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Preclinical assessment of dual CYP26[A1/B1] inhibitor, DX308, as an improved treatment for keratinization disorders - PubMed

  • ️Fri Jan 01 2021

Preclinical assessment of dual CYP26[A1/B1] inhibitor, DX308, as an improved treatment for keratinization disorders

J G S Veit et al. Skin Health Dis. 2021.

Abstract

Background: Retinoid-based therapies are commonly used in the treatment of disorders of keratinization and other skin disorders but can result in non-specific effects and adverse reactions. Use of retinoic acid metabolism blocking agents (RAMBAs) such as DX308 may address these shortcomings.

Objectives: Characterize the therapeutic potential of recently discovered, CYP26-selective RAMBA, DX308.

Materials and methods: Preliminary in vitro assessment of potential off-target activity, metabolic and toxicologic profiling. Studies to assess safety and efficacy of topical treatment in correcting abnormal skin morphology in rhino mice. Extensive gene expression profiling by RNA sequencing and qPCR in 3D epidermis grown with keratinocytes (KCs) from keratinization disorders and healthy controls, to investigate modulation of retinoid biopathways.

Results: In vitro, DX308 does not interact with off-target nuclear receptors or CYP450s, is not genotoxic, and is stable in skin, despite vigorous hepatic metabolism. In vivo, topical DX308 induces comedolysis and epidermal thickening without apparent adverse effects. Gene expression profiling shows potent modulation of retinoid-responsive genes by DX308 in both healthy and keratinization disorder KCs. Pathway analysis suggests DX308 may inhibit inflammatory and immune responses in KCs.

Conclusions: These preliminary studies suggest that DX308 is an efficacious topical therapeutic with a favourable metabolic and safety profiles. DX308 may present an improved therapeutic alternative for the treatment of keratinization disorders and other retinoid-responsive skin ailments.

© 2021 The Authors. Skin Health and Disease published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.

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Conflict of interest statement

P. Diaz is cofounder of DermaXon™ and inventor of the technology; he and The University of Montana are entitled to future royalty payments. D. Mendes, J. Kreitinger and L. Walker were employed by DermaXon™ during their contributions to the study. J.G.S. Veit was employed at DermaXon™ during a portion of this study.

Figures

FIGURE 1
FIGURE 1

Topical in vitro skin irritation testing and long‐term systemic in vivo effect on mouse weights. (a) In vitro skin irritation test in reconstructed human epidermis treated with topical DX308. Dosage of 0.07%–0.35% corresponds to approximately 2–10 mM (n = 3 per group; mean ± SD; *p ≤ 0.05, ***p ≤ 0.001; one‐way ANOVA with Dunnett's correction for multiple comparisons vs. vehicle control). (b) Weight of mice injected 3× per week with intraperitoneal DX308. SD shown in single direction to maintain figure clarity (n = 6–7 per group, mean ± SD). SD, standard deviation; SDS, sodium dodecyl sulphate

FIGURE 2
FIGURE 2

Efficacy of topical DX308 in rhino mouse model. (a) Representative image of H&E‐stained rhino mouse skin following treatment. Yellow inlay describes how comedonal profile (ratio of the comedo opening [d] to inner diameter [D]) and epidermal thickness (area of epidermis [yellow] divided by length of underlying basal layer [dotted green line]) were measured. Scale bars = 200 µm. (b) Treatment‐induced changes in epidermal thickness, comedonal profile, and comedones/cm are shown. Untreated samples are subject‐paired back tissue which received no vehicle or treatment (n = 5–6 mice per group; mean +  SD; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; two‐way repeated measures [subject‐paired] ANOVA with Bonferroni's correction vs. *vehicle or ^untreated skin). (c) AUC of TEWL over treatment period (n = 5–6 mice per group; mean ± SD; ***p ≤ 0.001; one‐way ANOVA with Dunnett's correction vs. vehicle). AUC, area under the curve; SD, standard deviation; TEWL, transepidermal water loss

FIGURE 3
FIGURE 3

RNA sequencing of healthy reconstructed human epidermis treated with DX308 and atRA. (a) Venn diagram of overlap in significantly DETs by treatment (values represent number of significantly expressed transcripts per group). (b) Hierarchical clustering of all significantly DETs. Expression shown in standardized TPM. Significantly DETs are those with a |fold‐change| ≥2 and FDR <0.05. atRA, all‐trans retinoic acid; DETs, differentially expressed transcripts; FDR, false discovery rate; TPM, transcripts per million

FIGURE 4
FIGURE 4

Predicted inhibition of inflammatory and immune response pathways in healthy reconstructed human epidermis treated with DX308 and atRA. Ingenuity pathway analysis of all significantly differentially expressed transcripts (|fold‐change| ≥2 and FDR <0.05) revealed a predicted inhibition of (a) inflammatory and (b) immune response by 1000 nM DX308 cotreated with 1 nM atRA. atRA, all‐trans retinoic acid; FDR, false discovery rate

FIGURE 5
FIGURE 5

Effects of DX308 and atRA on morphology and heparin‐binding EGF‐like growth factor (HBEGF) and keratin 10 (KRT10) localization in RHE. (a) H&E (left), HBEGF (middle) and KRT10 (right) staining in healthy RHE which were exposed to treatments for 4 days. Dotted white line illustrates the top of the basal keratinocyte layer. *Respective H&E and HBEGF stained RHE were treated at 100 nM DX308, KRT10 stained RHE were treated with 1000 nM DX308. Scale bars; black = 20 µm, white = 50 µm. (b) Fold‐change in HBEGF and KRT10 gene expression by RNA sequencing in healthy RHE. All non‐grey cells are significantly differentially expressed versus control (FDR ≤ 0.05). atRA, all‐trans retinoic acid; FDR, false discovery rate; RHE, reconstructed human epidermis

FIGURE 6
FIGURE 6

Expression of retinoid‐responsive genes CYP26A1, KRT10 and HBEGF in NAK and DD RHE; NAK, RXLI and LI monolayer keratinocytes. Values are shown as fold‐change in mRNA expression from vehicle control. *CYP26A1 mRNA was undetectable in control samples so a cycle threshold of 40 was assumed to calculate an estimate of the minimum fold‐change in expression from control for the treatment groups. NAK RHE, n = 3–5 (across three donors); DD RHE, n = 3; NAK ML, n = 2; RXLI ML, n = 3; LI‐1 ML, n = 3; LI‐2 ML, n = 3. atRA, all‐trans retinoic acid; DD, Darier disease; DX308+, DX308 + 1 nM atRA; LI, lamellar ichthyosis; Liarozole+, Liarozole + 1 nM atRA; ML, monolayer; NAK, Normal Adult Keratinocytes; RHE, reconstructed human epidermis; RXLI, recessive x‐linked ichthyosis

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