New Retinol Alternative

New Retinol Alternative: Next-Generation Sequencing Reveals Efficacy of Hydroxypinacolone Retinoate

Abstract

Retinol is an anti-aging ingredient with high efficacy in wrinkle reduction and skin renewal. Retinol, despite its benefits, causes side effects such as inflammation, spongiosis, redness and dryness, particularly with prolonged and high-dose use. In the past, pinacolone ester retinoids were proposed and developed, but their mechanical and effective advantages over classical retinol use remained unclear. To elucidate and compare their molecular mechanisms and advantages, hydroxypinacolone retinoate (HPR), a well-known pinacolone ester retinoid alternative, was comparatively used with next-generation sequencing in a whole gene expression analysis and an in vivo wrinkle prevention study. These data show that HPR activates a similar gene subset, but also enhances gene expression efficacy on gene sets involved in extracellular matrix regulation, cornified envelope, keratinization and protein digestion and absorption compared to retinol. In vivo efficacy in wrinkle reduction applied to half the face increases significantly and to the same extent as in retinol-treated skin, confirming it as a suitable retinol alternative without drawbacks.

Introduction

Retinoids are defined as beta-ionone-based vitamin A derivatives. They are included through nutrition, found as retinol and retinyl esters from animal sources or plant-based carotenoids. Retinoids have numerous functions depending on the tissue and active form. Specifically, their functions in different tissues are facilitated by the related retinoid derivative and retinoic acid stereoisomers, for example, visual function is facilitated by rhodopsin bound to retina in retinal cells, while tissue differentiation, for example, skin epidermis differentiation, is regulated by retinoic acid (RA) stereoisomers binding to related nuclear receptors in the relevant tissue and activating gene expression. Here, 9-cis RA acts primarily as a ligand for retinoid-X receptors RXRs, while retinoic acid receptors RAR bind 9-cis RA and all-trans RA (ATRA)^1;2. Since retinoids are hydrophobic, they require transport by retinoid binding proteins in aqueous tissue environments, namely plasma retinol binding protein (RBP 4), interstitial retinol binding protein (RBP 3) and cellular retinol-containing retinoid binding proteins (CRPs or RBP 1, RBP 2, RBP 5 and RBP 7). These transport proteins not only facilitate the dissolution of retinoids but also protect them from oxidation. Additionally, cellular retinoic acid binding proteins (CRABP) mediate the interaction between RA and RXR and RAR receptors, referring to CRABP co-activator properties. When RAR receptors bind to RA, this results in conformational changes allowing heterodimerization with other receptors such as RXRs, LXRs and PPARγ, among others. This heterodimerization of RAR receptors mediates the pleiotropic effects of RAs in various tissues. For example, heterodimer complex subunits are altered with the differentiation status of keratinocytes, with RARα/RXRα dominant in the basal layer and RARγ/RXRα dominant in the suprabasal layer. Beyond its function in regulating gene expression, RA exhibits non-transcriptional pathway activity through different RAR and RXR activation from its activity as transcription factors. For example, RARα forms a complex with a G protein-coupled receptor involving Gαq (G protein alpha Q) in lipid rafts in response to RA and activates p38 MAPK signaling³ and dependently binds PI3K subunits to activate RA to activate AKT and subsequently ERK1/2⁴. As mentioned above, retinoids affect differentiation processes, particularly keratinocyte differentiation where keratin and heparin-binding epidermal growth factor gene expression are required, thus regulating skin differentiation and barrier function. Clearly, KRT4 and KRT13 are upregulated in the suprabasal layer and KRT2 arising in the stratum spinosum is downregulated^5;6. Additionally, cell division of basal keratinocytes increases, leading to cell proliferation and subsequent epidermal thickening⁵. Thereby, vitamin A deficiency can cause skin problems, and retinoids are used in treating keratinization disorders, e.g. epidermolysis bullosa simplex and epidermolytic ichthyosis, as well as in treating psoriasis and acne vulgaris⁶. In aging processes, keratinization and epidermal thickness are impaired. Here, retinoids are widely used in topical formulations to slow skin phenotypes including wrinkles, roughness, laxity, dullness, scaling and dryness. In particular, improvement of UV-induced photoaging with retinoid use has been reported⁷. Mechanically, wrinkles, laxity and roughness are alleviated through regulation of collagen I, III and IV, which improve dermal-epidermal junctions and dermal tissue elasticity^8;9. In summary, retinoids have been shown to exhibit beneficial effects in topical treatment of aging phenotypes as well as psoriasis and acne vulgaris. However, recent reports focus attention on their limitations and side effects. One limitation of retinoids is their decreased bioavailability in long-term use due to increased inactivation by CYP26 family enzymes induced by retinoids and other cytochrome P450 members, creating a negative feedback loop with RAR/RXR signaling¹⁰. With this effect, vitamin A deficiency can occur, leading to unwanted skin conditions. Other adverse retinoid side effects are characterized as 'retinoid reaction', presenting with burning sensation, itching and erythema that occur at the beginning of retinoid use. This can be attributed to activation of the irritant receptor TRPV1 by retinoids and diminishes after adaptation to their use¹¹. However, customers and patients have also observed inflammation, peeling, redness and skin dryness following long-term retinoid use. With this effect, vitamin A deficiency can occur, leading to unwanted skin problems. Other adverse retinoid side effects are characterized as 'retinoid reaction', presenting with burning sensation, itching and erythema that occur at the beginning of retinoid use. This can be attributed to activation of the irritant receptor TRPV1 by retinoids and diminishes after adaptation to their use¹¹. However, customers and patients have also observed inflammation, peeling, redness and skin dryness following long-term retinoid use. Consequently, all these observed negative effects are multifaceted and their molecular causes have not been clarified in detail. For this reason, dermatologists and cosmetic industries considered the need to develop retinoid alternatives with good efficacy but reduced side effects. Here, Varani and colleagues developed retinoids such as pinacolone esters with less irritation and fewer side effects¹². Hydroxypinacolone retinoate (HPR) was the best-distributed derivative of this to market. However, to date, a detailed mechanical comparison was lacking to explain its advantages over all-trans retinol. Therefore, in this study, to elucidate and compare the molecular effects of a stabilized HPR mixture (referred to below as "ingredient"), we performed complete transcriptomic sequencing (RNA-Seq), in vitro 3D approach. Additionally, in an in vivo study we can demonstrate that the ingredient exhibits the same efficacy as all-trans retinol in wrinkle reduction.

