weeks, indicating a concentration-dependent effect of AOH. Overall, AOH improves wrinkles in a concentration-dependent manner, confirming its efficacy as a cosmetic ingredient for wrinkle improvement.

1. Introduction

The “fairy rings” phenomenon occurs when turfgrass growth is either accelerated or inhibited in a ring pattern, with mushrooms often emerging within this formation. According to western legends, these rings were believed to be spots where fairies danced, hence the term “fairy rings” [1,2,3]. 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH) have been identified as compounds responsible for the formation of fairy rings (Figure 1) [4,5,6]. After the title of a Nature article introducing our work, we refer to these compounds (AHX, ICA, and AOH) as fairy chemicals (FCs) collectively [7].

Preliminary studies with FCs showed that only AOH had a cytostatic effect on normal human epidermal keratinocytes (NHEKs). Therefore, we have focused on AOH among the three types of FCs to verify its usefulness and safety on skin. Safety evaluation studies of AOH have shown no adverse reactions in any tests, which supports its use in cosmetics [8,9]. Additionally, AOH exists endogenously in various plants, including major crops such as rice, wheat, and potatoes [10,11]. Previous studies have highlighted the potential of AOH among FCs as a functional cosmetic ingredient [12,13].

The efficacy of AOH was further substantiated by the finding that it did not exert any adverse effects on the viability of NHEKs [12]. Microarray-based expression profiling of genes related to skin aging and diseases in NHEKs revealed that AOH treatment generally upregulates genes involved in skin barrier function [12]. A clinical trial confirmed the effectiveness of AOH in enhancing skin barrier function, evidenced by increased stratum corneum water content and reduced trans-epidermal water loss (TEWL) [13]. Overall, the results of cellular-level experiments and clinical trials suggest that AOH plays a vital role in the regulation of skin barrier function. Another clinical trial focused on changes in L* values, an index of skin lightness, after applying a lotion containing 0.1% AOH for 8 weeks. After 8 weeks, L* values increased significantly, demonstrating the skin lightening effect of AOH [14].

Given that decreased stratum corneum water content from compromised skin barrier function can lead to wrinkle formation [15,16,17], potency in wrinkle improvement by AOH was suggested. Therefore, the present clinical study was conducted to prove the effect of AOH by assessing changes in stratum corneum water content and wrinkle parameters before and after prolonged AOH application.

2. Materials and Methods

2.1. Preparation of the Test Formulation

AOH was synthesized from 5-aminoimidazole-4-carboxamide, as previously described [18,19]. Methyl p-hydroxybenzoate (final concentration: 0.4%, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) was dissolved in 1,3-butanediol (final concentration: 5%, ITO Co., Ltd., Tokyo, Japan) by heating. Phenoxyethanol (final concentration: 0.3%; FUJIFILM Wako Pure Chemical Corporation), xanthan gum (KELCOL CG LAX-T, Sansho Co., Ltd., Osaka, Japan), and polyoxyethylene hydrogenated castor oil 60 (final 0.2%, Nikko Chemicals Co., Ltd., Tokyo, Japan) were then added to this 1.3-butanediol solution. Next, AOH (final concentration: 0.01% or 0.05%) in sodium phosphate buffer (pH 7.0, final 10 mM, FUJIFILM Wako Pure Chemical Corporation) and purified water (final 33.8%) were added to the solution. Prior to the clinical study, a human patch test was performed to evaluate the safety of the test formulation. The patch test was performed on the skin of 16 men and 6 women from Japan, aged 21–59 y (ave. 39 y). The test samples (0.01% or 0.05% AOH lotion) were placed on a Finn Chamber on Scanpor Tape and applied to each subject’s upper back for 24 h in an occlusive condition. Skin conditions were evaluated 1 and 24 h after the removal of the test sample. Skin reactions were evaluated according to the International Contact Dermatitis Research Group standards and the guidelines of the Japanese Dermatological Association [20].

2.2. Participant Selection for the Clinical Trial

The following selection criteria were applied: (1) women (yellow race) in their 30s to 50s at the time of consent; (2) participants who were concerned about dry skin and wrinkles at the corners of their eyes; (3) participants whose usual post-cleansing (or post-bathing) skin care routine consisted of only one or two products (i.e., lotion and emulsion); and (4) participants who received a full explanation of the study’s purpose and procedures, possessed the capacity to consent, and voluntarily agreed to participate by providing written informed consent. The exclusion criteria were as follows: (1) any skin condition at the evaluation site that may affect the study results, such as urticaria or inflammation; (2) any history or current history of atopic dermatitis or predisposition to atopy; (3) participants who had undergone cosmetic treatment on the evaluated area; (4) participants who had undergone skin care treatment at a beauty salon or esthetic clinic on the evaluated area within the past 4 weeks; (5) those who were deemed by the study investigator to be unsuitable for participation.

