Anti-Irritant Strategy Against Retinol Based On The Genetic Analysis Of Korean Population: A Genetically Guided Top–Down Approach Ⅱ

3.3. Anti-Irritation Efficacy of the Formula; Human Tests

Based on anti-irritant substances verified from the in vitro experiment guided by genetic analysis, we prepared the AF (Anti-irritant Formula) against retinol-induced irritation, which consisted of glucosamine 0.1%, trehalose 2%, ectoine 2%, sucralfate 0.1%, omega-9 1%, and 4-t-butyl cyclohexanol 0.7% with retinol. (standardized as an active concentration) First, we performed a pilot study with seven individuals to quantitatively verify the effectiveness of the AF. When the irritation score of each type of irritation for the individual was summed during the test period, it was shown that compared to the control retinol cream, AF effectively decreased retinol-induced irritation, especially desquamation (66.67% decrease), burning (68.42%), and stinging (68.97%), and this amelioration of symptoms was statistically signifificant (Figure 4a). Interestingly, the test subjects claimed they did not experience dryness during the test period, although they had undergone signifificant desquamation and dryness after 10 days, even over 2 weeks. This observation is consistent with a previous study that claimed that retinoic acid induces skin dryness after 9 days of treatment and persists until 18 days by showing a reduced turnover rate of the stratum corneum (15.8 days) compared with the placebo (18 days) [56]

Figure 4. Schematic and plots depicting the irritant efficacy of AF. Panel (a-d) indicates the results of the small pilot study (n = 7). A left schematic diagram shows the test schedule and procedure and test area (red dotted) results of the face that was evaluated for the measurement of TEWL and skin redness. (a) Average of the irritation scores that the test subject experienced during the test period. (b) Overall irritation score on each time point. Over irritation score " refers to the sum of scores of all types of irritation experienced by the individuals. (C) Total irritation score of the individuals. Total scores for each type of irritation experienced by the individual during the test period were averaged. Overall scores " on each timepoint were summed. (d) Skin redness and transepidermal water loss (IEWL ) measured by chromameter and tewameteiThe increased ratios (%) for individuals were averaged, (e) Occurrence rate of retinol-induced irritation. Test human subiects were asked a dichotomous question, whether "retinol-based cream is irritant or not" based on their own subject five criteria. A chi-square test was performed to investigate statistical significance. (p-value: 2.17906E-10): * p-value < 0.05** p-value< 0.001, AF, Anti-irritant Formula against retinol-induced irritation; error bars are shown.

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Next, the overall irritation score over time was investigated (Figure 4b). As may be expected, the irritation was observed within a few minutes after applying the retinol cream and decreased after a few hours. We found that our AF effectively reduced irritation, especially after the third application of the cream. While the level of irritation increased even after ceasing the application of retinol cream, the anti-irritation effect of AF remained and alleviated the retinol-induced irritation post-procedure. Collectively, AF reduced the total irritation score of individuals during the test period by 58.3% (Figure 4c). 

At the 7th day, we measured the TEWL and redness (Figure 4d). AF reduced both the increase in TEWL and the redness induced by retinol. The average increase in redness (a*) was 8.31% for AF and 8.31% for control. Skin redness, a clinical feature of cutaneous vasodilation, is an indicator of inflflammation [57]. Previous studies have shown that retinoids induce cutaneous inflflammation mediated by the release of MCP-1 and IL-8 from fibroblasts. It is noteworthy that as shown in the previous section, retinol activates macrophages (overexpression of IL-4R), which is the prime driver of cutaneous vasodilation [9]. 

In the TEWL analysis, the retinol cream based on AF increased TEWL by 19.07% for test subjects individually, while the control retinol cream increased TEWL by 42.54%. This clinical observation is similar to that of a previous report indicating that retinoids temporarily induce an increase in TEWL in vivo in mice and humans [42,58]. Although the statistical significance was low, the mitigation effect of AF on the increase in TEWL and redness may be expected.

We also measured the pressure–pain threshold using an algometer with a tip with a radius of 1 mm (Figure S2, see Supplementary Materials). The sensitive skin of psoriasis patients was reported to show a decreased pressure–pain threshold compared to normal individuals [59]. The differences in threshold values of the individuals before and after treatment with retinol cream were averaged. It was observed that retinol decreased the pressure–pain threshold (PPT), and AF increased the decreased PPT signifificantly compared to the control. It seems to be mediated by the anti-inflflammatory and antagonistic effects of AF on TRPV1. Earlier studies have shown that neurogenic inflflammation is highly related to the mechanical sensitization of cutaneous nociceptors [60], and mice lacking functional TRPV1 display attenuated mechanical hyperalgesia to noxious mechanical stimuli [61]. One human clinical study also demonstrated that a TRPV1 antagonist (V116517) signifificantly increased the PPT of the skin under various conditions [62]. 

To verify the anti-irritant effect of AF in the independent group, we grouped together all the human subjects, which consisted of 44 males and 47 females with ages ranging from 27 to 50 years. The test subjects were asked a dichotomous question, whether “retinol-based cream is irritant or not”, based on their own subjective criteria. This response ratio was compared to that of the first large-scale clinical evaluation of 173 individuals who had used a normal retinol cream in order to derive the anti-irritant effect of AF. The occurrence rate of retinol-induced irritation was calculated and compared (Figure 4e). 