Materials and Methods

RNA Sequencing of 3D Reconstructed Epidermal Skin Models

EpiDerm reconstructed 3D models (MatTek, Bratislava, Slovakia) were cultured for 72 hours. Then the research product SymRenewTM HPR 1 and the comparison benchmark all-trans retinol were applied daily for 5 days. This ingredient is a special hydroxypinacolone retinoate mixture that facilitates good stability. For each experimental condition, three biological replicates were analyzed. Epiderm models were collected in RNA lysis buffer. RNA was isolated using Quiagen RNAeasy Mini kit (Quiagen, Hilden, Germany). Samples were processed for Illumina TruSeq, stranded, poly(A) enriched RNA library preparation. Sequencing per sample was performed with Illumina NextSeq, v2.5 at 75 bp and 30 Mio reads.

RNA Sequencing Data Analysis

Following removal of Illumina adapter remnants, demultiplexing and trimming, data were mapped against the GRCh38 genome and FPKM obtained for each gene. FPKM was averaged and normalized relative to untreated control or sample treated with all-trans retinol. Genes with natural logarithmic fold change less than -1 ≤ and greater than ≥1 and p-value <0.05 (genes with FPKM <3 excluded) were considered significantly differentially expressed. Functional enrichment and clustering were performed using DAVID Bioinformatics Resources 6.8. Functional clusters with Bonferroni-corrected p value ≤ 0.05 were considered significant.

