Helmholtz Institute for Pharmaceutical Research Saarland

Saarbrücken, Germany

Helmholtz Institute for Pharmaceutical Research Saarland

Saarbrücken, Germany
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Schmidt C.,University Clinic Jena | Lautenschlaeger C.,University Clinic Jena | Collnot E.-M.,Saarland University | Collnot E.-M.,Helmholtz Institute for Pharmaceutical Research Saarland | And 6 more authors.
Journal of Controlled Release | Year: 2013

Most of the drugs used in the treatment of inflammatory bowel disease (IBD) become systemically bioavailable and potentially bear strong adverse effects. Targeting the inflamed areas of the intestine and keeping the drug localised at its site of action can reduce adverse effects. In animal studies, luminal uptake into inflamed mucosal areas has been shown to be size dependent. We investigated the potential of nano- and microparticle uptake into the rectal mucosa of human IBD patients. Fluorescently labelled placebo nanoparticles (NP) 250 nm in size and microparticles (MP) 3.0 μm in size were prepared. 2 h after rectal application to patients with Crohn's disease (CD) or ulcerative colitis (UC), confocal laser endomicroscopy was performed to visualise the particles in inflamed mucosal areas. In biopsies, ex vivo mucosal transport processes were investigated in miniaturised Ussing chambers. We examined 33 patients with IBD (19 patients with CD, 14 patients with UC) and 6 healthy controls. A significantly enhanced accumulation of MP in ulcerous lesions was observed (covered area = 1.28% (range 0.83%-3.45%) vs. 0% in controls; p = 0.011), while NP were visible only in traces on mucosal surfaces of all patients. The Ussing chamber experiments suggest persorption of particles through cellular voids; statistical significance was only reached for NP. Drug-containing particles may have great potential to more specifically target intestinal lesions to maximise therapeutic efficacy and minimise potential side effects. Nanoparticles may not be required for local drug delivery to intestinal lesions in humans, thereby minimising the risk of unintended translocation into the blood system. © 2012 Elsevier B.V.


Kann B.,Saarland University | Windbergs M.,Saarland University | Windbergs M.,Helmholtz Institute for Pharmaceutical Research Saarland | Windbergs M.,PharmBioTec GmbH
AAPS Journal | Year: 2013

Confocal Raman microscopy is an analytical technique with a steadily increasing impact in the field of pharmaceutics as the instrumental setup allows for nondestructive visualization of component distribution within drug delivery systems. Here, the attention is mainly focused on classic solid carrier systems like tablets, pellets, or extrudates. Due to the opacity of these systems, Raman analysis is restricted either to exterior surfaces or cross sections. As Raman spectra are only recorded from one focal plane at a time, the sample is usually altered to create a smooth and even surface. However, this manipulation can lead to misinterpretation of the analytical results. Here, we present a trendsetting approach to overcome these analytical pitfalls with a combination of confocal Raman microscopy and optical profilometry. By acquiring a topography profile of the sample area of interest prior to Raman spectroscopy, the profile height information allowed to level the focal plane to the sample surface for each spectrum acquisition. We first demonstrated the basic principle of this complementary approach in a case study using a tilted silica wafer. In a second step, we successfully adapted the two techniques to investigate an extrudate and a lyophilisate as two exemplary solid drug carrier systems. Component distribution analysis with the novel analytical approach was neither hampered by the curvature of the cylindrical extrudate nor the highly structured surface of the lyophilisate. Therefore, the combined analytical approach bears a great potential to be implemented in diversified fields of pharmaceutical sciences. © 2013 American Association of Pharmaceutical Scientists.


Siegl T.,Albert Ludwigs University of Freiburg | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2012

This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA and I-SceI systems). We will describe some new vectors recently developed for engineering of complex phenotypes in actinomycetes. Several site-specific recombinases, transposons, reporter genes and I-SceI endonuclease have been utilized for genome manipulation in actinomycetes. Novel molecular tools will help to overcome many technical difficulties and will encourage new efforts to address the function of actinomy-cete genes. © Springer Science+Business Media B.V. 2012.


Siegl T.,Albert Ludwigs University of Freiburg | Tokovenko B.,Helmholtz Institute for Pharmaceutical Research Saarland | Myronovskyi M.,Helmholtz Institute for Pharmaceutical Research Saarland | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Metabolic Engineering | Year: 2013

We developed a synthetic promoter library for actinomycetes based on the -10 and -35 consensus sequences of the constitutive and widely used ermEp1 promoter. The sequences located upstream, in between and downstream of these consensus sequences were randomised using degenerate primers and cloned into an integrative plasmid upstream of the gusA reporter gene. Using this system, we created promoters with strengths ranging from 2% to 319% compared with ermEp1. The strongest synthetic promoter was used in a proof-of-principle approach to achieve the overexpression of a natural type III polyketide synthase. We observed high correlation between the number of gusA reporter gene RNA-Seq reads and the GusA reporter protein activity, indicating that GusA is indeed a transcription-level reporter system. © 2013 Elsevier Inc.


