Thomas Schmidts

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.

Development and validation of an alternative disturbed skin model by mechanical abrasion to study drug penetration

Pharmaceuticals and cosmetics for dermal application are usually tested on healthy skin, although the primary permeation barrier, the stratum corneum, is often impaired by skin diseases or small skin lesions, especially on the hands. These skin conditions can considerably influence the permeation of chemicals and drugs. Furthermore, risk assessment for example of nanoparticles should be performed under various skin conditions to reflect the true circumstances. Therefore, an alternative and reproducible method for a high throughput of skin samples with impaired skin barrier was developed and verified by skin permeation studies (25 h) of caffeine, sorbic acid and testosterone compared to healthy (untreated) and tape-stripped skin. Skin barrier disruption was controlled by TEWL measurement. Skin permeation of the three substances was increased in tape-stripped and abraded skin compared to untreated skin due to the reduced barrier integrity. Enhancement of drug uptake was highest for the most hydrophilic substance, caffeine, followed by sorbic acid and lipophilic testosterone. No significant difference in drug uptake studies was observed between the new abrasion method with an aluminum-coated sponge and the tape-stripping method. The obtained results demonstrate that this abrasion method is an alternative way to achieve a disturbed skin barrier for drug and chemical uptake studies.

Development of a skin phantom of the epidermis and evaluation by using fluorescence techniques

The aim of this project was to develop a skin phantom that resembles the epidermis including the lipid matrix of the stratum corneum and the dermis. The main intent was to achieve optical properties similar to skin tissue. Therefore, two compartments of the skin, dermis and epidermis, were examined regarding their optical properties. Based on these results, the skin phantom was designed using relevant skin components. The scattering coefficient was measured by using Reflectance-based Confocal Microscopy (RCM) and the fluorescence spectrum was detected via confocal laser-scanning microscopy (CLSM). Prospective, the skin phantom can be used to incorporate various fluorescing chemicals, such as fluorescent dyes and fluorescent-labeled drugs to perform calibration measurements in wide-field and laser-scanning microscopes to provide a basis for the quantification of skin penetration studies.

Evaluation and quantification of spectral information in tissue by confocal microscopy

Abstract. A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

Feasibility of Monte Carlo simulations in quantitative tissue imaging

PURPOSE The feasibility of Monte Carlo simulations as a tool to facilitate quantitative image analysis is investigated by means of simulating light transport in skin phantoms. METHODS A Monte Carlo tool is used to compare if simulated fluorescent signals show agreement with measured data. The lipophilic fluorescent probe Nile Red and dedicated skin phantoms are also used in simulations to investigate the influence of the optical properties of the skin on the signal. RESULTS It is shown that the simulated and measured fluorescence signals show linear behavior up to a certain concentration of Nile Red. The simulations of the skin phantoms show the varying influence of single skin layers on the fluorescence signal. A calibration factor for quantitative analysis can be determined for the different skin layers. CONCLUSION Characterizing the influence of different media on imaging signals is a primary task in developing quantitative analysis methods. Monte Carlo simulations are a useful tool to investigate imaging properties of biological specimen where quantifying signals is important. However, detailed models must be provided.