ORIGINAL ARTICLE Isosorbide Di-(Linoleate/Oleate) Stimulates Prodifferentiation Gene Expression to Restore the Epidermal Barrier and Improve Skin Hydration Krzysztof Bojanowski1,2, William R. Swindell3, Shyla Cantor4 and Ratan K. Chaudhuri2,5 The breakdown of the epidermal barrier and consequent loss of skin hydration is a feature of skin aging and eczematous dermatitis. Few treatments, however, resolve these underlying processes to provide full symp- tomatic relief. In this study, we evaluated isosorbide di-(linoleate/oleate) (IDL), which was generated by esterifying isosorbide with sunflower fatty acids. Topical effects of IDL in skin were compared with those of ethyl linoleate/oleate, which has previously been shown to improve skin barrier function. Both IDL and ethyl linoleate/oleate downregulated inflammatory gene expression, but IDL more effectively upregulated the expression of genes associated with keratinocyte differentiation (e.g., KRT1, GRHL2, SPRR4). Consistent with this, IDL increased the abundance of epidermal barrier proteins (FLG and involucrin) and prevented cytokine- mediated stratum corneum degradation. IDL also downregulated the expression of unhealthy skin signature genes linked to the loss of epidermal homeostasis and uniquely repressed an IFN-inducible coexpression module activated in multiple skin diseases, including psoriasis. In a double-blind, placebo-controlled trial enrolling females with dry skin, 2% IDL lotion applied over 2 weeks significantly improved skin hydration and decreased transepidermal water loss (NCT04253704). These results demonstrate mechanisms by which IDL improves skin hydration and epidermal barrier function, supporting IDL as an effective intervention for the treatment of xerotic pruritic skin. Journal of Investigative Dermatology (2021) 141, 1416e1427; doi:10.1016/j.jid.2020.09.029 INTRODUCTION treatments and may be combined with topical corticosteroids Skin barrier defects may occur with aging or because of or oral antihistamines (Anderson and Dinulos, 2009; Elias chronic inflammatory skin diseases. This loss of barrier et al., 2019; Nolan and Marmur, 2012). Although formerly function can be accelerated by metabolic disorders (Aoki and viewed as inert and providing only a passive occlusive effect Murase, 2019; Okano et al., 2016; Yosipovitch et al., 2007) (Nygaard et al., 2017), more recent evidence suggests that or seasonal climate variation (Engebretsen et al., 2018) and petrolatum triggers the expression of genes encoding anti- may lead to increased systemic inflammation (Hu et al., microbial and epidermal barrier proteins (Czarnowicki et al., 2017). Epidermal barrier defects are also part of the patho- 2016). This has suggested new possibilities for next- genesis of atopic dermatitis, in which barrier defects com- generation occlusive treatments that alter skin physiology to bined with inflammation and environmental factors trigger restore skin’s endogenous barrier strength. symptoms, including intense pruritus that may lead to Phytochemicals from plant extracts can provide excellent lichenification and secondary infections (David Boothe et al., source material to develop novel occlusive agents with 2017). Symptomatic relief of dry skin (xerosis) is thus a barrier-building bioactivity. Sunflower oil, in particular, is common problem that must be addressed to prevent com- commonly used for topical applications because of its high plications. Petrolatum-based moisturizers are first-line linoleic acid (60e70%) content relative to oleic acid (20e30%) (Vaughn et al., 2018). Linoleic acid is an essential 1Sunny BioDiscovery, Santa Paula, California, USA; 2Symbionyx fatty acid for barrier recovery (Prottey et al., 1976), which can Pharmaceuticals, Boonton, New Jersey, USA; 3Department of Internal be generated from ethyl linoleate as a stable precursor Medicine, The Jewish Hospital, Cincinnati, Ohio, USA; 4Cantor Research (Hungund et al., 1995). In contrast, oleic acid disrupts the 5 Laboratories, Blauvelt, New York, USA; and Sytheon, Boonton, New skin barrier to enhance permeability (Mack Correa et al., Jersey, USA 2014). Sunflower oil therefore possesses a favorable bal- Correspondence: William R. Swindell, Department of Internal Medicine, The Jewish Hospital - Mercy Health, 4777 E. Galbraith Road, Cincinnati, Ohio ance of ingredients to improve hydration and stratum cor- 45236, USA. E-mail: [email protected] neum integrity in adults (Danby et al., 2013) and has been Abbreviations: CTL, control; DEM, differentially expressed module; EL, ethyl used to treat preterm infants with developmentally compro- linoleate/oleate; FDR, false discovery rate; IDL, isosorbide di-(linoleate/ mised barrier function (Darmstadt et al., 2002). Topical oleate); KC, keratinocyte; TEWL, transepidermal