Mechanism of action of ingenol mebutate gel in reconstructed human skin assessed by transcriptomics.

Abstract

e19056 Background: Ingenol mebutate (IM) gel topical applied once daily for 2-3 consecutive days, has recently been approved by FDA in treating actinic keratoses (AK), as a safe, tolerable and effective treatment. IM, a highly active biological diterpene ester, has shown in vitro and in vivo to induce a dual mechanism of action characterized by, first, cell death induced at high micromolar concentrations and, second, a protein kinase C dependent immune activation in the nanomolar range. The aim of this study was to investigate the mechanism of action of IM in reconstructed human skin (RHS). Methods: IM 0.05% gel or vehicle was applied topically to RHS (EpiDermFT) and normal pigskin and IM concentrations were measured after 2 and 24 hrs. In RHS, biopsies were obtained 1, 6 and 24 hrs after treatment and global transcriptomic profiling (Affymetrix HG 1.0 ST) was assessed. The analysis included transcription factor binding site (TFBS) and gene set enrichment analysis (GSEA). Results: We compared IM’s penetration through normal skin to RHS and found a 10-fold higher permeability in RHS, which likely models the increased permeability of AK lesions. Consequently we further investigated IM’s effects on RHS. Based on GSEA, we identified the major effects of IM to comprise: cell death, inflammation and wound healing responses, indicated by differential expression of e.g. caspases, DAPK1, chemokine ligands, HAS2, MMPs, SERPINs, ILs (and their receptors). A TFBS analysis identified E2F regulated genes as overrepresented, albeit in opposite directions in dermis (up-regulated) compared to epidermis (down-regulated). IM transiently activates PKC, and via this activation causes transcriptional silencing of E2F-responsive genes. The differential effect on dermis and epidermis could be due to different levels of PKC isoforms in the different skin layers. Indeed, a specific, inflammatory response was observed and several genes, including MMP10 and IL1R2, displayed a time-dependent increase in expression, reaching a maximum at 24h. Conclusions: In conclusion, IM displays pleiotropic effects, of which many are identified on a transcriptional level. Our data suggest involvement of in particular PKC, ERK2, and E2F regulated genes.