Accelerating topical formulation development for inflammatory dermatoses; an ex vivo human skin culture model consistent with clinical therapeutics.

Although animal models have been extensively used to evaluate human topical therapeutics, they exhibit marked physiological differences to human skin. Our objective was to develop a human ex vivo skin culture model to explore the pathophysiology of inflammatory dermatoses and for preclinical testing of potential therapeutic treatments. Ex vivo skin barrier integrity and metabolic activity was retained for 5 days and stimulation of T-helper cells (Th1), which produce proinflammatory cytokines, provided inflammatory responses similar to those reported from in vivo biopsy. Tissue responses to established therapies of pimecrolimus (Elidel) and clobetasol propionate (Dermovate) were evaluated using the human ex vivo skin culture, assessing pharmacodynamic changes in gene expression alongside the pharmacokinetics of drug penetration with both products showing time dependent efficacies. The translational utility of the human ex vivo skin culture model of inflammatory dermatoses was demonstrated through comparison with an in vivo clinical study, with similar reductions in inflammatory gene expression recorded for both drug treatments. Thus, this model can reduce, replace or refine animal testing and also mitigate the risk of failure in costly and time-consuming clinical trials associated with novel topical therapeutic development.

A new ex vivo skin model for mechanistic understanding of putative anti-inflammatory topical therapeutics.

Several in vitro models have been designed as test systems for inflammatory skin conditions, commonly using cell-culture or reconstructed human epidermis approaches. However, these systems poorly recapitulate the physiology and, importantly, the metabolism and biochemical activity of skin in vivo, whereas ex vivo skin culture models can retain these features of the tissue. Our objective was to develop a human ex vivo skin culture model to explore the pathophysiology of inflammatory dermatoses and for preclinical testing of potential therapeutic treatments. Following exogenous stimulation, tissue integrity and ability to induce inflammatory gene expression was retained, and stimulant concentrations and duration was optimised to mimic published data from inflammatory clinical biopsies of dermatitis and psoriasis patients. The validity and utility of the model was demonstrated when challenged with 5 drugs including a corticosteroid and vitamin D3 analogue, where inflammatory biomarkers were regulated in a manner consistent with the drugs' reported in vivo mechanisms of action. This model retains important inflammatory gene signals observed in human inflammatory dermatoses for preclinical evaluation of novel therapeutics.

RNA Aptamer Delivery through Intact Human Skin.

It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.