Lehmann A.D.,University of Bern | Daum N.,Helmholtz Institute for Pharmaceutical Research Saarland | Bur M.,Helmholtz Institute for Pharmaceutical Research Saarland | Lehr C.-M.,Helmholtz Institute for Pharmaceutical Research Saarland | And 2 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2011

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm2) was higher than in A549 (172 ± 59 Ω cm2) but similar to 16HBE14o- cells (1469 ± 156 Ω cm2). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm2) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm2) and 16HBE14o- (558 ± 267 Ω cm2). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall. © 2010 Elsevier B.V. All rights reserved.


Negri M.,Saarland University | Negri M.,Helmholtz Institute for Pharmaceutical Research Saarland | Recanatini M.,University of Bologna | Hartmann R.W.,Saarland University | Hartmann R.W.,Helmholtz Institute for Pharmaceutical Research Saarland
PLoS ONE | Year: 2010

Background: Bisubstrate enzymes, such as 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), exist in solution as an ensemble of conformations. 17β-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. Methodology/Principal Findings: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17β-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. Conclusions/Significance: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the βFαG′-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17β-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17β-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17β-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis. © 2010 Negri et al.


Van Koppen C.J.,ElexoPharm GmbH | Hartmann R.W.,ElexoPharm GmbH | Hartmann R.W.,Saarland University | Hartmann R.W.,Helmholtz Institute for Pharmaceutical Research Saarland
Expert Opinion on Therapeutic Patents | Year: 2015

Introduction: About 2% of the Western world population suffer from chronic wounds, resulting from underlying disorders (e.g., diabetes, excessive pressure, vascular insufficiencies and vasculitis), with a significant adverse effect on Quality of Life. Despite high incidence and economic burden, management of chronic wounds is still far from effective and novel therapies are in urgent need. Wound healing is a dynamic process of transient expression, function and clearance of mediators, enzymes and cell types. Failure to initiate, terminate or regulate leads to pathologic wound healing.Areas covered: The present review discusses patents of the seven most promising classes of biological agents, mostly published in 2009-2014 (CYP11B1 inhibitors, peptide growth factors, prolyl-4-hydroxylase and matrix metalloproteinase inhibitors, bone marrow-derived mesenchymal stem cells, elastase and connexin43 inhibitors). Relevant information from peer-reviewed journals is also presented.Expert Opinion: The aforementioned biological agents have different mechanisms of action, and considering the multifactorial pathogenesis of chronic wounds, they hold promise in treating chronic wounds. However, as administration of a certain biological agent may be beneficial in an early phase, it may slow down wound healing in a later phase. Basic and clinical research on chronic wound healing should therefore investigate the efficacy of these agents, alone and in concert, during the consecutive phases of wound healing. © Informa UK, Ltd.


Myronovskyi M.,Helmholtz Institute for Pharmaceutical Research Saarland | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Applied Microbiology and Biotechnology | Year: 2013

The rational modification of the actinomycetes genomes has a variety of applications in research, medicine, and biotechnology. The use of site-specific recombinases allows generation of multiple mutations, large DNA deletions, integrations, and inversions and may lead to significant progress in all of these fields. Despite their huge potential, site-specific recombinase-based technologies have primarily been used for simple marker removal from a chromosome. In this review, we summarise the site-specific recombination approaches for genome engineering in various actinomycetes. © 2013 Springer-Verlag Berlin Heidelberg.


Salomon J.J.,Trinity College Dublin | Muchitsch V.E.,Trinity College Dublin | Gausterer J.C.,Trinity College Dublin | Schwagerus E.,Trinity College Dublin | And 4 more authors.
Molecular Pharmaceutics | Year: 2014

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP + uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm2, after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP+ was time-and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP+ uptake into NCl-H441 cells, whereas the effect of d-and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture. © 2014 American Chemical Society.


Bilyk B.,Helmholtz Institute for Pharmaceutical Research Saarland | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Applied Microbiology and Biotechnology | Year: 2014

The φC31-encoded recombination system has become a widely used tool for genetic analysis of streptomycetes, gene therapy and generation of transgenic animals. However, the application of this system, even in the context of its natural host genus, Streptomyces, may require a specific approach for each species. In this study, we have identified a novel pseudo-attB site, called pseB4, for integration of vectors using the φC31 system. More than 90 % of clones contained two copies of pSET152- or pOJ436-based cosmids, after their introduction into S. albus. The efficiency of the integration of φC31-based vectors into pseB4 is therefore comparable to that of the integration into attB. Moreover, in contrast with integration into the native attB, integration into pseB4 is not polar and does not require a complementary sequence in the TT-core region. Furthermore, an analysis of conjugation frequency revealed mutual inhibition of plasmid integration into either site when both the attB and pseB4 sites were present in the genome. © 2014 Springer-Verlag.

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