PharmBioTec GmbH

Saarbrücken, Germany

PharmBioTec GmbH

Saarbrücken, Germany
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Franzen L.,Saarland University | Vidlarova L.,Saarland University | Vidlarova L.,Charles University | Kostka K.-H.,Caritas Krankenhaus | And 4 more authors.
Experimental Dermatology | Year: 2013

In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze-drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze-drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze-drying. Furthermore, we show that freeze-drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze-drying as a preserving preparation technique for in vitro testing of human skin. © 2013 John Wiley & Sons A/S.

Franzen L.,Saarland University | Mathes C.,Saarland University | Hansen S.,Saarland University | Hansen S.,Helmholtz Center for Infection Research | And 4 more authors.
Journal of Biomedical Optics | Year: 2013

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery. © 2013 SPIE.

Schmitt C.,Saarland University | Kail D.,PharmBioTec GmbH | Mariano M.,Saarland University | Empting M.,Helmholtz Institute For Pharmazeutische Forschung Saarland | And 5 more authors.
PLoS ONE | Year: 2014

The Dyrk family of protein kinases is implicated in the pathogenesis of several diseases, including cancer and neurodegeneration. Pharmacological inhibitors were mainly described for Dyrk1A so far, but in fewer cases for Dyrk1B, Dyrk2 or other isoforms. Herein, we report the development and optimization of 2,4-bisheterocyclic substituted thiophenes as a novel class of Dyrk inhibitors. The optimized hit compounds displayed favorable pharmacokinetic properties and high ligand efficiencies, and inhibited Dyrk1B in intact cells. In a larger selectivity screen, only Clk1 and Clk4 were identified as additional targets of compound 48, but no other kinases frequently reported as off-targets. Interestingly, Dyrk1A is implicated in the regulation of alternative splicing, a function shared with Clk1/Clk4; thus, some of the dual inhibitors might be useful as efficient splicing modulators. A further compound (29) inhibited Dyrk1A and 1B with an IC50 of 130 nM, showing a moderate selectivity over Dyrk2. Since penetration of the central nervous system (CNS) seems possible based on the physicochemical properties, this compound might serve as a lead for the development of potential therapeutic agents against glioblastoma. Furthermore, an inhibitor selective for Dyrk2 (24) was also identified, which might be are suitable as a pharmacological tool to dissect Dyrk2 isoform-mediated functions. © 2014 Schmitt et al.

Franzen L.,Saarland University | Windbergs M.,Saarland University | Windbergs M.,Helmholtz Center for Infection Research | Windbergs M.,PharmBioTec GmbH | And 2 more authors.
Skin Pharmacology and Physiology | Year: 2012

To perform accurate tape-stripping measurements and to control for site-specific and interindividual differences the amount of stratum corneum (SC) removed by each tape and the total SC thickness must be known. The purpose of this study was to evaluate the use of near-infrared (NIR) densitometry at λ = 850 nm for in situ determination of the total SC thickness. Quantitative tape stripping was performed on pig ear skin. The amount of SC removed by each tape was measured by NIR densitometry and by microprotein assay. Derived from the linear correlation between both measurements, a conversion factor was calculated that relates the individual NIR densitometry readings to the thickness of the SC on the corresponding tape (lSC-tape [μm] = (abs.850 - abs.850(blank))/23.9). The total SC thickness was determined based on the accumulated values of all tapes applied in quantitative tape stripping and compared to the values obtained from microscopic cross sections of biopsies. The total SC thickness was correctly determined by infrared densitometry independent of storage time and conditions (4°C up to 24 h; -21°C up to 3 months) in comparison with the standard histological evaluation. Copyright © 2012 S. Karger AG, Basel.

Gross P.C.,PharmBioTec GmbH | Burkart S.C.,PharmBioTec GmbH | Burkart S.C.,Saarland University | Muller R.,PharmBioTec GmbH | And 2 more authors.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2014

Purification and quality control of therapeutic peptides is often performed by one single method, RP-HPLC. As usage of an orthogonal technique is highly advisable for quality assurance, capillary electrophoresis (CE) employing a coated capillary coupled via a sheathless interface to a mass spectrometer was applied in parallel. The basic therapeutic peptide aviptadil served as a model substance to study the impurity profiles revealing 15 detectable impurities using CE-MS, two were detected by an appropriate nanoRP-HPLC-MS method. None of the impurities detected by CE were observed in LC and vice versa. The LOD in CE-MS was determined in the base peak electropherogram at ~1. fmol, a value 2500 times smaller than the LOD found in nanoRP-HPLC-MS (3. pmol). In nanoRP-HPLC-MS only 0.2% of the extrapolated CE-MS signal for a 25. ng aviptadil load was observed. We conclude that both, the LOD as well as the impurity profile of aviptadil, as analyzed by nanoRP-HPLC are influenced by both, the ligand-derivatized silica matrix and the flow-rate. Peptides may disappear completely and their variable emergence may lead to the determination of incorrect ratios as present in the sample. © 2013 .

