Dorota Dobler

Ionic liquids as ingredients in topical drug delivery systems.

Because of their properties, ionic liquids (ILs) (Ranke et al.) offer many advantages in topical drug delivery systems. For example, ionic liquids can be used to increase the solubility of sparingly soluble drugs and to enhance their topical and transdermal delivery. Furthermore, ILs can be used either to synthesize active pharmaceutical ingredients or as antimicrobial ingredients. In the present work, the conventional oil-in-water (O/W) and water-in-oil (W/O) emulsions containing the hydrophilic IL [HMIM] [Cl] and the hydrophobic IL [BMIM] [PF6] were prepared, and the influence of the ILs on emulsion properties was evaluated. It was found that ILs could be successfully incorporated into the emulsion structure, resulting in stable formulations. The antimicrobial activity of ILs in the formulations was estimated, and their application as preservatives was confirmed by performing preservative efficacy tests. Evaluation of the in vitro cytotoxicity of the emulsions containing hydrophilic or hydrophobic ILs showed the low cytotoxicity of the carriers. Finally, penetration enhancement of a fluorescent dye as a model drug in the presence of ionic liquids was shown.

Development of multiple W/O/W emulsions as dermal carrier system for oligonucleotides: effect of additives on emulsion stability.

Multiple water-in-oil-in-water (W/O/W) emulsions are of major interest as potential skin delivery systems for water-soluble drugs like oligonucleotides due to their distinct encapsulation properties. However, multiple emulsions are highly sensitive in terms of variations of the individual components. The presence of osmotic active ingredients in the inner water phase is crucial for the generation of stable multiple emulsions. In order to stabilize the emulsions the influence of NaCl, MgSO(4), glucose and glycine and two cellulose derivatives was investigated. Briefly, multiple W/O/W emulsions using Span 80 as a lipophilic emulsifier and different hydrophilic emulsifiers (PEG-40/50 stearate, steareth-20 and polysorbate 80) were prepared. Stability of the emulsions was analyzed over a period of time using rheological measurements, droplet size observations and conductivity analysis. In this study we show that additives strongly influence the properties stability of multiple emulsions. By increasing the concentration of the osmotic active ingredients, smaller multiple droplets are formed and the viscosity is significantly increased. The thickening agents resulted in a slightly improved stability. The most promising emulsions were chosen and further evaluated for their suitability and compatibility to incorporate a DNAzyme oligonucleotide as active pharmaceutical ingredient.

Development of drug delivery systems for the dermal application of therapeutic DNAzymes

DNAzymes are potent novel drugs for the treatment of inflammatory diseases such as atopic dermatitis. DNAzymes represent a novel class of pharmaceuticals that fulfil a causal therapy by interruption of the inflammation cascade at its origin. There are two challenges regarding the dermal application of DNAzymes: the large molecular weight and the sensitivity to DNases as part of the natural skin flora. To overcome these limitations suitable carrier systems have to be considered. Nano-sized drug carrier systems (submicron emulsions, microemulsions) are known to improve the skin uptake of drugs due to their ability to interact with the skins lipids. To protect the drug against degradation, the hydrophilic drug may be incorporated into the inner aqueous phase of carrier systems, such as water-in-oil-in-water multiple emulsions. In the present study various emulsions of pharmaceutical grade were produced. Their physicochemical properties were determined and the influence of preservation systems on stability was tested. Drug release and skin uptake studies using various skin conditions and experimental set-ups were conducted. Furthermore, cellular uptake was determined by flow cytometric analysis. The investigations revealed that the developed multiple emulsion is a suitable and promising drug carrier system for the topical application of DNAzyme.

Protective effect of drug delivery systems against the enzymatic degradation of dermally applied DNAzyme

DNAzymes are a group of RNA-cleaving DNA oligonucleotides that contain a catalytic domain and represent a novel class of antisense molecules. Although single-stranded DNAzymes may represent the most effective nucleic acid drug to date, the sensitivity to nuclease degradation is challenging. Therefore, it is important to develop a drug delivery system, which protects the molecule against degradation during dermal application. In the present study, the potential protective effect, regarding the dermal application of DNAzyme, of multiple (W/O/W) emulsions, W/O emulsions, submicron emulsion and microemulsions were investigated using a HPLC method. The HPLC method enables the quantitative analysis of DNAzyme as well as the detection of degradation products. The differences between the activity of DNase I and the activity of nucleases located in the porcine skin were compared. It was found that the degradation of an aqueous solution of DNAzyme is depending on the DNase I activity as well as on the incubation time. Furthermore, the activity of neutral and acid nucleases in skin tissue was determined to be 5.2 and 14.8 U per 1 g of porcine skin tissue, respectively. Investigation of the protective character of different delivery systems revealed that formulations containing DNAzyme in the outer water phase (submicron emulsion and microemulsion) did not exhibit any form of protective effect, whereas formulations containing DNAzyme in the inner water phase (multiple emulsion and W/O emulsion) were able to prevent the DNAzyme degradation to a considerable degree. Consequently, these formulations are promising candidates for the dermal drug delivery of oligonucleotides.