2.3. Clinical Trial Study

To target participants with dry skin, TEWL was measured in 40 subjects before the start of the clinical trial, and 24 subjects with high TEWL were selected. Twenty-four healthy Japanese women were enrolled in the study, which was conducted in Osaka from 19 January to 27 April 2023. This trial employed a double-blind, split-face, left–right randomized design. After washing their face twice in the morning and evening, participants applied a lotion containing 0.01% AOH to one side of their face and a lotion containing 0.05% AOH to the other side for twelve weeks. Approximately 0.3 g of each product was applied throughout the 12-week period, starting on day 1. After cleansing the face with water, the skin’s biophysical parameters were measured at baseline (the day before the first application) and again at 4, 8, and 12 weeks by the attending researcher. All participants completed a usability questionnaire at 4, 8, and 12 weeks after the start of the study. Skin evaluations were conducted following the “Anti-Wrinkle Evaluation Guidelines for Obtaining New Indications (Quasi-drugs)” [21].

2.4. Evaluation

All measurements were conducted under controlled environmental conditions (temperature, 21 ± 1 °C; relative humidity, 50 ± 10%), following at least a 30 min acclimatization period. Stratum corneum hydration was assessed by capacitance measurements using a Corneometer (Courage + Khazaka electronic GmbH, Cologne, Germany). Each measurement was taken at least three times, and the mean of three stable measurements was used. A replica of the wrinkle was taken using the replica agent SILFLO (Monaderm, Rue des Violettes, Monaco). A 4 cm × 4 cm area was sampled from the external canthus outward on both sides. Wrinkle data were collected using an ASA-03RXD reflective replica analysis system and reflective 3D skin analysis software Version 3.51 (Asch Japan Co., Ltd., Tokyo, Japan). The main analysis region was a 10 mm × 10 mm square located approximately 5 mm lateral to the external canthus. Parameters evaluated included wrinkle area percentage (%), maximum wrinkle depth (μm), mean maximum wrinkle depth (μm), and total mean wrinkle depth (μm).

For the analyses, measurements taken at baseline and 4, 8, and 12 weeks after application were compared among participants between the two groups, i.e., 0.01% AOH and 0.05% AOH lotion applications (within-county comparison). Additionally, changes from baseline to the subsequent time points (at 4, 8, and 12 weeks) were also compared between groups (between-county comparisons).

2.5. Statistical Analyses

All statistical analyses were conducted using IBM^® SPSS^® Statistics Version 23 (IBM Corp, New York, NY, USA). Two-tailed tests were used, and p-values were calculated using the corresponding t-tests. A Bonferroni correction was applied to account for multiple comparisons, setting the overall significance level at 5%.

3. Results

3.1. Evaluation of Stratum Corneum Water Content

Human patch tests showed no serious skin reactions in any of the subjects at 1 h and 24 h after removal of the test sample. In addition, 0.01% AOH lotion and 0.05% AOH lotion had a skin irritation index of 2.3 and 0.0, respectively. Based on these results, it was concluded that both lotions were safe products.

A total of 22 participants (16 males and 6 females; aged 21–59 years, mean: 39 years) underwent human patch testing with the test formulations. As a result, the skin irritation indices for the 0.01% and 0.05% AOH lotions were 2.3 and 0.0, respectively, indicating that both formulations were safe for use. Of the 24 participants initially enrolled, 23 participants (mean age 44 ± 8 years, range: 32–58 years) completed the clinical trial. They applied the test samples twice daily to their faces for 12 weeks. No participants reported adverse reactions on the usability questionnaire, and no adverse events related to the test product were observed.

Figure 2 shows the time course of the amount of change in stratum corneum water content compared to baseline (0 week). In the 0.01% AOH lotion group, an increasing trend in stratum corneum water content was observed, although the within-county comparison did not show statistical significance. Conversely, the within-county comparison for the 0.05% AOH lotion group revealed a statistically significant increase in stratum corneum water content at 8 weeks (p = 0.014) and 12 weeks (p = 0.004) compared to baseline. Furthermore, the between-county comparison showed that the 0.05% AOH lotion group had a statistically significant increase in stratum corneum water content at 4 weeks (p = 0.025), 8 weeks (p = 0.002), and 12 weeks (p = 0.012) compared to the 0.01% AOH lotion group, demonstrating a concentration-dependent effect.

In the current study, while the 0.01% AOH lotion group did not show a significant change, there was a trend toward increasing stratum corneum water content over time. Notably, the 0.05% AOH lotion group exhibited increases in stratum corneum water content of 2.7%, 7.3%, and 9.4% at 4, 8, and 12 weeks, respectively, compared to baseline. These findings underscore that continuous application of AOH lotion enhances stratum corneum water content in a concentration-dependent manner.