Franzen L.,Saarland University | Windbergs M.,Saarland University | Windbergs M.,Helmholtz Center for Infection Research | Windbergs M.,PharmBioTec GmbH
Journal of Raman Spectroscopy | Year: 2014

The opportunity of label-free and non-destructive detection of substances inside human skin by confocal Raman microscopy represents a novel approach for investigating drug penetration and permeation. Several studies already introduced confocal Raman microscopy for depth profiling in skin; however, the reported results show high deviations. Thus, analysis of the spectral variability of human skin itself is a necessary prerequisite for systematic quantitative investigations of drug penetration and permeation by confocal Raman microscopy. In our work, we acquired Raman depth profiles from excised human skin samples after abdominal plastic surgery and investigated the absolute intensity fluctuation of four major skin derived Raman peaks. The results prove the expected high variability in spectral intensity, but we could not detect dissimilarities between different skin donors. A detailed analysis of the major endogenous skin components revealed differences in spatial distribution which consequently affects their individual applicability as reference peaks for relative depth profiling. Furthermore, we discovered an increase in signal variability in deeper stratum corneum layers, which has to be considered in future substance depth profiling investigations. In addition, we discovered an exponential decay of the Raman signal for all major skin components accounting for signal attenuation inside biological tissue. Based on this mathematical description, quantitative follow-up of substances in human skin can be realized. All in all, the results of this study elucidate the necessity of substantial understanding of endogenous spectral characteristics inside human skin as essential prerequisite for rational depth profiling of substances in human skin. Copyright © 2013 John Wiley & Sons, Ltd.

Franzen L.,Saarland University | Anderski J.,Saarland University | Windbergs M.,Saarland University | Windbergs M.,Helmholtz Center for Infection Research | Windbergs M.,PharmBioTec GmbH
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2015

For rational development and evaluation of dermal drug delivery, the knowledge of rate and extent of substance penetration into the human skin is essential. However, current analytical procedures are destructive, labor intense and lack a defined spatial resolution. In this context, confocal Raman microscopy bares the potential to overcome current limitations in drug depth profiling. Confocal Raman microscopy already proved its suitability for the acquisition of qualitative penetration profiles, but a comprehensive investigation regarding its suitability for quantitative measurements inside the human skin is still missing. In this work, we present a systematic validation study to deploy confocal Raman microscopy for quantitative drug depth profiling in human skin. After we validated our Raman microscopic setup, we successfully established an experimental procedure that allows correlating the Raman signal of a model drug with its controlled concentration in human skin. To overcome current drawbacks in drug depth profiling, we evaluated different modes of peak correlation for quantitative Raman measurements and offer a suitable operating procedure for quantitative drug depth profiling in human skin. In conclusion, we successfully demonstrate the potential of confocal Raman microscopy for quantitative drug depth profiling in human skin as valuable alternative to destructive state-of-the-art techniques. © 2015 Elsevier B.V. All rights reserved.

Franzen L.,Saarland University | Windbergs M.,Saarland University | Windbergs M.,Helmholtz Center for Infection Research | Windbergs M.,PharmBioTec GmbH
Advanced Drug Delivery Reviews | Year: 2015

In the field of skin research, confocal Raman microscopy is an upcoming analytical technique. Substantial technical progress in design and performance of the individual setup components like detectors and lasers as well as the combination with confocal microscopy enables chemically selective and non-destructive sample analysis with high spatial resolution in three dimensions. Due to these advantages, the technique bears tremendous potential for diverse skin applications ranging from the analysis of physiological component distribution in skin tissue and the diagnosis of pathological states up to biopharmaceutical investigations such as drug penetration kinetics within the different tissue layers.This review provides a comprehensive introduction about the basic principles of Raman microscopy highlighting the advantages and considering the limitations of the technique for skin applications. Subsequently, an overview about skin research studies applying Raman spectroscopy is given comprising various in vitro as well as in vivo implementations. Furthermore, the future perspective and potential of Raman microscopy in the field of skin research are discussed. © 2015 Elsevier B.V.

PubMed | PharmBioTec GmbH and Saarland University
Type: Journal Article | Journal: Microbial cell factories | Year: 2017

Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules.To demonstrate an impact of the chromosomal position effect on the heterologous expression of genes and gene clusters in Streptomyces albus J1074, a transposon mutant library with randomly distributed transposon that includes a -glucuronidase reporter gene was generated. Reporter gene expression levels have been shown to depend on the position on the chromosome. Using a combination of the transposon system and a C31-based vector, the aranciamycin biosynthetic cluster was introduced randomly into the S. albus genome. The production levels of aranciamycin varied up to eightfold depending on the location of the gene cluster within the chromosome of S. albus J1074. One of the isolated mutant strains with an artificially introduced attachment site produced approximately 50% more aranciamycin than strains with endogenous attBs.In this study, we demonstrate that expression of the reporter gene and aranciamycin biosynthetic cluster in Streptomyces albus J1074 varies up to eightfold depending on its position on the chromosome. The integration of the heterologous cluster into different locations on the chromosome may significantly influence the titre of the produced substance. This knowledge can be used for the more efficient engineering of Actinobacteria via the relocation of the biosynthetic gene clusters and insertion of additional copies of heterologous constructs in a suitable chromosomal position.

May S.,PharmBioTec GmbH | Jensen B.,Boehringer Ingelheim | Wolkenhauer M.,Boehringer Ingelheim | Schneider M.,PharmBioTec GmbH | And 4 more authors.
Pharmaceutical Research | Year: 2012

Purpose To evaluate different dissolution testing methods and subsequently develop a simple to perform but reproducible and discriminating dissolution technique for inhalative powders. Methods From a dry powder a fraction of aerosolized particles with an aerodynamic particle size below 5 μm was collected on regenerated cellulose membranes using an abbreviated Andersen cascade impactor. The membrane was then transferred to the respective dissolution set up either paddle apparatus with membrane holder, flow through cell or Franz diffusion cell. Results All tested dissolution techniques could discriminate between good and poorly soluble substances, but only the paddle apparatus differentiated between small variations of solubility. We showed that membrane coverage and particle diameter play an important role for the dissolution rate. The profiles were fitted with mathematical models (e.g., Weibull, first order) choosing the best fit for determination of the mean dissolution time. Furthermore, a correlation between the dissolution profiles obtained with Franz cell compared to paddle apparatus could be shown. Conclusion The paddle apparatus with membrane holder has the best discrimination power with optimal reproducibility. © Springer Science+Business Media, LLC 2012.

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