water loss; USS, unhealthy application of linoleic aciderich sunflower oil also reverses skin signature scaling in cutaneous essential fatty acid deficiency (Prottey Received 7 February 2020; revised 16 September 2020; accepted 20 September 2020; accepted manuscript published online 9 November 2020; et al., 1975). Moreover, there is a ceramide subgroup that corrected proof published online 24 December 2020 contains linoleic acid esterified to the omega carbon of 1416 Journal of Investigative Dermatology (2021), Volume 141 ª 2021 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. K Bojanowski et al. Isosorbide Di-(Linoleate/Oleate) Restores Barrier omega-hydroxy fatty acids, which are found solely in the immunity and leukocyte proliferation (Figure 1g and i) and epidermis and form membrane sheets crucial for skin barrier decreased the expression of genes associated with cell function (Rabionet et al., 2014). Linoleic acid and/or EL thus adhesion, skin development, and cell-cell adhesion have a wide range of skin care benefits, with evidence sup- (Figure 1h and j). Because eukaryotic gene expression is porting anti-inflammatory effects (Zhao et al., 2005) and ef- known to be modular (Dong and Horvath, 2007; Wang et al., ficacy for the treatment of acne (Charakida et al., 2007), 2008), we evaluated the effects of EL on the expression of 235 wounds (Pereira et al., 2008), and hyperpigmentation (Ko and predefined modules, each containing genes coexpressed in Kim Cho, 2018). human epidermis (Swindell et al., 2013)(Supplementary We previously demonstrated that isosorbide dicaprylate, Figure S2a). A total of 22 differentially expressed modules obtained by reacting isosorbide with octanoic (caprylic) acid, (DEMs) were identified (false discovery rate [FDR] < 0.05; 10 is effective for improving the epidermal barrier to promote EL-decreased, 12 EL-increased; Supplementary Figure S2b skin hydration (Chaudhuri and Bojanowski, 2017). However, and d). The DEM most strongly downregulated by EL was although isosorbide dicaprylate increased the expression of EPHA-40 (FDR ¼ 3eÀ06; Supplementary Figure S2bed), some keratinocyte (KC) differentiation proteins (e.g., AQP3, which included genes associated with epidermal differenti- CD44), it did not upregulate FLG mRNA or protein ation (e.g., GRHL1, DSC1, DSG1; Supplementary (Chaudhuri and Bojanowski, 2017). We therefore recently Figure S2e). The DEM most strongly upregulated by EL was developed a different isosorbide diester compound, iso- MIF-44 (FDR ¼ 4.58eÀ04; Supplementary Figure S2bed). An sorbide di-(linoleate/oleate) (IDL), synthesized by esterifying IFN-induced DEM containing antiviral genes (STAT1-57), isosorbide (obtained from corn) with fatty acids from sun- previously shown to be activated in nonmelanoma cancers flower seed oil. The use of sunflower seed oil in this context, and psoriasis lesions (Swindell et al., 2013), was upregulated rather than semipurified linoleic acid, was advantageous by EL (FDR ¼ 0.015; Supplementary Figure S2a and b). because linoleic acid undergoes air oxidation and thus lacks the stability desired for esterification (Yang et al., 2009). IDL IDL upregulates genes associated with differentiation and keratinization but downregulates genes linked to is a lipophilic compound that can be formulated into stable inflammation and adaptive immunity emulsions, hydro-lipid dispersions, and anhydrous systems. DNA microarrays were next used to evaluate gene expression As a recently developed compound, however, potential differences between EpiDermFT cultures treated with IDL (50 benefits in skin have not been well explored and mechanisms mg/ml) and those treated with 0.1% DMSO (CTL). Changes in of action with respect to KC differentiation and barrier for- gene expression were stronger with IDL than with EL treat- mation have not been demonstrated. ment (Supplementary Figure S1j). We identified 316 IDL- This study evaluated the molecular and cellular effects of increased genes (P < 0.05; 164 IDL-increased, 152 IDL and its clinical efficacy in human subjects with xerotic IDL-decreased) from comparisons to the CTL treatment skin. We used DNA microarrays to identify gene expression (Supplementary Figure S1i). Genes most strongly upregulated responses to IDL in a multilayered in vitro model of human by IDL included C4orf19, PAX9, and KLK12 (Figure 2b, c, dermis and epidermis (EpiDermFT; MatTek Life Sciences, and e), whereas genes most strongly downregulated by IDL Ashland, MA), and we compared these responses to those included PCDH20, GSTA2, and BPIFB4 (Figure 2b, d, and f). observed in cultures treated with ethyl linoleate/oleate (EL) IDL upregulated genes associated with epidermal differenti- (approximately 70% ethyl linoleate