A 64.43% occurrence rate was observed in the group that had used normal retinol cream, whereas an occurrence rate of only 21.35% was observed in the group that had used AF-retinol cream, which indicated a decrease of 65.80%. 

3.4. Polygenic Risk Prediction Model for Retinol-Induced Irritation

As anti-irritants in AF were screened from the associated genes, we constructed a polygenic risk prediction model to evaluate the cumulative genetic effect of multiple location retinol-induced irritation. A polygenic risk prediction model was constructed using 26 signifificant SNPs selected after LD analysis based on |D’| and r2 values(coefficients for the degree to which an allele of one SNP is inherited or correlated to the allele of another SNP)between SNPs. 

Then, the risk scores of all 159 samples were visualized. The test subjects of the second large-scale clinical test were arbitrarily categorized into three groups: high (>75, 31 individuals), middle (65–75, 33 individuals), and low-risk score (<65, 27 individuals) (Figure S3, see Supplementary Materials). As characteristics of retinol-induced irritation in each group, the high-risk score group exhibited dryness and desquamation after the last third treatment of retinol compared to the other groups (Figure S4, see Supplementary Materials). This result supports our hypothesis that skin barrier disruption with increased TEWL plays an important role in retinoid-induced irritation, as SLS exhibited similar clinical features and anatomical changes [63].

As shown in Figure 5a, the group with a high-risk score consisted of the most irritationexperienced participants among the other groups. “Pruritus” was the most dominant type of irritation for the high-risk score group compared to the other groups. The participants in the high-risk score group experienced pruritus most frequently on the 1st day of retinol treatment, and the degree of pruritus decreased gradually over time. The high-risk score group seemed to experience allergy-like symptoms related to histamine and mast cell-mediated systems (Figure S4, see Supplementary Materials).

Figure 5. Bar plots illustrating polygenic risk score model and validation. 3300 IU retinol cream with AF to 91 individuals lest subjects were categorized into three groups: high-risk score (>75, 31 individuals), middle-risk score (65-75, 33 individuals), and low-risk score (<65,27 individuals) (a) irritation score of each type of irritation for three risk score groups. The scores for types of irritation of individuals during the test period were summed and averaged by risk score groups. (b) The total score during the test period. (c) Comparison of the occurrence rate of irritation in each risk-score group. The dichotomous question of whether retinol cream is irritant: Y/N was asked to test subjects. In the first clinical evaluation, retinol without AF was given. Retinol with AF was given in the second clinical evaluation.

The total score during the test period was calculated as 6.370 and 10.161 for the low-, middle-, and high-risk score groups, respectively (Figure 5b). The efficacy of AF was investigated according to the polygenic risk score model.

The results demonstrated that AF alleviated retinol-induced irritation, and the efficacy could be predicted using the polygenic risk score model (Figure 5c).

Consequently, these experimental results and observations not only demonstrate the anti-irritation effect of AF but also imply that proposing an appropriate dosage of retinol by estimating the susceptibility to retinol-induced irritation based on individual genetic information is applicable, which can finally offer genetically the most-optimized usage protocol to patients with higher compliance.

3.5. Further Consideration; Acute vs. Chronic Irritation? 

As mentioned before, allergy-like reactions, which include rapid burning and stinging sensation, itchiness, and rapid diffusive edema and rash were observed in retinol-sensitive individuals. These reactions, which occurred within a few minutes, sometimes even a few seconds, cannot be clearly explained by previous retinoid-mechanistic studies that have mainly focused on cytokine production by keratinocytes or fibroblasts [9], or the immune system in which T cells, B cells, or complementary activation are mainly involved. Although it is undeniable that skin barrier disruption is the main cause of irritation, it does not fully explain the acute irritation that promptly occurs.

In the first clinical evaluation, it was observed that about 5% of the test subjects experienced violent and severe above-mentioned reactions and were dropped from the test. However, detailed investigations have not been performed regarding the intolerance of retinoids depending on race, sex, or age, and it has been reported that the Asian population is more prone to these reactions, which urges the necessity for the consideration of rapidprovoked irritation for the further development of retinoid-based products. Our human test results suggest that neurogenic inflflammation, or a similar prompt mechanism, is also involved in retinol-induced irritation. The burning sensation and stinging were dominantly observed at each retinol treatment within a few minutes in all three groups and repressed over time.

Here, these are some points that deserve consideration. 

1. As reported earlier, retinol may be converted into retinaldehyde (retinal) and retinoic acid to possess the bioavailability at which RAR or RXR binding is involved. 

2. However, the homeostasis of retinoids in the skin is very tightly controlled [64], and paradoxically, it has not been experimentally proven that retinol converts into retinoic acid in vitro and ex vivo [65,66].

3. Under retinol treatment, fibroblasts or keratinocytes activate the circumjacent cutaneous immune system as quickly as within a few minutes by producing IL-1 or IL-6 through the interaction between RAR and retinoic acid, to which the retinol should be converted. 

Based on these considerations, it seems more reasonable that acute irritation within a few minutes may be driven by the histamine–mast cell system or neurogenic inflflammation promptly, which subsequently triggers extensive and pervasive inflflammation, leading to long-term irritation. 

4. Conclusions