Entwicklung innovativer Trägersysteme zur gezielten dermalen Applikation eines GATA-3-spezifischen DNAzyms als Therapeutikum der Atopischen Dermatitis

Atopic dermatitis (AD) is an inflammatory, chronically relapsing, non-contagious and pruritic skin disorder. AD affects 15–30% of children and 2–10% of adults in industrialized countries. AD is mediated by a strong Th2-cell response accompanied by the expression of various cytokines. The transcription factor GATA-3 which is mainly expressed in Th2-cells plays a key role in the regulation of these inflammatory processes. Inhibiting the activity of GATA 3 may therefore be a novel strategy for the treatment of allergic diseases such as AD. DNAzymes could be shown to be potent novel drugs for the treatment of inflammatory diseases such as atopic dermatitis. They represent a novel class of pharmaceuticals for causal therapies that are based on the interference with the specific pathway leading to the described inflammatory response. A DNAzyme-mediated cleavage of the GATA-3 specific mRNA aims for a suppression of the immune response leading to several known disorders like AD. There are two main challenges regarding the dermal application of DNAzymes: the relatively large molecular weight and the sensitivity to DNases which can be found on the skin surface as a part of the natural skin flora. A promising approach for the protection of DNAzymes is the development and application of suitable new carrier systems. In order to avoid degradation of the drug, the hydrophilic compound may be incorporated into the inner aqueous phase of carrier systems, such as water-in-oil-in-water (multiple) emulsions. In the present study, multiple emulsions and various other emulsions, such as small-scaled submicronemulsions, microemulsions and water-in-oil-emulsions were compared. Their physicochemical properties were determined and drug release as well as skin uptake studies using various skin conditions and experimental set-ups were conducted. Furthermore, cellular uptake was determined by flow cytometric analysis (FACS). DNAzyme uptake with finite dose set-up and application of the formulation under a gentle massage led to the superiority of the multiple emulsion over the submicronemulsion. Most likely the applied shear and partial evaporation of the outer water phase lead to a disruption of the oily membrane of the W/O/W emulsion, followed by the time dependent release of the DNAzyme and its successful penetration into the skin. In case of the submicronemulsion, the drug was reduced immediately by skin own DNases prior to penetration. DNAzymes target cells of the viable epidermis. In order to prove the uptake, FACS experiments using viable epidermal cells were performed. The data clearly demonstrates that DNAzyme uptake by the skin is not limited to the stratum corneum. Importantly, compared to the submicronemulsion, the specific drug uptake was increased using the W/O/W emulsion which is in line with the data obtained from skin penetration studies. The results could clearly show that antisense oligonucleotides protected in W/O/W emulsions represent a promising approach for a dermal therapy of atopic dermatitis.