In line monitoring of the preparation of water-in-oil-in-water (W/O/W) type multiple emulsions via dielectric spectroscopy

Multiple emulsions offer various applications in a wide range of fields such as pharmaceutical, cosmetics and food technology. Two features are known to yield a great influence on multiple emulsion quality and utility as encapsulation efficiency and prolonged stability. To achieve a prolonged stability, the production of the emulsions has to be observed and controlled, preferably in line. In line measurements provide available parameters in a short time frame without the need for the sample to be removed from the process stream, thereby enabling continuous process control. In this study, information about the physical state of multiple emulsions obtained from dielectric spectroscopy (DS) is evaluated for this purpose. Results from dielectric measurements performed in line during the production cycle are compared to theoretically expected results and to well established off line measurements. Thus, a first step to include the production of multiple emulsions into the process analytical technology (PAT) guidelines of the Food and Drug Administration (FDA) is achieved. DS proved to be beneficial in determining the crucial stopping criterion, which is essential in the production of multiple emulsions. The stopping of the process at a less-than-ideal point can severely lower the encapsulation efficiency and the stability, thereby lowering the quality of the emulsion. DS is also expected to provide further information about the multiple emulsion like encapsulation efficiency.

Development of a protective dermal drug delivery system for therapeutic DNAzymes

RNA-cleaving DNAzymes are a potential novel class of nucleic acid-based active pharmaceutical ingredients (API). However, developing an appropriate drug delivery system (DDS) that achieves high bioavailability is challenging. Especially in a dermal application, DNAzymes have to overcome physiological barriers composed of penetration barriers and degrading enzymes. The focus of the present study was the development of a protective and penetration-enhanced dermal DDS that was tailor made for DNAzymes. DNAzyme Dz13 was used as a potential API for topical therapy against actinic keratosis. In the progress of development and selection, different preservatives, submicron emulsions (SMEs) and the physiological pH range were validated with respect to the APIs integrity. A physicochemical stable SME of a pharmaceutical grade along with a high API integrity was achieved. Additionally, two developed protective systems, consisting of a liposomal formulation or chitosan-polyplexes, reduced the degradation of Dz13 in vitro. A combination of SME and polyplexes was finally validated at the skin and cellular level by in vitro model systems. Properties of penetration, degradation and distribution were determined. The result was enhanced skin penetration efficiency and increased cellular uptake with a high protective efficiency for DNAzymes due to the developed protective DDS.

Required HLB Determination of Some Pharmaceutical Oils in Submicron Emulsions

Different calculations of the hydrophilie-lipophilie balance (HLB) value of Sorbitan fatty acid ester are discussed in literature. Influence on the required HLB value of ethyl oleate was investigated using several emulsifier blends: Span 80/Tween 20, Oleth-3/Steareth-20, Oleth-5/Steareth-20, Oleth-3/PEG-40-Stearate. The calculations of the HLB value of Span 80 have a bearing influence on the determination of the required HLB value. The postulated transferability of the determined required HLB value by the Tween 20/Span 80 system compared to other emulsifier systems is depending on the used equation. Additionally, the required HLB values of the pharmaceutical oils coco-caprate/caprylate and cetearyl isononanoate were determined empirically by preparing submicron emulsions.

Degradation and protection of DNAzymes on human skin.

DNAzymes are catalytic nucleic acid based molecules that have become a new class of active pharmaceutical ingredients (API). Until now, five DNAzymes have entered clinical trials. Two of them were tested for topical application, whereby dermally applied DNAzymes had been prone to enzymatic degradation. To protect the DNAzymes the enzymatic activity of human skin has to be examined. Therefore, the enzymatic activity of human skin was qualitatively and quantitatively analyzed. Activity similar to that of DNase II could be identified and the specific activity was determined to be 0.59Units/mg. These results were used to develop an in vitro degradation assay to screen different kinds of protective systems on human skin. The chosen protective systems consisted of biodegradable chitosans or polyethylenimine, which forms polyplexes when combined with DNAzymes. The polyplexes were characterized in terms of particle size, zeta potential, stability and degree of complexation. The screening revealed that the protective efficiency of the polyplexes depended on the polycation and the charge ratio (ξ). At a critical ξ ratio between 1.0 and 4.1 and at a maximal zeta potential, sufficient protection of the DNAzyme was achieved. The results of this study will be helpful for the development of a protective dermal drug delivery systems using polyplexes.

Development of an insect metalloproteinase inhibitor drug carrier system for application in chronic wound infections

The insect metalloproteinase inhibitor (IMPI) represents the first peptide capable of inhibiting virulence‐mediating microbial M4‐metalloproteinases and is promising as a therapeutic. The purpose of this study was to develop a suitable drug carrier system for the IMPI drug to enable treatment of chronic wound infections. Specifically, we studied on poloxamer 407 hydrogels, examining the influence of several additives and preservatives on the rheological parameters of the hydrogels, the bioactivity and release of IMPI.

Effects of interference with GATA-3 expression by target-specific DNAzyme treatment on disease progression in a subacute oxazolone-induced mouse model of atopic dermatitis

Background DNAzymes represent a particular class of antisense molecules combining the specificity of antisense molecules with an inherent catalytic cleavage activity, which makes them an attractive tool for highly specific interference with target RNA molecules. In general, they are single-stranded DNA molecules with sequence-specific RNA-binding domains flanking a central catalytic domain. We developed and patented a DNAzyme named hgd40 – that targets the mRNA for GATA-3, the central transcription factor in T helper cell type 2 (Th2) differentiation and activation. For penetration enhancement and DNAzyme protection a specific water/oil/water emulsion for topical dermal application was developed and patented. Targeting GATA-3 might be a key for therapeutic intervention in predominantly Th2-driven diseases like atopic dermatitis.