In Vivo Study Wrinkle Reduction

This in vivo study was conducted according to the findings of National Health Council Regulatory Guidelines and Standards Decision 466/12. A total of 96 research subjects (mean age: 55 ± 5 years, Fitzpatrick phototype 3% phototype II and 97% phototype III) were instructed to discontinue use of any topical products on their faces 48 hours before study initiation. They were also instructed about laboratory testing and not using any products during the study period. The study was based on the hypothesis that treatment with research products according to instructions could reduce signs of aging in facial skin. The methodology used in this study evaluates reduction of wrinkles and facial expression lines through image analysis. Facial images were obtained at baseline, and after 14 and 28 days of home use. Research subjects applied the research products twice daily, once in the morning and once in the evening. Research subjects were instructed to apply one research product to the right side of the face and another research product to the left side of the face, or to keep one side of the face (right or left) as control. Application side (right or left) was randomly distributed between subjects. For each research subject, the area containing wrinkles and mimetic lines according to facial morphology, size and location was determined for analysis. Images were obtained at study baseline and 14 and 28 days after home use of research products at maximum resolution of 50 micrometers through Primos CR Large field (Canfield®) equipment. Obtained images were analyzed using VAM software, version 5.9.7, Canfield Scientific Inc, with parallel lines added over the determined area. Statistical method was performed using ANOVA - Dunnet test with 95% confidence interval. Intensity of wrinkles and lines was determined using roughness parameter Rv % values referring to deeper troughs of the profile analyzed according to ISO4287 standard. ¹ SymRenewTM HPR, produced by Symrise (https://www.symrise.com/scent-and-care/cosmetic-ingredients/, Holzminden)

Results

To explain and compare the transcriptional regulations of retinol, commonly used in cosmetics, relative to the newly proposed alternative ingredient, RNA-Seq was performed in 3D reconstructed epidermis models. RNA Seq analysis showed that the ingredient altered gene expression of 2,747 genes compared to untreated-untreated sample. All-trans retinol altered the expression of 3,893 genes compared to untreated control. Of these differentially regulated genes, 2,254 genes are regulated by both ingredient and all-trans retinol (Figure 1). Here, both retinol derivatives induce gene expression associated with keratinization, cornified envelope, keratinocyte differentiation, peptide cross-linking and sphingolipid biosynthesis, which are processes supporting skin barrier function and epidermal thickening through ceramide production and stratum corneum formation (Figure 2). Additionally, both inhibit gene expression linked to chemotaxis, ECM-receptor interaction, Golgi function, cell-cell junction organization, protein secretion processes. These regulations also point to increased keratinization and subsequently improved stratum corneum formation since it is suppressed here. These data show that gene expression induced by the ingredient is different from that induced by all-trans retinol. To demonstrate the advantages of the ingredient over all-trans retinol, we analyzed 493 genes regulated by the ingredient only and not by all-trans retinol (Figure 3). Among these, we found upregulation of extracellular matrix organization, genes associated with extracellular matrix and collagen, suggesting enhanced basal membrane formation and dermal-epidermal anchoring. Additionally, protein digestion and absorption were supported, which are critical for protecting skin cells from aging processes and improving stratum corneum formation during differentiation. In the next step, we examined whether the ingredient enhanced gene expression compared to all-trans retinol. Therefore, gene expression data for all-trans retinol was used as a control for normalization. With this, we found 843 genes differentially expressing compared to all-trans retinol (Figure 4). Here, the ingredient downregulates processes associated with extracellular matrix degradation, collagen catabolic processes and interferon-gamma signaling. This shows that the ingredient suppresses extracellular matrix and collagen degradation and is less irritating than all-trans retinol. On the other hand, it induces gene expression involved in cornified envelope, peptide cross-linking, keratinocyte differentiation and cell cycle, among others. Additionally, we compared gene expression of kallikreins, keratins and other genes related to keratinocyte differentiation. For example, keratin 1/10, keratin 4/13, filaggrin (Figure 5) were expressed at a higher rate in samples treated with retinol supporting the gene ontology data. This clearly demonstrates that the ingredient exhibits higher activity in terms of keratinocyte differentiation and thus improves epidermal thickening to a higher degree when used at equimolar concentration compared to all-trans retinol. After explaining the transcriptomic effects of the ingredient compared to all-trans retinol, its phenotypic efficacy was evaluated in an in vivo wrinkle study (Figure 6). The all-trans retinol formulation showed a significant reduction of 7.2% (P<0.05) reaching 9.5% in wrinkle intensity after 14 days of home use. Reduction in wrinkles and expression lines was observed in 100% of study subjects. For the ingredient, there was a significant reduction of 8.5% (P<0.05) reaching 11.0% in wrinkle intensity. It was possible to observe reduction in wrinkles and expression lines in 94% of study subjects. After 28 days of home use of the research product, compared to placebo-controlled skin, there was a significant reduction (P<0.05) of 12.6% reaching 15.6% in wrinkle intensity. It was possible to observe reduction in wrinkles and expression lines in 100% of study subjects. For the ingredient, compared to placebo-controlled skin, there was a significant reduction (P<0.05) of 13.4% reaching 17.0% in wrinkle intensity. It was possible to observe reduction in wrinkles and expression lines in 97% of study subjects. These data show the same efficacy in wrinkle reduction in vivo for both all-trans retinol and ingredient.