3.2. Image Analysis of Wrinkles Using Skin Replicas

Figure 3 illustrates the time course of the change in wrinkle area percentage from baseline. At the site where 0.01% AOH was applied, the wrinkle area percentage significantly decreased at 8 weeks (p = 0.015) and 12 weeks (p = 0.043). At the 0.05% AOH-applied site, there was a significant reduction at 12 weeks (p = 0.014).

Figure 4 presents the time course of the reduction in maximum wrinkle depth at the 0.01% AOH application site from baseline. The maximum wrinkle depth at the 0.01% AOH site was significantly reduced at 12 weeks (p = 0.042). In contrast, maximum wrinkle depth at the 0.05% AOH application site was significantly reduced at 4 weeks (p = 0.002), 8 weeks (p < 0.001), and 12 weeks (p < 0.001). Additionally, between-group comparisons indicated a statistically significant greater reduction in maximum wrinkle depth in the 0.05% AOH lotion group at 4, 8 and 12 weeks compared to the 0.01% group, demonstrating a concentration-dependent effect.

Figure 5 displays the time course of the change in mean maximum wrinkle depth relative to baseline. The mean maximum wrinkle depth at the 0.01% AOH-applied site displayed a decreasing trend, but no statistical significance was observed. In contrast, the mean maximum wrinkle depth at the 0.05% AOH application site was significantly reduced at 12 weeks (p = 0.005). Total average wrinkle depth did not change in either the 0.01% or the 0.05% AOH application site until 12 weeks.

Skin images of the representative participants taken by a Canon EOS 5D (Tokyo, Japan, Canon Inc.) are shown in Figure 6. In all the participants, wrinkles at the corners of the eyes were improved at 12 weeks, with greater enhancement in areas where the 0.05% AOH lotion was applied compared to the 0.01% AOH lotion.

Furthermore, in the analysis of wrinkle improvement by skin replicas, indices such as the wrinkle area ratio, maximum wrinkle depth, average wrinkle depth, and total average wrinkle depth were evaluated. In the 0.01% AOH lotion group, maximum wrinkle depth reductions were observed to be −4 µm, −7 µm, and −12 µm at 4, 8, and 12 weeks, respectively, compared to baseline. In the 0.05% AOH lotion group, the reductions were more pronounced, with maximum wrinkle depths decreasing to −14 µm, −24 µm, and −36 µm at the respective time points. This pattern further supports the concentration-dependent efficacy of AOH in wrinkle reduction.

4. Discussion

We have continuously explored the effects of AOH on skin at both cellular and clinical levels. This study presents the first report on the concentration-dependent effects of AOH on both increasing stratum corneum water content and wrinkle reduction.

It is recognized that decreases in stratum corneum water content and increases in TEWL are factors in wrinkle formation [15,16,17]. Hyaluronic acid plays a crucial role in maintaining or regulating the water content of the stratum corneum and epidermis. Research indicates that the concentration of hyaluronic acid in the epidermis diminishes with age, and this age-related dryness may also contribute to wrinkle development [22,23]. DNA microarray analysis of NHEK has shown increased expression of hyaluronic acid synthase 3 (HAS3) in the epidermis [12]. Additionally, AOH boosted hyaluronic acid production in in vitro experiments with human fibroblasts (unpublished data), suggesting that AOH application may enhance hyaluronic acid levels in the skin, thereby aiding in wrinkle reduction.

Another factor contributing to decreased stratum corneum water content is the increased water evaporation associated with impaired skin barrier function. Tight junctions (TJs), essential for skin barrier integrity to prevent water loss, are intercellular connections in the granular layer, and they are crucial not only for preventing water evaporation but also for the formation of mature stratum corneum and intercellular lipids [24,25]. Furthermore, the protein components of TJs diminish with aging and exposure to UV light [26]. Previous DNA microarray analysis using NHEK demonstrated that AOH upregulates a set of genes that enhance skin barrier function [12]. The results of this DNA microarray and our clinical study suggested that AOH may stimulate hyaluronic acid production and improve skin barrier function, resulting in increasing stratum corneum water content and a subsequent reduction in wrinkles in humans.

5. Conclusions

In this study, a long-term continuous-use study was conducted using 0.01% and 0.05% AOH lotion to verify the concentration-dependent effect of AOH on wrinkle improvement. The results showed that water content increased in a concentration-dependent manner, and the wrinkle area ratio, maximum wrinkle depth, and mean maximum wrinkle depth improved.

Considering that no adverse reactions or events were observed in this study, we concluded that AOH is a safe cosmetic ingredient with the potential to reduce the appearance of fine lines and wrinkles caused by dryness of the skin.