Discussion and Conclusion

This study compared the molecular and phenotypic effects of all-trans retinol and an alternative derivative based on pinacolone esters, the ingredient. Here, we were able to show that the ingredient demonstrates enhanced transcriptomic activity on keratinocyte differentiation and barrier formation compared to all-trans retinol. For example, the ingredient offers advantages over all-trans retinol because it increases extracellular matrix organization and collagen formation more to strengthen the basal membrane. It is less irritating because it suppresses interferon-mediated signaling. Additionally, it exhibits greater activity regarding keratinocyte differentiation and therefore improves epidermal thickening more when used at equimolar concentrations compared to all-trans retinol. In the in vivo wrinkle study, the ingredient and all-trans retinol cause significant wrinkle reduction with similar efficacy. With this study, we concluded that the ingredient is a safe and effective alternative ingredient for use in anti-aging cosmetic formulations. References 1 Heyman, R A, Mangelsdorf, D J, Dyck, J Aet al (1992) 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68(2) 397-406 2 Levin, A A, Sturzenbecker, L J, Kazmer, Set al (1992) 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature 355(6358) 359-361 3 Piskunov, A, & Rochette-Egly, C (2012) A retinoic acid receptor RAR pool present in membrane lipid rafts forms complexes with G protein Q to activate p38MAPK. Oncogene 31(28) 3333-3345 4 Masiá, S, Alvarez, S, de Lera, A Ret al (2007) Rapid, Nongenomic Actions of Retinoic Acid on Phosphatidylinositol-3-Kinase Signaling Pathway Mediated by the Retinoic Acid Receptor. Molecular Endocrinology 21(10) 2391-2402 5 Szymański, Ł, Skopek, R, Palusińska, Met al (2020) Retinoic Acid and Its Derivatives in Skin. Cells 9(12) 2660 6 Törmä, H (2011) Regulation of keratin expression by retinoids. Dermato-endocrinology 3(3) 136-140 7 Hubbard, B A, Unger, J G, & Rohrich, R J (2014) Reversal of Skin Aging with Topical Retinoids. Plastic and reconstructive surgery 133(4) 8 Baumann, L (2007) Skin ageing and its treatment. J Pathol 211(2) 241-251 9 Puizina-Ivic, N, Miric, L, Carija, Aet al Modern approach to topical treatment of aging skin. Coll Antropol 34(3) 1145-1153 10 Isoherranen, N, & Zhong, G (2019) Biochemical and physiological importance of the CYP26 retinoic acid hydroxylases. Pharmacology & Therapeutics 204 107400 11 Yin, S, Luo, J, Qian, Aet al (2013) Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity. Journal of Clinical Investigation 123(9) 3941- 3951 12 Varani, J, Fay, K, & Perone, P (2007) MDI 301, a non-irritating retinoid, induces changes in human skin that underlie repair. Archives of Dermatological Research 298(9) 439-448 Authors Maria Reichenbach, Symrise AG, Germany Michele Massironi, Cutech Srl, Italy Mickaël Larnicol, Symrise SAS, France Translated and Compiled By Tuğba Bayazıt Technical Application Specialist Africa, Middle East, Turkey Team Leader Symrise AG